EMBO/soda-nel huggingface dataset:
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 6

Figure 6. Excess of HA tagged Tsp96F blocks Ptc MVBs polarized exocytosis: A) 3D reconstruction of wing disc immuno-labelled for endogenous Ptc and dorsally over-expressing the Tsp96F-HA construct, keeping the ventral side of the disc in wild type condition as internal control. Note the dorsal accumulation of Ptc in very large punctae across the apico-basal axis, but mainly at the basal side of the epithelium. On the right, graphs represent fluorescence values from apical to basal, comparing dorsal and ventral sides and showing a shift of the highest values towards the basal side after Tsp96-HA expression.

B) Confocal image of a wing disc with a clone expressing Tsp96F-HA and immuno-labelled with anti-Ptc and anti-HA antibodies, which co-localize in the large basolateral punctae (yellow arrows).

C) Electron microscopy image from immunolabelled (anti-GFP) Hh-receiving cells of a wing disc expressing Ptc-GFP and Tsp96F-HA. The detailed image shows large sac structures that accumulate several dense MVBs positive labelled for Ptc.

Data Information: Scale bars (B), 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 7

Figure 7. Ptc is secreted in extracellular vesicles from wing disc cultured cells: A) Ptc is mainly secreted in small EVs (P100K) from Cl8 cells.

B) Large EVs (lEVs), small EVs (sEVs) and very small EVs (vsEVs) were analysed in triplicate by Western blotting. Note that sEVs are enriched in Ptc and in the EV-associated proteins Syntaxin1 (Synt1A), Late bloomer (Lbm) and Hsp70.

C) Fractionation of sEVs by Sucrose Density Gradient: Ptc partially co-fractioned with Synt1A, Lbm and Hsp70. Note that while unprocessed form of co-receptor Ihog does not co-fraction with Ptc, the processed form does in the same subpopulation.

D) Fractionation of sEVs by Size Exclusion Chromatography: Ptc perfectly co-fractioned with Synt1A, Lbm and Hsp70. In agreement with the density gradient analysis, unprocessed form of co-receptor Ihog does not co-fraction with Ptc in the same subpopulation of sEVs.

E) Effect on EV-associated Ptc secretion after gene silencing on Cl8 cells by dsRNAs against the SNARE proteins Syb and α-Snap, the tetraspanin Tsp96F and the ESCRT components Hrs, Tsg101, Shrub and Vps4. Hsp70 in cells and EVs is detected by different exposition time.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 8

Figure 8. ESCRT machinery and SNARE-mediated exocytosis are required for Ptc exposure at basal membrane:

A) Projection images of three confocal sections (1 μm) at the basal side of wing discs expressing the Bac constructs for Hh-GFP and Ptc-cherry, used to analyse co-localization between the ligand and the receptor in wild type conditions and after inhibition of vesicle fusion (Syb RNAi), vesicle trafficking (Hrs RNAi) or Ptc protein degradation (Dor RNAi). Boxplot shows co-localization rates using the Manders coefficient for proportion of red covered by green in projected images for wing discs temporally expressing RNAi against ESCRT proteins Hrs and Vps4, SNAREs Syb and α-Snap, and Dor as control for effects after Ptc degradation-blocking, as well as temporally over-expressing the Tsp96F-HA. Central horizontal lines show median values of N=8-12 (see Apendix Table S2), box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding. Note the significant reduction in co-localization rates (1= total coverage of red by green) after RNAi expression for both ESCRT and SNARE proteins as well as after Tsp96F-HA over-expression, while RNAi expression for Dor presents greater co-localization. Significance levels for pairwise test (Wilcoxon) ***P<0.001, **P<0.01, *P<0.05.

B) Projection images of three confocal sections (1 μm) from the apical side of the wing discs. Quantification of co-localization rates as in (A) but using apical projection images; central horizontal lines show median values of N=8-12 (see Apendix TableS2), box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note that at the apical side no clear significant reduction in co-localization rates (1= total coverage of red by green) is observed after RNAi expression or in Tsp96F-HA over-expression. Significance levels for pairwise test (Wilcoxon) ***P<0.001, **P<0.01, *P<0.05, +P<0.1.

