figf1a , Sections of the thymus and muscle of fed and starved GFP - LC3 mice were stained with anti - GFP Alexa 488 antibody ( green ) and 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Images are representative of n = 6 . b , Thymic stromal cells from GFP - LC3 mice were fixed onto glass slides and stained with anti - GFP Alexa 488 and DAPI . Typical autophagy - positive cTECs and mTEChi are shown . The average frequencies ± s . d . of cTECs and mTEChi scoring autophagy positive ( that is , ≥ 5 autophagosomes ) in two independent preparations are indicated . Less than 1 % of dendritic cells ( DCs ) and mTEClo were positive . c , Relative distribution of the number of autophagosomes in autophagy - positive cTECs and mTEChi ; a . u . , arbitrary units .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

figf1a, Sections of the thymus and muscle of fed and starved GFP-LC3 mice were stained with anti-GFP Alexa 488 antibody (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of n = 6.b, Thymic stromal cells from GFP-LC3 mice were fixed onto glass slides and stained with anti-GFP Alexa 488 and DAPI. Typical autophagy-positive cTECs and mTEChi are shown. The average frequencies ± s.d. of cTECs and mTEChi scoring autophagy positive (that is, ≥5 autophagosomes) in two independent preparations are indicated. Less than 1% of dendritic cells (DCs) and mTEClo were positive.c, Relative distribution of the number of autophagosomes in autophagy-positive cTECs and mTEChi; a.u., arbitrary units.


figf2a , Thymi from wild - type ( WT ) or Atg5 - / - embryos were transplanted under the kidney capsule of wild - type recipients and analysed 6 weeks later . The average cellularities ± s . d . are shown ; n = 5 . b , Normal compartmentalization of Atg5 - / - grafts in cytokeratin - 8 - positive ( red ; K8 ) cortical and cytokeratin - 5 - positive ( green ; K5 ) medullary regions ; n = 8 . c , Expression of MHC class I and II on cTECs or mTECs from Atg5 - / - to wild - type ( WT ) grafts ( red ) , or wild - type to wild - type grafts ( blue ) . Staining controls are depicted in grey ; data are representative of three experiments . dd , RT - PCR analysis of autoimmune regulator ( Aire ) and TRA expression in mTECs isolated from Atg5 - / - to wild - type , or wild - type to wild - type grafts . Expression of β - actin ( Actb ) , intestinal trefoil factor ( Tff3 ) , casein 2 ( Csn2 ) and salivary protein 1 ( Spt1 ) are also shown ( 5 - fold serial dilutions ) . Data are representative of two experiments .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2a, Thymi from wild-type (WT) or Atg5-/- embryos were transplanted under the kidney capsule of wild-type recipients and analysed 6 weeks later. The average cellularities ± s.d. are shown; n = 5.b, Normal compartmentalization of Atg5-/- grafts in cytokeratin-8-positive (red; K8) cortical and cytokeratin-5-positive (green; K5) medullary regions; n = 8.c, Expression of MHC class I and II on cTECs or mTECs from Atg5-/- to wild-type (WT) grafts (red), or wild-type to wild-type grafts (blue). Staining controls are depicted in grey; data are representative of three experiments. dd, RT-PCR analysis of autoimmune regulator (Aire) and TRA expression in mTECs isolated from Atg5-/- to wild-type, or wild-type to wild-type grafts. Expression of β-actin (Actb), intestinal trefoil factor (Tff3), casein 2 (Csn2) and salivary protein 1 (Spt1) are also shown (5-fold serial dilutions). Data are representative of two experiments.


figf3a , Polyclonal T - cell development in Atg5 - / - to wild - type ( WT ) , or wild - type to wild - type grafts . The top dot plots show CD4 and CD8 staining ; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes ; n ≥ 5 . b , Atg5 - / - or wild - type lobes were grafted into TCR - HA transgenic recipients . Graft cellularity ± s . d . and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of TCR - HA + cells among CD4 SP cells was 32 . 1 % ± 3 . 2 % in wild - type to TCR - HA , and 19 . 6 % ± 4 . 3 % in Atg5 - / - to TCR - HAlobes ; P = 1 . 8 × 10 - 8 ( n ≥ 14 ) . c , Atg5 - / - or wild - type lobes were grafted into AND transgenic mice . Graft cellularity ± s . d . and the expression of the transgenic TCR ( Vb3 - Va11 ) on CD4 SP cells were analysed after 6 weeks . The frequency ± s . d . of Vb3 - Va11 + CD4 SP cells was 93 . 5 % ± 0 . 2 % in wild - type to ANDlobes ( n = 3 ) and 97 . 9 % ± 1 . 1 % ( n = 6 ) in Atg5 - / - to ANDlobes ( P = 9 × 10 - 4 ) . d , Thymi from wild - type or Atg5 - / - embryos were grafted into P14 , OT - I or HY TCR - transgenic recipients . The dot plots show the expression of the transgenic TCR V - segments on CD8 SP cells . Thymus cellularities ± s . d . are shown in the respective bar diagrams . Data are representative of n = 4 ( P14 ) , n ≥ 4 ( OT - I ) and n ≥ 2 ( HY ) . e , Thymi from wild - type or ( BALB / c3C57BL / 6 ) F1 Atg5 - / - embryos were transplanted into ( BALB / c3C57BL / 6 ) F1 recipients . Six weeks after grafting , expression of the I - Ea52 - 68 - I - Ab complex on cTECs was analysed using the monoclonal antibody Y - Ae . Atg5 - / - cTECs are shown in red ; wild - type cTECs are shown in blue . Control stainings are shown in orange ( Atg5 - / - ) and green ( wild type ) . Data are representative of two independent experiments .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3a, Polyclonal T-cell development in Atg5-/- to wild-type (WT), or wild-type to wild-type grafts. The top dot plots show CD4 and CD8 staining; the bottom dot plots show expression of CD25 and Foxp3 by CD4 SP thymocytes; n ≥ 5.b, Atg5-/- or wild-type lobes were grafted into TCR-HA transgenic recipients. Graft cellularity ± s.d. and the expression of the transgenic TCR on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of TCR-HA+ cells among CD4 SP cells was 32.1% ± 3.2% in wild-type to TCR-HA, and 19.6% ± 4.3% in Atg5-/- to TCR-HAlobes; P = 1.8 × 10-8 (n ≥ 14).c, Atg5-/- or wild-type lobes were grafted into AND transgenic mice. Graft cellularity ± s.d. and the expression of the transgenic TCR (Vb3-Va11) on CD4 SP cells were analysed after 6 weeks. The frequency ± s.d. of Vb3-Va11+ CD4 SP cells was 93.5% ± 0.2% in wild-type to ANDlobes (n = 3) and 97.9% ± 1.1% (n = 6) in Atg5-/- to ANDlobes (P = 9 × 10-4).d, Thymi from wild-type or Atg5-/- embryos were grafted into P14, OT-I or HY TCR-transgenic recipients. The dot plots show the expression of the transgenic TCR V-segments on CD8 SP cells. Thymus cellularities ± s.d. are shown in the respective bar diagrams. Data are representative of n = 4 (P14), n ≥ 4 (OT-I) and n ≥ 2 (HY).e, Thymi from wild-type or (BALB/c3C57BL/6)F1 Atg5-/- embryos were transplanted into (BALB/c3C57BL/6)F1 recipients. Six weeks after grafting, expression of the I-Ea52-68-I-Ab complex on cTECs was analysed using the monoclonal antibody Y-Ae. Atg5-/- cTECs are shown in red; wild-type cTECs are shown in blue. Control stainings are shown in orange (Atg5-/-) and green (wild type). Data are representative of two independent experiments.


figf4Atg5 - / - or wild - type ( WT ) lobes were grafted into athymic ( nu / nu ) mice ( nu / nuAtg5 - / - or nu / nuWT , respectively ) . Data are representative of three experiments with n ≥ 4 . a , Expression of activation markers on lymph node CD4 T cells in nu / nuAtg5 - / - or nu / nuWT chimaeras . b , The changes in body weight over time are shown . c , The appearance of nu / nuAtg5 - / - or nu / nuWT chimaeras . The colon ( arrow ) and uterus ( arrowhead ) are highlighted ; note the absence of fat pads ( asterisk ) in nu / nuAtg5 - / - chimaeras . d , Haematoxylin and eosin staining of organs from nu / nuAtg5 - / - or nu / nuWT chimaeras . Arrows indicate infiltrates .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

figf4Atg5-/- or wild-type (WT) lobes were grafted into athymic (nu/nu) mice (nu/nuAtg5-/- or nu/nuWT, respectively). Data are representative of three experiments with n ≥ 4. a, Expression of activation markers on lymph node CD4 T cells in nu/nuAtg5-/- or nu/nuWT chimaeras.b, The changes in body weight over time are shown.c, The appearance of nu/nuAtg5-/- or nu/nuWT chimaeras. The colon (arrow) and uterus (arrowhead) are highlighted; note the absence of fat pads (asterisk) in nu/nuAtg5-/- chimaeras.d, Haematoxylin and eosin staining of organs from nu/nuAtg5-/- or nu/nuWT chimaeras. Arrows indicate infiltrates.


