EMBO/soda-nel huggingface dataset:
Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection (2018)
Figure 4

Figure 4: During MCMV infection, the TLR9/Myd88-dependent pathway is necessary for pDC cytokine production, irrespective of their IFN-I responsiveness

(A) Dot plots represent IFN-I vs GFP staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-GFP-infected MBMC. One staining representative of 6 animals from 2 independent experiments is shown.

(B, C) Percentages of GFP+ cells within 5.2+ pDC (B) or of GFP+ (green) vs GFP- (white) cells within 5.2+ IFN-I-producing pDC (C) isolated from indicated MCMV-GFP-infected MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.

(D) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV-infected MBMC. CTR MBMC (black) and Myd88-/- TST MBMC (dark green) were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.

(E-G) CD86 MFI in 5.2+ pDC of each indicated MBMC type (E) and 5.2/ 5.1 ratio of cytokine-producing pDC (F, G). Data (mean±SEM) are representative of 2 pooled independent experiments each with 2-3 mice per group.

(H-L) CD86 MFI (H, K) and percentages of cytokine-producing cells (I, J and L) within pDC of each indicated mouse strain. Black, C57BL6; red, Ifnar1-/-; orange, Ifnar1-/-x Myd88-/-; green, Tlr9-/- and pale green, Tlr7-/-. In (K-L) indicated mice were treated with IC (square) or blocking α-IFNAR1 mAbs (triangle). Data depicted (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.

Data information: ns, not significant (p>0.05); *, p<0.05; **, p<0.01; ***, p<0.001; nonparametric Mann-Whitney test for (B), non parametric Kruskal-Wallis test combined with Dunn's multiple correction test for (D-L).
Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection (2018)
Figure 5

Figure 5: IRF7 is necessary for IFN-I production in pDC, but low protein levels are sufficient to promote this function during MCMV infection

(A) IRF7 protein expression in pDC isolated from Irf7-/- (pink, bottom), C57BL/6 (WT, black, top) and Ifnar1-/- (red, middle) uninfected (empty histograms, Ø, left) vs MCMV-infected mice (filled histograms, MCMV, right). For each condition histograms are representative of one out of 4-6 mice from 2 independent experiments.

(B) IRF7 protein expression in pDC (left), cDC (middle) and conventional monocytes (cMo, right) isolated from uninfected (Ø, empty) or MCMV-infected (MCMV, filled) C57BL/6 (black) or Ifnar1-/- (red) mice. Data are shown as net IRF7 MFI, after subtraction of the MFI obtained using isotype control mAbs. Data shown (mean±SEM) are pooled from 2 independent experiments with 5-6 mice for each group.

(C-E) The expression of CD86 (C) or of indicated cytokines (D-E) was analyzed, as described in Fig 2. Black, CTR MBMC; pink, Irf7-TST MBMC. Mice were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.

Data information: ns, not significant (p>0.05); *, p<0.05; **, p<0.01; ***, p<0.001; non parametric Kruskal-Wallis test combined with Dunn's multiple correction test for (B-E).
Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection (2018)
Figure 6

Figure 6: IKKβ is required for pDC IFN-I production

(A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected (A) or CpG-stimulated (B) MBMC of each indicated type.

(C) Ratio of IL-12- or TNF-producing cells in 5.2+ vs 5.1+ pDC of indicated MCMV-infected MBMC mice.

Data information: Black, CTR MBMC; Brown, Ikbkb∆Itgax TST MBMC; pale brown, IkkαAA TST MBMC. Data shown (mean±SEM) are from 2-3 pooled independent experiments each with 2-3 mice per group. ns, not significant (p>0.05); *, p<0.05; **, p<0.01; non parametric Kruskal-Wallis test combined with Dunn's multiple correction test for panels (A-C).
Molecular dissection of plasmacytoid dendritic cell activation in vivo during a viral infection (2018)
Figure 7

Figure 7: pDC IFN-I production is promoted by LFA1 engagement and occurs in the vicinity of infected cells during MCMV infection.

(A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected (A) or CpG-stimulated (B) MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group. ns, not significant (p>0.05); *, p<0.05; **, p<0.01; nonparametric Mann-Whitney test.

(C) EYFP expression in splenocytes isolated from uninfected (NI) vs MCMV-infected (MCMV) IFNbtm1(EYFP) reporter mice. The expression of indicated markers was analyzed on YFP+ cells. Plots are representative of one out of 6 mice from 2 independent experiments.