C) Correlation coefficients between the maximum value of quantified integrated density (integrating both fluorescence intensity and signal area) for Ptc-GFP values (Yac construct) and distance from the basal side (-1=basal, +1=apical). Box plot shows quantification data of discs after RNAi expression for the vacuole sorting protein Dor (necessary for Ptc degradation), the ESCRT Hrs, the SNARE Syb as well as after over-expression of the Tsp96F-HA construct and wild type condition. Central horizontal lines show median values of N=8-12, box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note the apical localization of Ptc-GFP highest values after degradation impairment (Dor RNAi) in contrast to the basal shift resulting from exocytosis deficiency (Syb RNAi).

Data Information: Scale bars 10 μm.
Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc (2020)
Figure 9

Figure 9. Hindering traffic and basal exocytosis of Ptc results in flattening of the Hh signalling gradient:

A) Confocal images at the lateral plane showing the immunostaining of the medium threshold Hh target Collier (Col) of wild type wing discs and discs after inhibition of vesicle fusion (α-Snap RNAi), vesicle trafficking (Hrs RNAi) or in over-expression of the Tsp96F-HA. Quantification of gradient lengths (μm) using Collier signal and normalized to the wing pouch size, showing the proportion of wing pouch covered by signal (1=total coverage). Quantification was performed for wild type condition as well as in temporally regulated RNAi expression for the ESCRT complex proteins Hrs, Tsg101, Shrub and Vps4 and for the SNARE proteins Syb, Snap24, α-Snap, as well as in transient over-expression of the Tsp96F-HA. Central horizontal lines show median values of N=7-22 (see Apendix TableS2), box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a slight but significant increase in the length of Col signal for all treatment compared to the wild type condition. Significance levels for pairwise test (Wilcoxon) ***P<0.001, **P<0.01, *P<0.05.

B) Lateral confocal images and quantification of the extension of immuno-labeled Engrailed as the high threshold Hh target. Quantification shows number of cell diameters covered by signal from the posterior/anterior border marked by Hh-GFP (Bac construct); central horizontal lines show median values of N=6-10 (see Apendix TableS2), box shows lower and upper quartiles and the whiskers show the maximum and minimum excluding outliers. Note a significant reduction after RNAi expression, suggesting Hh reception impairment and flattening of the signal gradient. Significance levels for pairwise test (Wilcoxon) ***P<0.001, **P<0.01, *P<0.05.

C) Lateral confocal images and quantification of Ptc transcription as a Hh target through immunolabeling of the reporter β−galactosidase (Ptc-LacZ). Middle graph shows the extension covered by signal normalized to wing pouch size, in wild type condition (N=6) or after ectopic expression of the Tsp96F-HA construct (N=9). Green shaded areas show data variability and dotted line gives the average values. Note the greater distance covered after Tsp96F-HA transient expression. Boxplot (right) shows distance quantifications with central horizontal lines showing median values of N=6-9 (see Appendix Table S2), box showing lower and upper quartiles and whiskers showing the maximum and minimum excluding outliers. Note a significant extension under Tsp96F-HA expression, suggesting again flattening of the signal gradient. Significance levels for pairwise test (Wilcoxon) ***P<0.001.

Data Information: Scale bars 10 μm.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 1

PR-SET7 is required for proper liver organogenesis during embryonic development

A
Representative pictures of embryos at 18.5 days postcoitum (E18.5) and hematoxylin and eosin staining of whole-mount embryo sections from PR-SET7loxp/Alfp-Cre (PR-SET7ΔHepE) mice and control littermates (WT).

B
Hematoxylin and eosin staining of liver sections.

C
Immunohistochemical staining of liver sections with albumin antibody.

D
qPCR analysis of Albumin,Afp and HNF4 mRNA levels. Bars represent mean values of mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and SEM from samples of four individual mice. *P-value < 0.01.

E
Immunohistochemical staining of liver sections with cyclin B1 antibody.

F
Immunohistochemical staining of liver sections with γH2AX antibody.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 2

Postnatal inactivation of PR-SET7 in hepatocytes leads to cell death

A
Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers.