Figure 1A . Representative images of immunofluorescence of α - smooth muscle actin ( α - SMA ) , pan Cytokeratin , Pan - hMENA , hMENA11a and E - cadherin expression in CAFs and autologous cancer cells ( Ep - PDAC ) obtained from enzymatically digested primary PDAC tissue of patient # 36 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 20 μm . B . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal fibroblasts derived from transplant donor ( P - NF ) , pancreatic serous cystadenoma # 71 and cancer associated fibroblasts ( n = 10 ) obtained from primary PDAC tissues . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression . Quantification of P # 110 is relative to the sample shown in the WB on the left . C . Representative immunoblot ( top ) of hMENA / hMENA ∆ v6 expression levels ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) in normal lung fibroblasts ( L - NF ) , cancer associated fibroblasts obtained from NSCLC tissues ( n = 4 ) and normal dermal fibroblasts ( HNF ) . Densitometry quantified data ( bottom ) of hMENA ∆ v6 expression .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Representative images of immunofluorescence of α-smooth muscle actin (α-SMA), pan Cytokeratin, Pan-hMENA, hMENA11a and E-cadherin expression in CAFs and autologous cancer cells (Ep-PDAC) obtained from enzymatically digested primary PDAC tissue of patient #36. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.B. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal fibroblasts derived from transplant donor (P-NF), pancreatic serous cystadenoma #71 and cancer associated fibroblasts (n=10) obtained from primary PDAC tissues. Densitometry quantified data (bottom) of hMENA∆v6 expression. Quantification of P#110 is relative to the sample shown in the WB on the left. C. Representative immunoblot (top) of hMENA/hMENA∆v6 expression levels (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) in normal lung fibroblasts (L-NF), cancer associated fibroblasts obtained from NSCLC tissues (n=4) and normal dermal fibroblasts (HNF). Densitometry quantified data (bottom) of hMENA∆v6 expression.


Figure 2A . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in the primary PDAC tissue of patient # 138 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . αSMA ‑ positive CAFs are also positive for Pan - hMENA ( arrow ) . B . Representative images of immunofluorescence of Pan - hMENA ( yellow ) and α - SMA ( red ) in NSCLC case # 484 from whom high hMENA ∆ v6 CAFs were obtained . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 50 μm . The inset of the dashed area is provided on the right as a zoomed - in and cropped fluorescence image . Scale bar : 20 μm . As in A , α - SMA signal is evident in stromal cells which are also positive for Pan - hMENA ( arrow ) . C . Boxplots showing the mean mRNA expression of ENAH gene ( hMENA ) for 22 patients in the 10 groups of cell types ( Peng et al . , 2019 ) ( total number of cells : 38487 , mean of cells for patient : 1603 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Expressions from each cell - type group were compared to all other groups by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method . Stromal cell - type groups with significantly up - regulated ENAH expression respect to other stromal groups are : Fibroblast , * * * q = 0 . 0002 ; Stellate , * * * q = 1 . 5e - 07 . Non - stromal cell - type groups with significantly up - regulated ENAH expression with respect to other non - stromal groups are : Ductal 1 , * * q = 0 . 0013 . D . Boxplots showing the mRNA expression of ENAH gene ( hMENA ) in the 7 groups of cell types from ( Lambrechts et al . , 2018 ) ( total n = 52 ) . Shown in each boxplot are the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) . Cell expression from each group were compared to all other stromal / not - stromal cells by using Mann - Whitney U - test ( two - sided ) and P - values were adjusted for multiple testing using the Benjamini - Hochberg method ( Fibroblast group vs other stromal groups , * * q = 0 . 0022 ; Other comparisons , q > 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in the primary PDAC tissue of patient #138 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. αSMA‑positive CAFs are also positive for Pan-hMENA (arrow).B. Representative images of immunofluorescence of Pan-hMENA (yellow) and α-SMA (red) in NSCLC case #484 from whom high hMENA∆v6 CAFs were obtained. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 μm. The inset of the dashed area is provided on the right as a zoomed-in and cropped fluorescence image. Scale bar: 20 μm. As in A, α-SMA signal is evident in stromal cells which are also positive for Pan-hMENA (arrow).C. Boxplots showing the mean mRNA expression of ENAH gene (hMENA) for 22 patients in the 10 groups of cell types (Peng et al., 2019) (total number of cells: 38487, mean of cells for patient: 1603). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Expressions from each cell-type group were compared to all other groups by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Stromal cell-type groups with significantly up-regulated ENAH expression respect to other stromal groups are: Fibroblast, ***q=0.0002; Stellate, ***q= 1.5e-07. Non-stromal cell-type groups with significantly up-regulated ENAH expression with respect to other non-stromal groups are: Ductal 1, **q=0.0013.D. Boxplots showing the mRNA expression of ENAH gene (hMENA) in the 7 groups of cell types from (Lambrechts et al., 2018) (total n=52). Shown in each boxplot are the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line). Cell expression from each group were compared to all other stromal/not-stromal cells by using Mann-Whitney U-test (two-sided) and P-values were adjusted for multiple testing using the Benjamini-Hochberg method (Fibroblast group vs other stromal groups, **q=0.0022; Other comparisons, q>0.05


Figure 3A . Quantification of in vitro matrigel invasion assay ( bottom ) of P - CAF and L - CAF ( P - CAF # 36 , 138 and L - CAF # 189 , 484 ) transfected with control siRNA ( CNT ) or hMENA siRNA ( hMENA ( t ) ) indicating that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 reduces the invasive ability of CAFs with respect to siCNT CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot showing hMENA / hMENAΔv6 expression ( detected by Pan - hMENA mAb and by the specific anti - hMENAΔv6 antibody ) of the CAFs employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 . B . Quantification of in vitro matrigel invasion assay ( bottom ) of P - NF and L - NF and P - CAF # 110 and L - CAF # 400 transfected with control or hMENA ∆ v6 expressing vectors , demonstrating that the overexpression of hMENA ∆ v6 isoform induced the invasiveness of P - NFs and L - NFs and / or P - and L - CAFs . Number of invading cells was measured by counting 6 random fields . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Immunoblot of hMENAΔv6 expression ( detected by the specific anti - hMENAΔv6 antibody ) in fibroblasts employed is reported ( top ) . TUBULIN was used as loading control . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Quantification of in vitro matrigel invasion assay (bottom) of P-CAF and L-CAF (P-CAF # 36, 138 and L-CAF #189, 484) transfected with control siRNA (CNT) or hMENA siRNA (hMENA(t)) indicating that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 reduces the invasive ability of CAFs with respect to siCNT CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot showing hMENA/hMENAΔv6 expression (detected by Pan-hMENA mAb and by the specific anti-hMENAΔv6 antibody) of the CAFs employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.B. Quantification of in vitro matrigel invasion assay (bottom) of P-NF and L-NF and P-CAF#110 and L-CAF#400 transfected with control or hMENA∆v6 expressing vectors, demonstrating that the overexpression of hMENA∆v6 isoform induced the invasiveness of P-NFs and L-NFs and/or P-and L-CAFs. Number of invading cells was measured by counting 6 random fields. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Immunoblot of hMENAΔv6 expression (detected by the specific anti-hMENAΔv6 antibody) in fibroblasts employed is reported (top). TUBULIN was used as loading control. P values were calculated by two‐sided Student's t‐test. *P<0.05, ***P<0.001.