(D-F) Immunohistological analysis of splenic sections stained with mAb against MOMA1/CD169 (blue), GFP/EYFP (green), whose expression correlates with that of IFN-β, and MCMV IE-1 (red). Scale bars, 100µm. The image shown is one representative of 15 obtained from 5 different mice. Optical magnification was 20x, and images were scanned on the microscope at zoom x2 for (E, F). White dotted lines delimit marginal zone. Yellow squares in (D) defines zoomed region in (E, F).

Expanded View Figures Legends
Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy. (2016)
Fig 3

Autophagy evaluation in motor cortex and lumbatr spinal cord

A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively. B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.
PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. (2014)
Figure 1

PINK1 accumulates upon knockdown of Lon.

(A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4. The RNAi constructs used were as follows: the non-specific control mCherry-RNAi (Control-R), Lon-RNAi1 (Lon-R1), and Lon-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio. (C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B. (G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.
PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. (2014)
Figure 2

PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.

(A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V β (Comp V β), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. Results shown are representative of at least three independent subcellular fractionation experiments per genotype. Error bars represent s.e.m. *p<0.05 by Student t test.
PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. (2014)
Figure 3

PINK1 accumulates on the outer membrane and in the matrix upon knockdown of Lon.

(A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane–associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown. (C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison. (E) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.
PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. (2014)
Figure 4

PINK1 accumulates in the mitochondrial matrix upon knockdown of Lon.

Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged Mitochondrial Rho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A–E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.
PINK1-Parkin pathway activity is regulated by degradation of PINK1 in the mitochondrial matrix. (2014)
Figure 5

Lon deficiency stimulates PINK1 activity <italic>in vivo</italic>.

(A) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver. (B) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L)Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 1

Figure 1: Tgif1 inactivation deepens KrasG12D-driven PDAC

A Formalin-fixed paraffin-embedded (FFPE) sections from control or Tgif1KO mice were immunostained with anti-pSmad2 antibody and revealed by immunofluorescence (IF) and DAPI. Representative pictures at 40x are shown (n=30). Scale bars, 100 μM.

B FFPE pancreatic sections from control or Tgif1KO mice were stained with hematoxylin and eosin (H&E) or immunostained with antibodies to CK19 or Amylase and revealed by IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.

C Kaplan-Meyer survival analysis of control, Tgif1KO, KC and KTC mice. A regular mosaic two-colors line was used to discriminate between control and Tgif1KO mice.

D Pictures of whole pancreas and spleen tissues from control, Tgif1KO, KC or KTC mice (left). Weight of pancreas was measured and presented as dot plot (n=30).

E FFPE pancreatic sections from control, Tgif1KO, KC or KTC were stained with H&E or subjected to IHC analysis using antibodies to CK19 or α-SMA. Yellow arrows indicate PanINs in KC mice. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 2

Figure 2: Tgif1 inactivation promotes PDAC metastasis

A Representative pictures of dissected abdominal region of KC and KTC mice. Yellow arrows indicate metastatic nodules in liver. Yellow dotted lines show the pancreas.

B FFPE sections of pancreas, liver, lungs or lymph-nodes from control, KC or KTC mice were stained with H&E or immunostained with anti-Vimentin antibody and revealed by IF. Representative pictures at 4x (Lymph node, H&E), 10x (Liver, H&E) and 20x (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin) are shown (n=18 to 30). Scale bars, 1,000 μM (Lymph node, H&E), 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Lung, H&E; Lymph node, IF-Vimentin).

C Tumor volume and metastasis in live KCLuc and KTCLuc mice were analyzed by IVIS bioluminescence. Following imaging live animals, pancreas, brain, liver and lung were dissected and analyzed again by IVIS bioluminescence (n= 6, 5 out of 6 mice developed metastasis to liver and lung).

D Total cell lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to pSmad2, Smad2 or JunB (n=6).

E Expression of JunB in pancreas from control, KC or KTC mice (same as in D) was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; based on a two-tailed Student's test.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 3

Figure 3: TGIF1 associates and with and inhibits Twist1 expression

A-C Protein extracts from MIAPaCa-2 cells (left) and control or Tgif1KO mice (right) were immunoprecipitated using normal IgG, anti-TGIF1 or anti-Twist1 antibody and analyzed by immunoblotting using antibodies to Twist1 or TGIF1 (A-C). In C, cell lysates were treated with DNase I before coimmunopreciptation. To control for loading, total cell lysates were analyzed by immunoblotting using antibodies to Twist1, TGIF1 or β-Actin.

D Panc-1 cells were treated with TGF-β for 30 min, and protein extracts were immunoprecipitated with anti-TGIF1 antibody and analyzed by immunoblotting using anti-Twist1 antibody. To control for activation of TGF-β signaling, total cell lysates were analyzed by immunoblotting using anti-pSmad2 antibody.