B
Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A—'normal zone', to Area-B—'necrotic zone' and to Area-C—'regenerative zone'.

C
Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody.

D
TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative.

E
Immunohistological staining with γH2AX and albumin (Alb) antibodies.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 3

PR-SET7-deficient hepatocytes die via necrosis in a cell division-dependent manner

A
Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes.

B
Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei.

C–F
Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 4

Compensatory proliferation of neighboring hepatocytes and ductal progenitor cells in FGF7 signal-containing microenvironment

A, B
Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with Ki67, albumin (Alb) and A6 or HNF4 antibodies as indicated. Arrows depict the different areas in the sections as indicated above (Fig.1B). Asterisks (*) indicate Ki67+ hepatocytes. Small arrows (>) indicate Ki67+ non-hepatic cells. Note the lack of co-staining of cells with HNF4 and A6 antibodies.

C
Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with FGF7 and A6 antibodies.

D
Relative mRNA levels of FGF7. Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120-day-old mice. The data are presented as fold over values obtained with wild-type P45 samples. *P-value < 0.01.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 5

ROS accumulation, STAT3 activation and late-onset spontaneous development of hepatocellular carcinoma in PR-SET7ΔHepA mice

A
Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).

B
Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody.

C
Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies.

D
Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT).

E
Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01.

F
Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown.

G
Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.

Source data are available online for this figure.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 6

Hepatocellular carcinoma in P240 PR-SET7-deficient mice is composed of cells expressing hepatic progenitor cell markers

A, B
Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies.

C–E
Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.
Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers. (2015)
Figure 7

Quantitative assessment and xenograft tumor formation potential of progenitor-derived carcinoma cells

A
P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.

B
Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture.

C
Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1–6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection.

D, E
Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.
TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria (2021)
Figure 1

Figure 1 - Tpl2[D270A] mutation impairs phagosome maturation

A. Phagocytic uptake of fluorescently-labelled latex beads by WT and Tpl2[D270A] (Tpl2D270/D270A) BMDMs. Intracellular fluorescence was monitored following uptake of AF488 latex beads by BMDMs, (n = 4 wells).

B. Intra-phagosomal proteolysis in WT and Tpl2[D270A] BMDMs was assayed following uptake of DQ Green BSA / AF594 latex beads. As positive assay control, BMDMs were separately pre-treated with 100 μg/ml leupeptin for 1 h to inhibit serine-cysteine proteases (n = 4 wells).

C. Cathepsin activity assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells).

D. Intra-phagosomal acidification in WT and Tpl2[D270A] BMDMs was monitored following uptake of BCECF-coupled latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit V-ATPases (n = 4 wells).

E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the Lysotracker Red probe per cell was quantified (n = 95-126 cells).

F. Intra-phagosomal oxidative burst in WT and Tpl2[D270A] BMDMs was assayed following uptake of OxyBURST Green BSA / AF594 latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit ROS production (n = 4 wells)

G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells)

Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Panels (B, D, F) Paired Mann-Whitney t-test; All differences relative to WT are ****. Panels (C, E, G) Student's unpaired t-test.
TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria (2021)
Figure 2

Figure 2 - Tpl2[D270A] mutation promotes phagosome maturation independently of MAP kinase activation

A-F. Experiments were performed using murine BMDMs. (A) Intra-phagosomal proteolysis in WT, Nfkb1[SSAA] (Nfkb1SSAA/SSAA) and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as Fig 1B (n = 4 wells). (B) Intra-phagosomal acidification in WT, Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as in Fig 1C (n = 4 wells). (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. (D) Intra-phagosomal proteolysis in WT and inhibitor-treated BMDMs (n = 4 wells) was monitored as in Fig 1B. BMDMs were pre-treated with 0.1 μM PD0325901 (10 min) to inhibit MEK1, pre-treated with 1 μM VX-745 (1 h) to inhibit p38α and pre-treated with 10 μM C34 (1 h) to inhibit TPL-2. (E). Intra-phagosomal acidification in WT and inhibitor-treated BMDMs (see (D) for conditions) was assayed as in Fig 1C (n = 4 wells). (F) Cell extracts from LPS-stimulated, inhibitor-treated WT and Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. 1 μM bafilomycin A1 was added to inhibit V-ATPases.