Figure 4A . Quantification of in vitro matrigel invasion assay of PANC - 1 cells cultured for 48 h with conditioned media ( CM ) of NFs ( P - NFs - CM ) , CAF low # 44 and # 110 and CAFs high # 36 and 138 . Histograms show the number of invading cells measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates , performed at least in duplicate each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . B . Quantification of in vitro matrigel invasion assay of PANC - 1 cultured for 48 h with CM derived from control P - CAFs # 36 ( siCNT - P - CAF - CM # 36 ) and hMENA / hMENA ∆ v6 silenced P - CAFs ( sihMENA ( t ) - P - CAF - CM # 36 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects PANC - 1 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 . C . Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media ( culture medium ) or conditioned media ( CM ) of L - CAF low # 400 and CAFs high # 189 , as described above . Data are presented as the mean ± SD of two biological replicates , performed in triplicates each . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 . D . Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L - CAFs # 484 ( siCNT - L - CAF - CM # 484 ) and hMENA / hMENA ∆ v6 silenced L - CAFs ( sihMENA ( t ) - L - CAF - CM # 484 ) , showing that the siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 affects H1975 invasive ability mediated by CAF - CM . Culture medium ( DMEM ) was used as control . Cells invading matrigel were counted in 6 random fields . Data are presented as the mean ± SD of six replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 006 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 . E . Representative immunoblot ( top ) and densitometry quantification ( bottom ) of hMENA ∆ v6 expression level in P - NFs grown in RPMI control medium ( - ) or PANC - 1 - CM for 24 h ( n = 3 ) . Data are represented as fold increase with respect to control medium ± SD ( set as 1 ) . Data were analyzed using two ‐ sided Student ' s t ‐ test . * P < 0 . 05 . F . Representative images ( top ) and quantification ( bottom ) of collagen gel - contraction ability of CAFs ( monoculture ) or CAFs co - cultured with siCNT ( co - siCNT ) or sihMENA ( t ) PANC - 1 cells ( co - sihMENA ( t ) ) . Dashed white circles illustrate the margins of the gel area . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 005 , followed by Bonferroni ' s multiple comparison test ANOVA : * P < 0 . 05 , * * P < 0 . 01 . G . Representative immunoblot ( top ) and quantification ( bottom ) of hMENA ∆ v6 and α - SMA expression in P - CAFs # 110 treated with DMEM medium ( - ) or with conditioned medium ( CM ) derived from siCNT ( siCNT PANC - 1 ) or sihMENA ( t ) PANC - 1 cells ( ( sihMENA ( t ) PANC - 1 ) ( n = 3 ) . Data are represented as fold increase with respect to DMEM ( set as 1 ± SD ) . Data presented were analyzed using two ‐ sided Student ' s t ‐ test : * P < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Quantification of in vitro matrigel invasion assay of PANC-1 cells cultured for 48 h with conditioned media (CM) of NFs (P-NFs-CM), CAF low #44 and #110 and CAFs high #36 and 138. Histograms show the number of invading cells measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates, performed at least in duplicate each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. * P <0.05, **P<0.01, ***P<0.001.B. Quantification of in vitro matrigel invasion assay of PANC-1 cultured for 48 h with CM derived from control P-CAFs#36 (siCNT-P-CAF-CM#36) and hMENA/hMENA∆v6 silenced P-CAFs (sihMENA(t)-P-CAF-CM#36), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects PANC-1 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.C. Quantification of in vitro matrigel invasion assay of H1975 cells cultured for 48 h with control media (culture medium) or conditioned media (CM) of L-CAF low #400 and CAFs high #189, as described above. Data are presented as the mean ± SD of two biological replicates, performed in triplicates each. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.D. Quantification of in vitro matrigel invasion assay of H1975 cultured for 48 h with CM derived from control L-CAFs#484 (siCNT-L-CAF-CM#484) and hMENA/hMENA∆v6 silenced L-CAFs (sihMENA(t)-L-CAF-CM#484), showing that the siRNA‐mediated knock‐down of hMENA/hMENA∆v6 affects H1975 invasive ability mediated by CAF-CM. Culture medium (DMEM) was used as control. Cells invading matrigel were counted in 6 random fields. Data are presented as the mean ± SD of six replicates. Statistical analysis was performed with one-way ANOVA P=0.006, followed by Bonferroni's multiple comparison test. *P < 0.05, **P<0.01.E. Representative immunoblot (top) and densitometry quantification (bottom) of hMENA∆v6 expression level in P-NFs grown in RPMI control medium (-) or PANC-1-CM for 24 h (n = 3). Data are represented as fold increase with respect to control medium ± SD (set as 1). Data were analyzed using two‐sided Student's t‐test. *P < 0.05.F. Representative images (top) and quantification (bottom) of collagen gel-contraction ability of CAFs (monoculture) or CAFs co-cultured with siCNT (co-siCNT) or sihMENA(t) PANC-1 cells (co-sihMENA(t)). Dashed white circles illustrate the margins of the gel area. Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P=0.005, followed by Bonferroni's multiple comparison test ANOVA: *P<0.05, **P<0.01.G. Representative immunoblot (top) and quantification (bottom) of hMENA∆v6 and α-SMA expression in P-CAFs #110 treated with DMEM medium (-) or with conditioned medium (CM) derived from siCNT (siCNT PANC-1) or sihMENA(t) PANC-1 cells ((sihMENA(t) PANC-1) (n = 3). Data are represented as fold increase with respect to DMEM (set as 1± SD). Data presented were analyzed using two‐sided Student's t‐test: *P<0.05.


