EMBO/soda-nel huggingface dataset:
Intra-epithelial non-canonical Activin A signalling safeguards prostate progenitor quiescence (2022)
Figure 2

Figure 2. Intra-epithelial non-canonical Activin A signalling mediates the Tgf-β induced cytostatic response in mouse prostate organoids

A Cell cycle analysis by flow cytometry (EdU vs. TO-PRO-3) in complete medium (ENRAD) or in the absence of A83-01 (24 hours).

B Detection of proteins secreted by mouse prostate organoids in culture based on click-chemistry enrichment and mass spectrometry analysis (n=4 biological replicates; data points are shown with crossing line representing mean value).

C IF analysis for selected markers in mouse prostate tissue and organoid sections (scale bar = 50 μm).

D Representative stereoscopic images of mouse prostate organoids cultured in the absence of A83-01, with or without Follistatin (Fst, 500 ng/mL, 6 days), or with or without the Activin A neutralizing antibody MAB3381 (1 μg/mL, 6 days). Scale bar = 1 mm

E Western blot (WB) analysis in mouse prostate organoids for selected canonical and non-canonical Tgf-β signalling mediators, and the cell cycle inhibitor p21 (Activin A, 50 ng/mL; Tgfb1, 500 ng/mL; 24 hours).

F Schematic view of the non-canonical Activin A pathway, including inhibitors used for the experiments described in this figure.

G Representative stereoscopic images of mouse prostate organoids following treatment with Takinib (Tak1 inhibitor; 5 μM, 6 days), SB203580 (p38α/β inhibitor; 10 μM, 6 days) or the structurally unrelated Ralimetinib (p38α/β inhibitor; 1 μM, 7 days). Scale bar = 1 mm

H Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected signalling mediators in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 hours).

I Gene set enrichment analysis (GSEA) plots displaying significant enrichment for inflammatory response and Tumor Necrosis Factor (TNF) signalling in mouse prostate organoids cultured without A83-01 (24 hours) versus complete medium (ENRAD).

J Nuclear/Cytoplasmic fractionation and western blot analysis in mouse prostate organoids for selected cell cycle regulators, in the presence or absence of SB203580 (p38α/β inhibitor; 10 μM, 24 or 48 hours).

Data information: In (A) quantification of cells in S-phase for n = 3 biological replicates are presented in Appendix Figure S2A.
Intra-epithelial non-canonical Activin A signalling safeguards prostate progenitor quiescence (2022)
Figure 3

Figure 3. Evasion from the Tgf-β induced cytostatic response leads to DNA replication stress and genomic instability

A Diagram depicting the expansion of mouse prostate organoid cultures in the absence of the Tgf-β ligand inhibitor A83-01 (n = 3 biological replicates per lobe-specific organoid culture; arrow = continuous expansion; dot = passage; cross = culture loss).

B Representative stereoscopic images of C57BL/6J DLP #1 and #3 mouse prostate organoid lines during adaptation in the absence of A83-01. Scale bar = 1 mm

C Principal Component Analysis (PCA) based on bulk RNA-sequencing of C57#1 DLP and C57#3 DLP mouse prostate organoids cultured in normal conditions (ENRAD) (n = 3 biological replicates, C57#1 DLP; n = 3 biological replicates, C57#3 DLP), upon acute A83-01 depletion (-A83-01, 24 hours) (n = 3 biological replicates, C57#1 DLP only) or adaptation (-A83-01, long-term) (n = 3 biological replicates, C57#1 DLP; n = 3 biological replicates, C57#3 DLP).

D Gene set enrichment analysis (GSEA) plot displaying significant enrichment for activation of ATR signalling in mouse prostate organoid lines (C57#1 DLP, top; C57#3 DLP, bottom) adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD).

E Western blot analysis in C57#1 and C57#3 DLP mouse prostate organoid lines adapted to grow in the absence of A83-01 vs. normal control organoids cultured in complete medium (ENRAD). Immunoblots are displayed for Chek1 (ATR signalling mediator), Trp53 and p21.

F Representative spectral karyotype (SKY) images of metaphase spreads obtained from C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread genomic instability is observed following adaptation. Scale bars = 5 µm.

G-H Representative telomere FISH images - and quantification (n = 2 biological replicates, 20 metaphases per condition) - in C57#1 DLP mouse prostate organoids cultured in normal conditions (ENRAD) or upon adaptation without A83-01. Widespread telomeric instability is observed following adaptation (arrows indicate telomere doublets, One-way ANOVA, scale bars = 10 µm).

Data information: F, quantification of spectral karyotype (SKY) for 25 metaphases per condition in n = 2 biological replicates are presented in Expanded Figure EV5B.
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 1

Figure 1 - Loss of Zuc and Gasz impacts the cellular localization of Armi and Piwi in OSCs.

A. Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control.

B. Western blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively.

C. Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei. D. Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 2

Figure 2 - Mitochondrial Gasz interacts with Armi and Piwi bound with piRNA precursor.

A. Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control.

B. Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD). Armi signal with a longer exposure is shown in Fig EV2A.

C. Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD). A gel image of the control lanes (control) with a long exposure is shown in Fig EV2B.

D. Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 3

Figure 3 - Departure of Armi from Yb bodies depends on Piwi.

A and B. Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.

C. The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05.

D. The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31.

E. In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 4

Figure 4 - Departure of Armi from Yb bodies requires piRNA precursor loading onto Piwi.

A. Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting.

B. Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.

C. Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 5

Figure 5 - Translocation of Piwi−pre-piRISC from Yb bodies requires the RNA-binding activity of Armi

A. Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT bound with Piwi and Gasz. The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz. Luciferase-Flag (Luc-F) was used as a negative control.

B. Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.

C. In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.
Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation (2019)
Figure 6

Figure 6 - The N-terminal end of Armi plays an important role in the departure of Piwi−pre-piRISC from Yb bodies.

A. Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control.

B. Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.

C. In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.

D. Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 1

FIGURE 1. Expression of FOXA1 and FOXA2 in human PDACs.

A) FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade. Immunohistochemistry analysis of FOXA1 (top) and FOXA2 (bottom) in human PDAC. Representative G1, G2 and G3 areas from one patient are shown. Red dotted lines indicate the borders of poorly differentiated (G3) tumor areas.

B) Percent of FOXA1- and FOXA2-positive nuclei in PDAC cells from areas of different grade. Tissue microarrays were automatically acquired and quantified using QuPath. The number (n) of analyzed areas of each grade is indicated. P‐values were calculated using a Welch's two-samples t-test. Means + SD are shown. Each dot represents a single area.

C) Expression of FOXA1 and FOXA2 in low-grade and high-grade PDAC cell lines. Vinculin is shown as loading control.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 2

FIGURE 2. Contribution of FOXA1/2 to the transcriptional programs of PDAC cells.

A) Western blot showing siRNA-mediated depletion of FOXA1 and/or FOXA2 in the low-grade CFPAC1 cell line. The three biological replicates used in the subsequent experiments are shown.

B) Heatmap showing row-normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1/2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.

C) FOXA2 and FOXA1 expression in wild type and genome-edited high-grade PANC1 cells. Three different bulk populations were used for RNA-seq experiments.

D) Heatmap showing row-normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.

E) Venn diagram showing the overlap between FOXA1/2-regulated genes in CFPAC1 and PANC1 cells.

F) Representative Gene Ontology (GO) terms enriched in genes differentially expressed upon FOXA1/2 depletion in CFPAC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA1/2 depletion on cell morphology and adhesion to multiple substrates. Three replicates per condition were used: circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition. P‐values were calculated using a Welch's two-samples t-test. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.

G) Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition. P‐values were calculated using a Welch's two-samples t-test. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 3

FIGURE 3. Genomic distribution of FOXA2 in well- and poorly-differentiated PDAC cells.

A) Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated.

B) Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.

C) Distribution of the positions of the FOXA motif relative to the summit of different sets of FOXA2 peaks. The PWM used to perform CentriMo analysis and the E-value for each dataset are shown.

D) Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific.

E) GO terms retrieved by GREAT analysis associated with distal FOXA2 peaks specific of CFPAC1 (left) or PANC1 (right). Terms are ranked by the negative log10 p-value from binomial correct test.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 4

FIGURE 4. Identification of FOXA2 partner transcription factors in well-differentiated PDAC cells.

A) The five top transcription factor motifs over-represented in TSS-distal CFPAC1-specific FOXA2 peaks. TFs were grouped into families or subfamilies and ranked based on the best PWM q-value. The POU domain TF family includes HNF1β. Top scoring motifs are shown on the right.

B) Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP-seq peaks.

C) Distribution of the positions of the best HNF1β motif relative to the summit of the CFPAC1-specific FOXA2 peaks. The graph indicates the average density in 20 bp bins in a window of 500 bp.

D) Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS (left) and JUNB (right) ChIP-seq peaks.

E) Distribution of the positions of the AP-1 motif relative to the summit of the TSS-distal, CFPAC1-specific FOXA2 peaks. The graph indicates the average density in 20 bp bins in a window of 500 bp.

F) Two representative ChIP-seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 5

FIGURE 5. Expression of HNF1b in PDAC and correlation with FOXA2.

A) Representative areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown. P‐values were calculated using a Welch's two-samples t-test. Means + SD are shown. Each dot represents a single area.

B) Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 6

FIGURE 6. Impact of HNF1β loss on FOXA2 binding and chromatin organization.

A) FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks.

B) Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models.

C) (Left) Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.

D) Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.
FOXA2 controls the cis-regulatory networks of pancreatic cancer cells in a differentiation grade-specific manner (2019)
Figure 7

FIGURE 7. Expression of HNF1β promotes recruitment of FOXA2 to chromatin.

A) Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control.

B) Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right.

C) TF motif over-represented in gained or lost FOXA2-bound genomic regions.

D) Representative snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.