G-K. Experiments were performed using human primary monocyte-derived macrophages. (G) Intra-phagosomal proteolysis in human macrophages was monitored following uptake of DQ Green BSA / AF594 latex beads. TPL-2 catalytic kinase activity was blocked by pre-treatment with 10 μM C34 for 1 h (n = 4 wells). (H) Intra-phagosomal acidification in human macrophages was monitored following uptake of BCECF-coupled latex beads (n = 4 wells). (I) Intra-phagosomal proteolysis in human macrophages was assayed as in Fig 2G. MAP kinase activity was inhibited by combinatorial pre-treatment with 0.1 μM PD0325901 (MEK1 inhibition) for 10 min and 1 μM VX-745 (p38 inhibition) for 1 h (n = 4 wells). (J) Intra-phagosomal acidification in human macrophages was monitored as in Fig 2H (n = 4 wells). (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2, ERK1/2, p-p38, p38 and HSP90.

Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Panels (A, B, D, E, G, H, I, J) Paired Mann-Whitney t-test; all differences relative to WT are ****.
TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria (2021)
Figure 3

Figure 3 - Tpl2[D270A] mutation alters the protein composition of phagosomes

WT and Tpl2[D270A] BMDMs were incubated with latex beads for 0.5h. Latex bead phagosomes were purified from Tpl2[D270A] and WT BMDMs and analysed by mass spectrometry. Biological triplicates were analysed for each genotype.

A. Heatmap of selected proteins that were significantly downregulated in BMDMs from Tpl2[D270A] mice relative to WT (P < 0.05). Selected hits were grouped into clusters according to their molecular functions.

B. Gene Set Enrichment Analysis (GSEA) of significantly downregulated biological processes in phagosomal fractions. Changes of Tpl2[D270A] phagosomes relative to WT. Dot colour; enrichment score. Dot size; statistical significance.

C. Protein intensities of V-ATPase subunits from phagosomes purified from WT and Tpl2[D270A] BMDMs, (n = 3 biological replicates).

D. Protein intensities of RAB5 and LAMP-1 from phagosome proteome analysis of WT and Tpl2[D270A] BMDMs (n = 3 biological replicates) (left). Immunoblot of isolated phagosomes from WT and Tpl2[D270A] BMDMs probed for RAB5, LAMP-1 and vimentin. Phagosomal fractions of two biological replicates were pooled. One representative experiment out of two shown (right) (n = 2).

Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria (2021)
Figure 5

Figure 5 - Tpl2[D270A] mutation impairs maturation of Staphylococcus aureus phagosomes

A-K. BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (A) Phagocytic uptake (CFU) of YFP-labelled S. aureus into BMDMs of indicated genotypes 1h post-infection (n = 12; 2 biological and 6 technical replicates per condition per experiment). (B) Representative images of YFP+ BMDMs labelled with Magic Red cathepsin L substrate (red) and DAPI (nuclear stain) 1h post-infection. (C) Quantification of Magic Red cathepsin L staining from panel (A). Average fluorescence intensity of Magic Red cathepsin L substrate per cell relative to fluorescence intensity of YFP-S.aureus (n = 26-29 cells). (D) Quantification of Magic Red cathepsin L staining co-localised with YFP-S. aureus from panel (A) (n = 26-29 cells). (E) Representative images of YFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with YFP-S. aureus (green).(F) Quantification of LysoTracker Red signal from panel (D). Average fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity of YFP-S. aureus (n = 66-98 cells). (G) Quantification of LysoTracker Red co-localisation with YFP-S. aureus from panel (D) (n = 66-98 cells). (H) Representative images of BMDMs stained with an anti-EEA1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (I) Quantification of EEA1+ S. aureus-containing phagosomes from panel (G) (n = 27 cells). (J) Representative images of BMDMs stained with an anti-LAMP-1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (K) Quantification of LAMP-1+ S. aureus phagosomes from panel (I) (n = 25 cells).

Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.