Figure 5A . Heat map of proteins identified by LC - MS / MS analysis as differentially secreted in CM of normal fibroblasts ( P - NF # 1 and P - NF # 2 ) , CAFs with low level of hMENA ∆ v6 expression ( P - CAFs low # 44 and # 49 ) , CAFs with high level of hMENA ∆ v6 expression ( P - CAFs high # 67 and # 36 ) . The boxed hMENA ∆ v6 associated signature represents proteins exclusively present in the CM derived from CAFs high . Color code is shown in the upper left corner . Co - variance = 0 . 2 , q value = 0 . 1 ( 10 % false discovery rate ) , adjusted P value = 0 . 0074542 . N = 36 . The values of mass spec identified for each protein were plotted in log2 scale . B . Quantification of GAS6 secretion levels , as detected by ELISA , in the CM of P - NF ( # 1 ) , P - CAFlow ( # 44 and # 49 ) and P - CAF high ( # 67 and # 36 ) . Data are presented as the mean ± SD of two biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 . C . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in NFs ( P - NF # 1 ) , P - CAF low and P - CAF high , as described above . Data are presented as the mean ± SD of three replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 001 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 . D . Real time qRT - PCR analysis of the relative GAS6 mRNA expression level in PANC - 1 and P - CAF ( n = 3 ) , showing a low GAS6 expression in PANC - 1 cells . Data are presented as the mean ± SD . P values were calculated by two ‐ sided Student ' s t ‐ test . * * * P < 0 . 001 . E . Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts ( white ) ( n = 15 ) versus primary NSCLC fibroblasts ( light blue ) ( n = 15 ) ( GSE22862 data set ) . In each boxplot the median value ( horizontal line ) , 25th - 75th percentiles ( box outline ) , and highest and lowest values within 1 . 5x of the inter - quartile range ( vertical line ) are shown . Statistical significance was calculated by Mann - Whitney U - test ( two - sided ) ( P = 0 . 0208 ) . F , G . Real time qRT - PCR analysis of P # 138 CAF ( F ) and L # 189 CAF ( G ) transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) as representative cases . The siRNA ‐ mediated knock ‐ down of hMENA / hMENA ∆ v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells ( set as 100 ) . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test * * P < 0 . 01 , * * * P < 0 . 001 . H . Quantification of matrigel invasion assay of PANC - 1 cultured for 48 h with DMEM ( culture medium ) , or conditioned medium ( CM ) derived from control siRNA P # 106 CAFs ( siCNT P - CAF - CM ) , GAS6 siRNA ( siGAS6 P - CAF - CM ) , hMENA ( t ) siRNA ( sihMENA ( t ) P - CAF - CM ) , hMENA ( t ) siRNA plus rGAS6 ( sihMENA ( t ) P - CAF - CM + rGAS6 ) . Number of invaded PANC - 1 cells after 48 h of treatment was measured by counting 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P = 0 . 004 , followed by Bonferroni ' s multiple comparison test . * P < 0 . 05 , * * P < 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Heat map of proteins identified by LC-MS/MS analysis as differentially secreted in CM of normal fibroblasts (P-NF#1 and P-NF#2), CAFs with low level of hMENA∆v6 expression (P-CAFs low#44 and #49), CAFs with high level of hMENA∆v6 expression (P-CAFs high #67 and #36). The boxed hMENA∆v6 associated signature represents proteins exclusively present in the CM derived from CAFs high. Color code is shown in the upper left corner. Co-variance = 0.2, q value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log2 scale.B. Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P-NF (#1), P-CAFlow (#44 and #49) and P-CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. ***P<0.001.C. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in NFs (P-NF#1), P-CAF low and P-CAF high, as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one-way ANOVA P=0.001, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.D. Real time qRT-PCR analysis of the relative GAS6 mRNA expression level in PANC-1 and P-CAF (n=3), showing a low GAS6 expression in PANC-1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t‐test. ***P<0.001.E. Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) (n=15) versus primary NSCLC fibroblasts (light blue) (n=15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25th-75th percentiles (box outline), and highest and lowest values within 1.5x of the inter-quartile range (vertical line) are shown. Statistical significance was calculated by Mann-Whitney U-test (two-sided) (P=0.0208).F, G. Real time qRT-PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENA∆v6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test **P<0.01, ***P<0.001.H. Quantification of matrigel invasion assay of PANC-1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P-CAF-CM), GAS6 siRNA (siGAS6 P-CAF-CM), hMENA(t) siRNA (sihMENA(t) P-CAF-CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P-CAF-CM +rGAS6). Number of invaded PANC-1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P=0.004, followed by Bonferroni's multiple comparison test. *P< 0.05, **P<0.01.


Figure 6A . Immunoblot of AXL expression in PANC - 1 and A549 cancer cells upon transfection of control siRNA ( CNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a reduction of AXL protein expression . B . Real time qRT - PCR analysis of the relative AXL mRNA expression in PANC - 1 and A549 cancer cells transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA ( sihMENA ( t ) ) , indicating that the knock ‐ down of hMENA isoforms , resulted in a significant reduction of mRNA AXL expression . Data are presented as the mean ± SD of three replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * * P < 0 . 01 , * * * P < 0 . 001 . C . Real time qRT - PCR analysis of the relative levels of mature AXL mRNA or AXL pre - mRNA in PANC - 1 ( left ) and A549 ( right ) cell lines , transfected with control siRNA ( siCNT ) or hMENA ( t ) siRNA , sihMENA ( t ) . Data represent percent of AXL mRNA or pre - mRNA levels in hMENA ( t ) silenced cells relative to siCNT control cells ( set at 100 ) . Data are presented as the mean ± SD of two biological replicates . P values were calculated by two ‐ sided Student ' s t ‐ test . * P < 0 . 05 , * * * P < 0 . 001 . D . Immunoblot analysis with the indicated antibodies of PANC - 1 cells transfected with control siRNA ( CNT ) or hMENA ( t ) siRNA , showing that the knock ‐ down of total hMENA isoforms , ( hMENA ( t ) ) inhibits GAS6 - mediated pAXL and pAKT expression . Cells were serum starved overnight and subsequently stimulated with DMSO ( 0 . 02 % ) in control culture medium ( - ) or rGAS6 ( 200 ng / mL ) , for 30 and 60 min . The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported . E . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) toward rGAS6 as chemo - attractant ( siCNT + rGAS6 and sihMENA ( t ) + rGAS6 ) , showing that the knock ‐ down of hMENA ( t ) reduced cancer cell invasion toward GAS6 . The number of invading cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * P < 0 . 01 . F . Quantification of in vitro matrigel invasion assay of PANC - 1 siCNT cells ( siCNT ) and hMENA / hMENA ∆ v6 silenced cells ( sihMENA ( t ) ) , untreated ( - ) or treated with conditioned media derived from CAFs ( P - CAF # 36 - CM ) for 48 h . The number of invaded cells were counted in 6 random fields . Data are presented as the mean ± SD of three biological replicates . Statistical analysis was performed with one - way ANOVA P < 0 . 0001 , followed by Bonferroni ' s multiple comparison test . * * * P < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Immunoblot of AXL expression in PANC-1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression.B. Real time qRT-PCR analysis of the relative AXL mRNA expression in PANC-1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t‐test. **P<0.01, ***P<0.001.C. Real time qRT-PCR analysis of the relative levels of mature AXL mRNA or AXL pre-mRNA in PANC-1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre-mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t‐test. *P< 0.05, ***P<0.001.D. Immunoblot analysis with the indicated antibodies of PANC-1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6-mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (-) or rGAS6 (200 ng/mL), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported.E. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)) toward rGAS6 as chemo-attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. **P<0.01.F. Quantification of in vitro matrigel invasion assay of PANC-1 siCNT cells (siCNT) and hMENA/hMENA∆v6 silenced cells (sihMENA(t)), untreated (-) or treated with conditioned media derived from CAFs (P-CAF#36-CM) for 48 h. The number of invaded cells were counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one-way ANOVA P<0.0001, followed by Bonferroni's multiple comparison test. *** P< 0.001.


Figure 7A . Overall survival ( OS ) curves in Pancreatic Adenocarcinoma patients ( PDAC ) ( n = 172 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in PDAC . B . Overall survival ( OS ) curves in Lung Squamous cancer patients ( LUSC ) ( n = 501 ) from The Cancer Genome Atlas ( TCGA ) , showed that combined expression of AXL , GAS6 and ENAH ( hMENA ) , is a prognostic signature in LUSC .

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Figure 7A. Overall survival (OS) curves in Pancreatic Adenocarcinoma patients (PDAC) (n=172) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in PDAC.B. Overall survival (OS) curves in Lung Squamous cancer patients (LUSC) (n=501) from The Cancer Genome Atlas (TCGA), showed that combined expression of AXL, GAS6 and ENAH (hMENA), is a prognostic signature in LUSC.


Figure 6Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2 / eGFP mice 21 days after tamoxifen treatment Double - positive cells are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 20 µm ( B ) , 75 µm ( C ) , 30 µm ( D ) , Histograms showing quantifications ( B , n = 6 animals ; 5 region of interest ( ROI ) covering the diencephalon on a total of 3 slides analyzed per animal Data information : data are presented as mean ± SEM . Each data point represents one animal . Micrographs of maximum intensity projections of forebrain sections from 3 mo old C57BL / 6J mice after EdU application for 4 weeks in drinking water ( F ) immunostained as indicated on top of the panels . Double - positive cells are indicated by arrowheads . CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 10 µm ( F ) . G ) Histograms showing quantifications for n = 5 animals ; 5 ROI covering the diencephalon on a total of 2 slides analyzed per animal ) . Data information : data are presented as mean ± SEM . Each data point represents one animal .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Micrographs of maximum intensity projections of forebrain sections from 3 mo old GLASTCreERT2/eGFP mice 21 days after tamoxifen treatment Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 20 µm (B), 75 µm (C), 30 µm (D), Histograms showing quantifications (B, n = 6 animals; 5 region of interest (ROI) covering the diencephalon on a total of 3 slides analyzed per animal Data information: data are presented as mean ± SEM. Each data point represents one animal.Micrographs of maximum intensity projections of forebrain sections from 3 mo old C57BL/6J mice after EdU application for 4 weeks in drinking water (F) immunostained as indicated on top of the panels. Double-positive cells are indicated by arrowheads. CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 10 µm (F).G) Histograms showing quantifications for n = 5 animals; 5 ROI covering the diencephalon on a total of 2 slides analyzed per animal). Data information: data are presented as mean ± SEM. Each data point represents one animal.