E PL45 stably expressing Dox-inducible control or sh-RNA targeting TGIF were treated with vehicle or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6). In left panel, the intensity of the bands was assessed by densitometry, and the result was presented as a ratio of Twist1/TGIF1. In right panel, data are expressed as mean ± SEM. ***p< 0.001; ns, not significant; based on a two-tailed Student's test.

F PL45 stably expressing Dox-inducible TGIF1 were treated with vehicle or Dox for 48 hr and analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.

G Pancreatic chromatin from control or Tgif1KO mice was analyzed for binding of TGIF1 to the Twist1 promoter by ChIP and agarose gel.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 4

Figure 4: TGIF1 inhibits Twist1 transcriptional activity

A PL45 stably expressing both Dox-inducible TGIF1 and Tam-inducible Twist1 (Twist1ER) were treated with Tam and/or Dox for 48 hr and analyzed for Twist1 expression by immunoblotting or qRT-PCR (n=6).

B MIAPaCa-2 cells were transfected with wild-type or mutated (Twist1 binding site) Twist1 luciferase reporters (TGTLuc) together with increasing amounts of Twist1 expression vector in the presence of empty vector (EV) or TGIF1 expression vector. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).

C Total lysates from pancreas of control, KC or KTC mice were pooled and analyzed by immunoblotting using antibodies to E-Cadherin, Vimentin, p16Ink4A and β-Actin (n=6).

D-F Expression of Cdh1, Vimentin, and p16Ink4A in pancreas from control, KC or KTC mice (same as in C) was analyzed by qRT-PCR (n=6).

G FFPE pancreatic sections from control, KC or KTC mice were stained with H&E or immunostained with antibodies to E-Cadherin, Vimentin or p16Ink4a and revealed by IF or IHC. Representative pictures at 20x are shown (n=30). Scale bars, 200 μM.

Data information: For (A, B, D, E and F), data are expressed as mean ± SEM. *p< 0.05; **p< 0.01; ***p< 0.001; ns, not significant; based on a two-tailed Student's test.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 5

Figure 5: Twist1 inactivation blocks PDAC formation and metastasis in KTC mice

A Kaplan-Meyer survival analysis of control, KTC and KTWC mice.

B FFPE sections of pancreas or liver from control, KTC or KTWC mice were stained with H&E or immunostained with anti-CK19 antibody and revealed by IHC. Representative pictures at 10x (Liver, H&E) or 20x (Pancreas, H&E; Pancreas, IHC-CK19) are shown (n=30). Scale bars, 400 μM (Liver, H&E) and 200 μM (Pancreas, H&E; Pancreas, IHC-CK19).

C-E Expression of Cdh1, Vimentin and p16Ink4A in pancreas from control, KTC or KTWC mice was analyzed by qRT-PCR (n=6). Data are expressed as mean ± SEM. **p< 0.01; ***p< 0.001; based on a two-tailed Student's test.
TGIF1 functions as a tumor suppressor in pancreatic ductal adenocarcinoma (2019)
Figure 6

Figure 6: Increased TGIF1 phosphorylation in human PDAC

A Specificity of anti-phospho-TGIF1 (pTGIF1) antibody. Cell extracts from Tgif1-/- MEFs cells reconstituted with empty vector (EV), wild-type (TGIF1) or non-phosphorylated form (TGIF1.2TA) of Flag-TGIF1 were immunoblotted with anti-phospho-TGIF1 or β-Actin as a loading control.

B,C Expression of pTGIF1 and Twist1 in human tissue microarrays of human PDAC samples was analyzed by immunohistochemistry. Representative pictures at different stages (40x) are shown. The percentages of samples with high versus low expression of pTGIF1 and Twist1 in normal tissues versus PDAC tissues are shown. Scale bars, 100 μM.

D,E MIAPaCa-2 cells were transfected with TGIF1 or TGIF1.2TD expression vectors, selected with G418 and resistance colonies were pooled (n=6). Expression of Twist1 was examined by qRT-PCR (D). Binding of TGIF1 to the Twist1 promoter was examined by ChIP and agarose gel.

F BxPC3 cells were transfected with TGTLuc together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D. Luciferase activity was measured 48 hr following transfection and normalized on the basis of co-transfected Renilla luciferase (n=6).

G,H BxPC3 cells were transfected with CDH1Luc or p16Luc reporter together with TGIF1 or TGIF1.2TA in the presence or absence of KrasG12D and luciferase activity was measured 48 hr following transfection as described in (E) (n=6).

Data information: For (D, F, G and H), data are expressed as mean ± SEM. **p< 0.01; ***p< 0.001; ns: not significant; based on a two-tailed Student's test.