Figure 7A - E Micrographs of maximum intensity projections ( A - D ) or single optical section ( E ) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo ( A , B , E ) and 3 mo ( C , D ) GLASTCreERT2 / Confetti mice at 21 dpt . Note that clusters of duplets ( D ) and triplets ( C ) labelled in the same color can be found only in the DIE . Also note that astrocyte somata are often very close together , as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles . Data information : CTX : cerebral cortex ; DIE : diencephalon ; HC : hippocampus . Scale bars : 300 µm ( A ) , 100 µm ( B ) , 50 µm ( C , D ) , 20 µm ( E ) .

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Figure 7A-E Micrographs of maximum intensity projections (A - D) or single optical section (E) of forebrain sections stained as indicated for fluorescent proteins in astrocytes labelled in 8 mo (A, B, E) and 3 mo (C, D) GLASTCreERT2/Confetti mice at 21 dpt. Note that clusters of duplets (D) and triplets (C) labelled in the same color can be found only in the DIE. Also note that astrocyte somata are often very close together, as also observed after live in vivo imaging of dividing astrocytes Somata of single cells are highlighted in circles. Data information: CTX: cerebral cortex; DIE: diencephalon; HC: hippocampus. Scale bars: 300 µm (A), 100 µm (B), 50 µm (C, D), 20 µm (E).


Figure 8B Micrographs of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP 21 dpt mice stained for HA show maximum intensity projections of HA + expressing cells with astroglial morphology . CTX GM : cerebral cortex grey matter ; DIE : diencephalon . Scale bars : 100 µm . C Micrographs of forebrain sections of 3 mo old GLASTCreERT2 / RPL22HA / eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β + astrocytes . DIE : diencephalon . Scale bar : 70 μm . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was pooledD Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE ( blue bars ) and CTX GM ( red bars ) samples . Data information : CTX GM : cerebral cortex grey matter ; DIE : diencephalon ; n = 3 biological replicates , tissue from 3 mice was

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8B Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP 21 dpt mice stained for HA show maximum intensity projections of HA+ expressing cells with astroglial morphology. CTX GM: cerebral cortex grey matter; DIE: diencephalon. Scale bars: 100 µm. C Micrographs of forebrain sections of 3 mo old GLASTCreERT2/RPL22HA/eGFP mice 21dpt stained for HA and S100β showing the specificity of the mouse line with HA detected only in S100β+ astrocytes. DIE: diencephalon. Scale bar: 70 μm. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooledD Histogram showing expression levels of the top 10 astrocyte specific markers genes in DIE (blue bars) and CTX GM (red bars) samples. Data information: CTX GM: cerebral cortex grey matter; DIE: diencephalon; n=3 biological replicates, tissue from 3 mice was pooled


Figure 9C Example of a Smad4 / GFP double - positive astrocyte ( indicated by arrowhead ) in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top of the panel . Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel . Scale bar : 15 µm . D Example of a proliferating ( PCNA + , arrowhead ) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2 / eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels . Scale bars : 15 µm . E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x - axis ( n = 6 animals per genotype ; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal ; PCNA negative and positive cells comparing wt / wt vs . fl / fl animals , adjusted P - value = 0 . 002 ) . Data information : data are presented as mean ± SEM . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons testF , G Histograms depicting percentage of the neurosphere - generating cells obtained from the DIE ( F ) or SEZ ( G ) of GLASTCreERT2 / Smad4fl / fl / eGFP or GLASTCreERT2 / Smad4WT / WT / eGFP , as indicated on the x - axis ( n = 3 - 4 animals for SEZ , 4 to 8 technical replicates analyzed per animal ; 4 animals for DIE , 8 technical replicates analyzed per animal ) . Data information : data are presented as mean ± SEM . F : GFP negative and GFP positive cells comparing wt / wt vs fl / fl animals , adjusted P - value = 0 . 0408 ; G : GFP negative and GFP positive cells comparing wt / wt vs fl / fl animals , ns ) . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric 2way ANOVA followed by Sidak ' s multiple comparisons testH Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f / f neurosphere cells at 7 days after infection with MLV - based retrovirus containing either CAG - NLS - Cre or only CAG - GFP ( n = 11 cultures / animals , 3 technical replicates per condition analyzed ; control vs . Cre , P - value = 0 . 0149 ) . Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information : data are presented as mean ± SEM . Each dot represents one n . * P < 0 . 05 , * * P < 0 . 01 nonparametric Mann - Whitney test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9C Example of a Smad4/GFP double-positive astrocyte (indicated by arrowhead) in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top of the panel. Orthogonal projections of the area highlighted in left panel of C shown at higher magnification in the right panel. Scale bar: 15 µm.D Example of a proliferating (PCNA+, arrowhead) astrocyte in a single optical section of DIE from 3 mo old GLASTCreERT2/eGFP mice 21 dpt immunostained as indicated on top with single channels shown in middle and right panels. Scale bars: 15 µm.E Histogram depicting percentage of PCNA positive cells of all GFP positive cells in the DIE of the genotype indicated on the x-axis (n = 6 animals per genotype; 5 ROI covering the diencephalon on a total of 3 slides were analyzed per animal; PCNA negative and positive cells comparing wt/wt vs. fl/fl animals, adjusted P-value = 0.002). Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons testF, G Histograms depicting percentage of the neurosphere-generating cells obtained from the DIE (F) or SEZ (G) of GLASTCreERT2/Smad4fl/fl/eGFP or GLASTCreERT2/Smad4WT/WT/eGFP, as indicated on the x-axis (n = 3-4 animals for SEZ, 4 to 8 technical replicates analyzed per animal; 4 animals for DIE, 8 technical replicates analyzed per animal). Data information: data are presented as mean ± SEM. F: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, adjusted P-value = 0.0408; G: GFP negative and GFP positive cells comparing wt/wt vs fl/fl animals, ns). Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric 2way ANOVA followed by Sidak's multiple comparisons testH Histogram depicting the number of secondary neurospheres generated from 10000 Smad4f/f neurosphere cells at 7 days after infection with MLV-based retrovirus containing either CAG-NLS-Cre or only CAG-GFP (n = 11 cultures / animals, 3 technical replicates per condition analyzed; control vs. Cre, P-value = 0.0149). Note the decrease in DIE astrocyte proliferation in vivo and in neurosphere formation upon Smad4 deletion Data information: data are presented as mean ± SEM. Each dot represents one n. * P < 0.05, ** P < 0.01 nonparametric Mann-Whitney test).


Figure 1Antibacterial competition assay . E . coli K12 recipient cells ( W3110 gfp + , kanR ) were mixed with the indicated EAEC attacker cells ( 1 : 4 ratio ) : WT , ∆ T6SS - 1 and ∆ tle1 - tli1 carrying pBAD18 and pBAD33 vectors , or producing the indicated proteins , and spotted on SIM 0 . 02 % arabinose agar plates for 4 - h at 37 ° C . The image of a representative bacterial spot is shown . The number of recovered prey cells is indicated in the upper graph ( in log of colony - forming units ( cfu ) ) . The black , dark grey and light grey circles indicate values obtained from three different spots , and the average is indicated by the bar . Western blot analysis of the production of Tle1V and Tle1 ∆ 1 - 26 V is shown in the inset . The experiment was performed in triplicate and a representative result is shown . Co - immunoprecipitation assay . Lysates from E . coli K - 12 W3110 cells producing VSVG - tagged Tle1 ( Tle1V ) or VSVG - tagged Tle1 ∆ 1 - 26 truncated variant ( Tle1 ∆ 1 - 26 V ) were mixed with lysates from W3110 cells producing FLAG - tagged VgrG ( VgrGFL ) or not ( - , empty vector ) and subjected to immunoprecipitation on anti - FLAG - coupled beads . The mixed soluble lysates ( Input ) and the immunoprecipitated material ( IP ) were subjected to 12 . 5 % SDS - PAGE and immunodetected with anti - FLAG ( upper panel ) and anti - VSVG ( lower panel ) monoclonal antibodies . Molecular weight markers ( in kDa ) are indicated on the left . This experiment was performed in triplicate and a representative result is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Antibacterial competition assay. E. coli K12 recipient cells (W3110 gfp+, kanR) were mixed with the indicated EAEC attacker cells (1:4 ratio): WT, ∆T6SS-1 and ∆tle1-tli1 carrying pBAD18 and pBAD33 vectors, or producing the indicated proteins, and spotted on SIM 0.02 % arabinose agar plates for 4-h at 37°C. The image of a representative bacterial spot is shown. The number of recovered prey cells is indicated in the upper graph (in log of colony-forming units (cfu)). The black, dark grey and light grey circles indicate values obtained from three different spots, and the average is indicated by the bar. Western blot analysis of the production of Tle1V and Tle1∆1-26 V is shown in the inset. The experiment was performed in triplicate and a representative result is shown.Co-immunoprecipitation assay. Lysates from E. coli K-12 W3110 cells producing VSVG-tagged Tle1 (Tle1V) or VSVG-tagged Tle1∆1-26 truncated variant (Tle1∆1-26 V) were mixed with lysates from W3110 cells producing FLAG-tagged VgrG (VgrGFL) or not (-, empty vector) and subjected to immunoprecipitation on anti-FLAG-coupled beads. The mixed soluble lysates (Input) and the immunoprecipitated material (IP) were subjected to 12.5% SDS-PAGE and immunodetected with anti-FLAG (upper panel) and anti-VSVG (lower panel) monoclonal antibodies. Molecular weight markers (in kDa) are indicated on the left. This experiment was performed in triplicate and a representative result is shown.


Figure 5Specific phospholipase A1 ( PLA1 ) activity measurements of Tle1H , SVgrG - Tle1H complex , and isolated SVgrG . Thermomyces lanuginosus lipase ( TLL ) was used as positive standard for PLA1 activity . Mean values and standard deviation from at least three independent assays are shown . Statistical analysis relative to the Tle1 activity is indicated . * * * P < 0 . 001 ( P = 0 , 0001998 ) , unpaired two - sample Fisher - Pitman permutation test . VgrG inhibition of Tle1H . Specific phospholipase A1 ( PLA1 ) activity measurements of purified Tle1H in the presence of 1 : 10 molar ratio of lysozyme from chicken egg white ( Lyso ) and increasing molar ratio ( 0 , 1 , 5 , and 10 ) of SVgrG or SVgrG1 - 490 . Inhibition experiments with SVgrG were repeated three times with independent protein preparations , each measured in triplicate . Inhibition experiments with SVgrG1 - 490 were repeated twice with two independent protein preparations , each measured in triplicate . Mean values and standard deviation are shown . Statistical analysis relative to the Tle1 activity is indicated . ns , non - significant ( P > 0 . 05 ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - sample Fisher - Pitman permutation test . ( P values : Lyso , 0 . 7006 ; VgrG 1 : 1 , 0 . 004946 ; VgrG 1 : 5 , 1 . 448e - 07 ; VgrG1 : 10 , 1 . 448e - 07 ; VgrG1 - 490 1 : 1 , 0 . 5627 ; VgrG1 - 490 1 : 5 , 0 . 2977 ; VgrG1 - 490 1 : 10 , 0 . 002631 ) . Representative SDS - PAGE / Coomassie blue staining of the Tle1H , SVgrG and SVgrG1 - 490 purified proteins used in B ( 5 μg / well ) .

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Figure 5Specific phospholipase A1 (PLA1) activity measurements of Tle1H, SVgrG-Tle1H complex, and isolated SVgrG. Thermomyces lanuginosus lipase (TLL) was used as positive standard for PLA1 activity. Mean values and standard deviation from at least three independent assays are shown. Statistical analysis relative to the Tle1 activity is indicated. ***P<0.001 (P=0,0001998), unpaired two-sample Fisher-Pitman permutation test.VgrG inhibition of Tle1H. Specific phospholipase A1 (PLA1) activity measurements of purified Tle1H in the presence of 1:10 molar ratio of lysozyme from chicken egg white (Lyso) and increasing molar ratio (0, 1, 5, and 10) of SVgrG or SVgrG1-490. Inhibition experiments with SVgrG were repeated three times with independent protein preparations, each measured in triplicate. Inhibition experiments with SVgrG1-490 were repeated twice with two independent protein preparations, each measured in triplicate. Mean values and standard deviation are shown. Statistical analysis relative to the Tle1 activity is indicated. ns, non-significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, unpaired two-sample Fisher-Pitman permutation test. (P values: Lyso, 0.7006; VgrG 1:1, 0.004946; VgrG 1:5, 1.448e-07; VgrG1:10, 1.448e-07; VgrG1-490 1:1, 0.5627; VgrG1-490 1:5, 0.2977; VgrG1-490 1:10, 0.002631).Representative SDS-PAGE / Coomassie blue staining of the Tle1H, SVgrG and SVgrG1-490 purified proteins used in B (5 μg/well).


Figure 1 ( G ) . GFP - tagged ERMES complex proteins retain their characteristic punctate structure in Δlam6 , suggesting that Lam6 is not an essential complex member . Scale bar represents 5 μm .

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Figure 1(G). GFP-tagged ERMES complex proteins retain their characteristic punctate structure in Δlam6, suggesting that Lam6 is not an essential complex member. Scale bar represents 5 μm.


Figure 2 ( A ) Fluorescence microscopy demonstrates that Lam6 - GFP co - localizes with ERMES ( Mdm34 - Cherry ) ( yellow arrows ) and also localizes to non - ERMES locations in the cell ( red arrows ) . These additional locations co - localized with the NVJ ( Nvj1 - GFP ) as well as with the vCLAMP ( GFP - Vps39 ) . Scale bar represents 5 μm . ( B ) Overexpression and tagging of Lam6 confirmed that it co - localizes with ERMES ( Mdm34 - GFP ) ( yellow arrows ) as well as to additional contact sites ( red arrows ) , NVJ ( marked by Nvj1 ) and vCLAMP ( marked by Vps39 ) . Scale bar represents 5 μm .

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Figure 2(A) Fluorescence microscopy demonstrates that Lam6-GFP co-localizes with ERMES (Mdm34-Cherry) (yellow arrows) and also localizes to non-ERMES locations in the cell (red arrows). These additional locations co-localized with the NVJ (Nvj1-GFP) as well as with the vCLAMP (GFP-Vps39). Scale bar represents 5 μm.(B) Overexpression and tagging of Lam6 confirmed that it co-localizes with ERMES (Mdm34-GFP) (yellow arrows) as well as to additional contact sites (red arrows), NVJ (marked by Nvj1) and vCLAMP (marked by Vps39). Scale bar represents 5 μm.


Figure 3 ( A ) Fluorescence microscopy demonstrates that the overexpression of Cherry - Lam6 ( OE - Cherry - LAM6 ) results in an expansion of the following three contact sites : ERMES ( Mdm34 - GFP ) , NVJ ( Nvj1 - GFP ) , and vCLAMP ( GFP - Vps39 ) . This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion . The numbers represent the average contact site size ( 120 cells per sample ) . Scale bar represents 5 μm . ( B ) Immuno - EM verified that GFP - Lam6 overexpression indeed causes an expansion of contact site size . While vCLAMP was hardly visible in WT cells , it could be detected easily in the OE strain ( albeit often had ER tubules invading it ) . The NVJ underwent expansion as well as evoked PMN in OE strains , and the ER - mitochondria contact became large and elongated instead of small and distinct . N , nucleus ; M , mitochondria ; V , vacuole . Scale bar represents 200 nm ( see also Figure S3I ) .

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Figure 3(A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm.(B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).


Figure 4 ( A ) Fluorescent microscopy shows that the ERMES contact ( as measured by the number of Mdm34 - GFP puncta per cell ) expanded in the Δvps39 background relative to WT . However , the expansion did not occur on the background of Δvps39 Δlam6 , demonstrating that Lam6 is necessary for ERMES expansion under these conditions . Scale bar represents 5 μm . ( B ) Quantitation of ( A ) . Bars represent the percentage of cells containing the specific number of puncta / cell out of total cells counted for the strain ( for WT , n = 234 cells ; for Δvps39 , n = 246 ; for Δvps39 Δlam6 , n = 271 ) . ( C ) Fluorescent microscopy demonstrates that downregulating ERMES contacts ( by growing GALp - MDM34 strains in glucose ) indeed caused expansion of vCLAMP ( GFP - Vps39 ) . However , this expansion was diminished in a Δlam6 background . Scale bar represents 5 μm ( see also Figure S4 ) .

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Figure 4(A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm.(B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271).(C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4).


Figure 1C . Quantification of Cre activity by dual luciferase assays ( mean ± SD , n = 3 ) . Data information Npu : Nostoc punctiforme , Ssp : Synechocystis sp . , CDS : coding sequence . D . Quantification of tTA activity by dual luciferase assays ( mean ± SD , n = 3 ( N and C ) or n = 7 ( N / C ) ) . Data information : Npu : Nostoc punctiforme CDS : coding sequence .

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Figure 1C. Quantification of Cre activity by dual luciferase assays (mean±SD, n=3). Data information Npu: Nostoc punctiforme, Ssp: Synechocystis sp., CDS: coding sequence.D. Quantification of tTA activity by dual luciferase assays (mean±SD, n=3 (N and C) or n=7 (N/C)). Data information: Npu: Nostoc punctiforme CDS: coding sequence.


Figure 2A - B . Scheme of engineered Spc and Ccsp loci after integration of split - Cre and split - tTA effectors ( red mark = stop codon ) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections . Blue : DAPI . Scale bar : 100 µm . C - D . β - galactosidase and H & E staining of lung sections from BASC tracer and BASC viewer animals . Scale bars : 500 µm ( overview ) and 20 µm ( boxed magnification ) . E . Localization of β - gal + cells in adult BASC viewer animals . BADJ - associated and " alveolar " β - gal + cells are expressed as percentage of all lineage - labeled cells ( means ± SD n = 3 ) . F . Number of β - gal + cells in adult BASC tracer and BASC viewer animals , normalized to the number of terminal bronchioles ( TBs ) ( means ± SD n = 3 ) .

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Figure 2A-B. Scheme of engineered Spc and Ccsp loci after integration of split-Cre and split-tTA effectors (red mark = stop codon) and comparison of endogenous SPC or CCSP expression and YFP or mCherry reporter expression in lung sections. Blue: DAPI. Scale bar: 100 µm.C-D. β-galactosidase and H&E staining of lung sections from BASC tracer and BASC viewer animals. Scale bars: 500 µm (overview) and 20 µm (boxed magnification).E. Localization of β-gal+ cells in adult BASC viewer animals. BADJ-associated and "alveolar" β-gal+ cells are expressed as percentage of all lineage-labeled cells (means±SD n=3).F. Number of β-gal+ cells in adult BASC tracer and BASC viewer animals, normalized to the number of terminal bronchioles (TBs) (means±SD n=3).


Figure 3A . Sequential microscopic imaging of epifluorescence and β - galactosidase activity in lung sections of adult BASC viewer animals . Arrow highlights labeled BASC at BADJ ( magnified in box ) . Blue : DAPI . Scale bars : 100 µm ( overview ) and 20 µm ( boxed magnification ) . B . β - galactosidase staining of AT2 cells ( YFP + ) , Club cells ( mCherry + ) and BASCs ( mCherry + / YFP + ) isolated from BASC viewer mice by fluorescence - activated cell sorting ( FACS ) . Scale bar : 50 µm . C . Doxycycline - dependent reversible labeling of BASCs in BASC viewer mice . Scale bar : 200 µm .

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Figure 3A. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of adult BASC viewer animals. Arrow highlights labeled BASC at BADJ (magnified in box). Blue: DAPI. Scale bars: 100 µm (overview) and 20 µm (boxed magnification).B. β-galactosidase staining of AT2 cells (YFP+), Club cells (mCherry+) and BASCs (mCherry+/YFP+) isolated from BASC viewer mice by fluorescence-activated cell sorting (FACS). Scale bar: 50 µm.C. Doxycycline-dependent reversible labeling of BASCs in BASC viewer mice. Scale bar: 200 µm.


Figure 4A . Heatmaps for Spearman correlation . C . MA and Volcano plots , and Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression analysis of BASCs and Club cells . D . MA and Volcano plots , and Top50 DEGs ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells . E . Heatmap of RNA - seq z - scores computed for the Top50 DEGs in BASC , AT2 , and Club cells ( FDR < 0 . 05 , Log2FC > ± 0 . 585 , Means > 5 ) .

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Figure 4A. Heatmaps for Spearman correlation.C. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and Club cells. D. MA and Volcano plots, and Top50 DEGs (FDR<0.05, Log2FC>±0.585, Means>5) for visualization of pairwise differential gene expression analysis of BASCs and AT2 cells. E. Heatmap of RNA-seq z-scores computed for the Top50 DEGs in BASC, AT2, and Club cells (FDR<0.05, Log2FC>±0.585, Means>5).


Figure 5B . Quantification of β - gal + cells in FACS - purified AT2 and Club cell fractions isolated from 3 months old BASC v - race mice ( mean ± SD , n = 3 ) and example of β - galactosidase label distribution in lung sections . Scale bar : 100 µm . C . BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life . β - galactosidase staining of lung sections derived from BASC v - race animals collected at indicated time points . Arrow highlights β - gal + cell at P6 . Scale bar : 200 µm .

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Figure 5B. Quantification of β-gal+ cells in FACS-purified AT2 and Club cell fractions isolated from 3 months old BASC v-race mice (mean±SD, n=3) and example of β-galactosidase label distribution in lung sections. Scale bar: 100 µm.C. BASCs emerge at early postnatal stages and contribute to epithelial turnover throughout life. β-galactosidase staining of lung sections derived from BASC v-race animals collected at indicated time points. Arrow highlights β-gal+ cell at P6. Scale bar: 200 µm.


Figure 6A . β - galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v - race animals 21 days ( bleomycin , naphthalene ) or 35 days ( influenza ) following injury . Scale bar : 5 mm ( whole lungs ) and 1 mm ( magnification ) . B . Sequential microscopic imaging of epifluorescence and β - galactosidase activity in lung sections of control and injured BASC v - race mice . Clusters of BASC - derived AT2 ( YFP + ) and Club cells ( mCherry + ) are highlighted . Blue : DAPI . Scale bar : 200 µm . C . Contribution of BASCs to different cellular compartments . Bronchiolar and alveolar accumulations of lineage - traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β - gal + cells . Data depicted as mean ± SD , n = 4 ( bleomycin , naphthalene ) or n = 5 ( influenza ) .

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Figure 6A. β-galactosidase staining of cleared whole lung preparations isolated from control and injured BASC v-race animals 21 days (bleomycin, naphthalene) or 35 days (influenza) following injury. Scale bar: 5 mm (whole lungs) and 1 mm (magnification).B. Sequential microscopic imaging of epifluorescence and β-galactosidase activity in lung sections of control and injured BASC v-race mice. Clusters of BASC-derived AT2 (YFP+) and Club cells (mCherry+) are highlighted. Blue: DAPI. Scale bar: 200 µm.C. Contribution of BASCs to different cellular compartments. Bronchiolar and alveolar accumulations of lineage-traced BASC derivatives are expressed as percentage of all clusters containing ≥ 10 β-gal+ cells. Data depicted as mean±SD, n=4 (bleomycin, naphthalene) or n=5 (influenza).


Figure 7A . β - galactosidase and H & E staining of lung sections from control ( non - infected ) and injured BASC viewer mice 7 days post infection ( 7 dpi ) with influenza virus . Scale bar : 200 µm . B . Abundance of β - gal + cells in control ( non - infected ) and injured ( 7 dpi ) BASC viewer animals . Data depicted as mean ± SD , n = 4 , * p = 0 . 0286 ( Mann - Whitney test ) , TB - terminal bronchiole . C . Club cell depletion / recovery after naphthalene treatment . Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3 , 7 and 21 days post naphthalene ( dpn ) . Blue : DAPI . Scale bar : 200 µm . D . β - galactosidase staining of lung sections from control and injured BASC viewer animals 3 , 7 and 21 days post naphthalene ( dpn ) . Scale bar : 200 µm . E . Abundance of β - gal + cells in control and injured BASC viewer animals . The percentage of terminal bronchioles containing 0 , 1 , 2 , 3 , 4 , ≥ 5 β - gal + cells is shown . Data depicted as mean ± SD , n = 3 .

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Figure 7A. β-galactosidase and H&E staining of lung sections from control (non-infected) and injured BASC viewer mice 7 days post infection (7 dpi) with influenza virus. Scale bar: 200 µm. B. Abundance of β-gal+ cells in control (non-infected) and injured (7 dpi) BASC viewer animals. Data depicted as mean±SD, n = 4, *p = 0.0286 (Mann-Whitney test), TB - terminal bronchiole. C. Club cell depletion/recovery after naphthalene treatment. Microscopic imaging of epifluorescence in lung sections from control and injured BASC viewer animals isolated 3, 7 and 21 days post naphthalene (dpn). Blue: DAPI. Scale bar: 200 µm.D. β-galactosidase staining of lung sections from control and injured BASC viewer animals 3, 7 and 21 days post naphthalene (dpn). Scale bar: 200 µm.E. Abundance of β-gal+ cells in control and injured BASC viewer animals. The percentage of terminal bronchioles containing 0, 1, 2, 3, 4, ≥ 5 β-gal+ cells is shown. Data depicted as mean±SD, n=3.


Figure 8A . Quantification of lineage - traced Club cells in distal TBs and β - galactosidase staining of cleared accessory lobe preparation ( scale bar : 250 µm ) isolated from injured BASC v - race mice at 21 dpn ( mean ± SD , n = 4 ) . Bottom panel : example of sequential microscopic imaging to quantify β - gal + mCherry + cells in distal TBs . Arrows denote last airway bifurcation before bronchioalveolar duct junction ( BADJ ) . Scale bar : 200 µm . B . β - galactosidase staining of FACS - purified Club cells from control and injured BASC v - race animals . Scale bar : 50 µm . The percentage of lineage - traced Club cells is shown ( mean ± SD , n = 4 ( oil ctrl . ) or n = 5 ( 21 dpn ) , * p = 0 . 0159 ( Mann - Whitney test ) . C . Combined β - galactosidase ( blue ) and acetylated tubulin staining ( cilia , brown color ) of lung sections from BASC v - race animals at 21 dpn . Red : DAPI ( pseudocolor ) . Examples of lineage - traced ciliated cells ( arrowheads ) and Club cells ( asterisks ) are highlighted . Scale bar : 50 µm . D . β - galactosidase staining of cleared accessory lobe preparations ( scale bar : 5 mm ) and quantification of β - gal + bronchioles in lung sections from adult BASC v - race ( ctrl . ) and BASC - ablated mice ( DTA ) . Data depicted as mean ± SD , n = 3 . E . β - galactosidase staining of cleared accessory lobe preparations ( scale bar : 5 mm ) and fluorescence microscopy of lung sections from uninjured and naphthalene - exposed BASC v - race ( ctrl . ) and BASC - ablated ( DTA ) mice 12 days post naphthalene ( dpn ) . Dotted lines depict bronchiolar boundaries . Scale bar : 100 µm . F . Quantification of Club cell recovery in injured BASC v - race ( ctrl . ) and BASC - ablated ( DTA ) animals , expressed as percentage of bronchiolar circumference re - populated by mCherry + cells at 12 dpn . Data depicted as mean ± SD , n = 5 , * * p = 0 . 0079 ( Mann - Whitney test ) . DTA - diphtheria toxin fragment A .

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Figure 8A. Quantification of lineage-traced Club cells in distal TBs and β-galactosidase staining of cleared accessory lobe preparation (scale bar: 250 µm) isolated from injured BASC v-race mice at 21 dpn (mean±SD, n=4). Bottom panel: example of sequential microscopic imaging to quantify β-gal+ mCherry+ cells in distal TBs. Arrows denote last airway bifurcation before bronchioalveolar duct junction (BADJ). Scale bar: 200 µm.B. β-galactosidase staining of FACS-purified Club cells from control and injured BASC v-race animals. Scale bar: 50 µm. The percentage of lineage-traced Club cells is shown (mean±SD, n=4 (oil ctrl.) or n=5 (21 dpn), *p = 0.0159 (Mann-Whitney test).C. Combined β-galactosidase (blue) and acetylated tubulin staining (cilia, brown color) of lung sections from BASC v-race animals at 21 dpn. Red: DAPI (pseudocolor). Examples of lineage-traced ciliated cells (arrowheads) and Club cells (asterisks) are highlighted. Scale bar: 50 µm.D. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and quantification of β-gal+ bronchioles in lung sections from adult BASC v-race (ctrl.) and BASC-ablated mice (DTA). Data depicted as mean±SD, n=3. E. β-galactosidase staining of cleared accessory lobe preparations (scale bar: 5 mm) and fluorescence microscopy of lung sections from uninjured and naphthalene-exposed BASC v-race (ctrl.) and BASC-ablated (DTA) mice 12 days post naphthalene (dpn). Dotted lines depict bronchiolar boundaries. Scale bar: 100 µm. F. Quantification of Club cell recovery in injured BASC v-race (ctrl.) and BASC-ablated (DTA) animals, expressed as percentage of bronchiolar circumference re-populated by mCherry+ cells at 12 dpn. Data depicted as mean±SD, n=5, **p = 0.0079 (Mann-Whitney test). DTA - diphtheria toxin fragment A.


Figure 1Assessment of mtDNA damage by quantitative PCR . HEK293 cells infected with control ( empty vector ) or shATG5 for 5 days . Cells were then incubated with DMSO or 4 mM 3 - NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h . Cells with or without washout were used for the extraction of total DNA . All DNA samples were used for amplification of 8 . 9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment . PCR products were quantitated by PicoGreen staining using Micro Plate Reader . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . The data in ( A ) were further calculated for the frequency of mtDNA damage . The equation was seen in " Materials and Methods " . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 . Representative images show 8 - oxo - dG staining . Control or shATG5 HeLa cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and then either fixed immediately or washed with fresh medium and incubated for another 1 h . Cells were immunostained with DAPI and anti - 8 - oxo - dG antibody and analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in ( C ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . Control or shATG5 HEK293 cells stably expressing mito - Keima . Control cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and shATG5 cells were treated with 200 µM H2O2 as a negative control . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Right panels show the pixel intensity of red ( mitochondria within lysosomes ) and green ( mitochondria in the cytoplasm ) from a line . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( E ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * p < 0 . 01 . HeLa cells expressing GFP - LC3 were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were stained with anti - Tom20 and anti - DNA antibodies , then analyzed by confocal microscopy . The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA / Tom20 . Scale bar , 10 µm . Quantification of the LC3 punta colocalizing or contacting with mtDNA / Tom20 in a cell . n = 30 cells from 3 coverslips , Data are presented as mean ± SD ( n = 30 ) , statistical significance was assessed by a one - way ANOVA with Tukey ' s multiple comparisons test , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.