EMBO/soda-nel huggingface dataset:
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 1

Figure 1: TGFβ/ActivinB pathway is activated in Group 3 MB patients and cell lines. (A) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) and PMEPA1 (high and low exposures displayed) in MB patient sample lysates from different groups: WNT (blue), SHH (red), Group 3 (yellow) or Group 4 (green). β-actin was used as a loading control. Relative quantification of P-Smad2 signal to β-actin (P-S2/β-Actin) and total Smad2 (P-S2/Tot-S2) are indicated below the blots. (B) Boxplots summarizing the expression of INHBB, TGFB1 and TGFB3 ligands of the TGFβ/Activin pathway in the different groups of MB (blue WNT, red SHH, yellow Group 3 and green Group 4) and in fetal and adult cerebellum (grey) in the dataset of "Data ref: Cavalli et al, 2017". (C) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in non-Group 3 (blue) and Group 3 (yellow) MB cell lines on the left panel. The level of total Smad2 (Smad2) was assessed and β-actin was used as a loading control. On the right panel, relative level of P-Smad2 (P-S2) were quantified to total β-actin. P-Smad2 to total Smad normalization is also provided on Appendix Figure S5. (D) RT-qPCR were performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBB (left) and TGFB3 (right). Wilcoxon rank-sum tests were performed to determine p-values for panel B. The remaining p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5. Detailed statistics are presented in Appendix Table S1 for B and Appendix Table S2 for D.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 2

Figure 2: An autocrine stimulation by ActivinB in the 1603MED cell line. (A-C, E) The level of phosphorylated Smad2 (P-Smad2) and total Smad2 (Smad2) was assessed by immunoblotting and β-actin was used as a loading control. Lower bar graphs show WB quantification of P-Smad2 (P-S2) normalized to β-actin. (A) Activation of the pathway was assessed in non-Group 3 (blue) and Group 3 (yellow) MB cell lines in response to TGFβ or ActivinB stimulation for 1h. (B) 1603MED cells were treated with PBS (vehicle) or a blocking antibody targeting ActivinB (Ab α-ActB) or Follistatin. (C) Conditioned media experiments were performed on the HDMB03 MB cell line. Phosphorylation of Smad2 was analyzed by immunoblot upon treatment with either non conditioned media (NCM), media conditioned with HDMB03 cells (CM-HDMB03) or media conditioned with 1603MED cells (CM-1603). Pre-incubation with blocking antibody against ActivinB (Ab α-ActB) or vehicule (PBS) was performed before HDMB03 cell-line treatment as indicated. Relative level of P-Smad2 (P-S2) were quantified to β-actin (below). (D) RT-qPCR were performed on total RNA extracted from 1603MED cells 48 hours after transfection with siRNA targeting INHBB. Relative INHBB expression was assessed. siCTRL condition was set at 1. (E) 1603MED cells were transfected with the indicated control siRNA (siCTRL, blue) or targeting INHBB (siINHBB, red). Lysates were prepared 48 hours after transfection. Lower bar graphs represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. (F) Growth curve of 1603MED cells after transfection with either siCTRL (blue) or siINHBB (red). P-Smad2 to total Smad normalization is provided on Appendix Figure S5. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 3

Figure 3: ActivinB promotes cell proliferation in Group 3 MB cell lines. (A-D) D458 or (E-H) D283 cell lines were treated with PBS (vehicle, black) or with ActivinB (green). (A and E) Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin in response to ActivinB stimulation for 24 hours. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panels. (B and F). P-Smad2 to total Smad normalization is provided on Appendix Figure S5. Growth curve experiments showing cell proliferation upon ActivinB treatment. (C and G) Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon ActivinB stimulation. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). (D and H) Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48h hours after treatment with ActivinB. The p-values were determined by unpaired t-test and two-way ANOVA for B and F. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 4

Figure 4: TGFβ/ActivinB signaling promotes cell proliferation in Group 3 MB cell lines. (A-D) 1603MED or (E-H) D283 cells were treated with DMSO (vehicle, black), with LY364947 (red) or with SB431542 (orange). (A and E) Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon inhibition of TGFβ/Activin signaling using LY364947 and SB431542 inhibitors for 24 hours. Bar graphs on the right panel represent the quantification of the relative level of P-Smad2 (P-S2) to β-actin. (B and F). P-Smad2 to total Smad normalization is provided on Appendix Figure S5. Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition. (C-G) Cell cycle analysis by FACS measuring BrdU incorporation and 7AAD labelling at 48 hours upon inhibition. The percentage of cells in the different phases of the cell cycle is represented (G0/G1, S and G2/M phases). (D and H) Percentage of apoptotic cells measured by FACS analysis of cleaved caspase3 48 hours after TGFβ/Activin signaling inhibition. The p-values were determined by unpaired t-test and two-way ANOVA for B and F. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 5

Figure 5: PMEPA1 is a target gene involved in the response to Activin signaling

(A) Ranking of top genes whose expression is correlated with INHBB in Group 3 MB patient samples. Spearman's rank correlation coefficient ρ and p-value are indicated. (B) Boxplots representing PMEPA1 expression levels in the different MB groups (WNT in blue, SHH in red, Group 3 in yellow and Group 4 in green) and in fetal and adult cerebellum (grey) in the dataset " Data ref: Cavalli et al, 2017". Only p-values corresponding to comparisons between Group 3 and the other groups are indicated. Full statistics can be found in Appendix Table S1 (C) Scatter plot of INHBB and PMEPA1 gene expression levels in all MB groups. Colored dots represent each patient samples and colors represent the MB groups (WNT in blue, SHH in red, Group 3 in yellow and Group 4 in green). (D) Boxplot represents the quantification of PMEPA1 protein level normalized to β-actin levels across groups. (E) Scatter plot represents Log2 relative protein level of P-Smad2 (x axis) and PMEPA1 (Y axis) normalized to β-actin in each individual samples. The color code is the same as for B, C and E. (F) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from 1603MED or D283 or D458 cells treated with either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB (ActB) for 24 hours. Blot quantification to β-actin is presented in Appendix Figure S3D. (G and I) Immunoblot analysis of PMEPA1 levels in 1603MED (G) and D283 (I) cells 48 hours after transfection with either siCTRL (blue) or siPMEPA1 (red). Bar graphs on the right represent the quantification of the relative level of PMEPA1 protein level normalized to β-actin. P-Smad2 to total Smad normalization is provided on Appendix Figure S5. (H and J) Growth curves of 1603MED (H) and D283 (J) cells after transfection with either siCTRL (blue) or siPMEPA1 (red). The p-values were determined by Spearman rank correlation test for A and C, by unpaired t-test for D, G and I, by two-way ANOVA forH and J, and by Wilcoxon rank-sum test for panel B and E. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 6

Figure 6: Activated TGFβ/ActivinB signaling in group 3 MB PDXs.

(A) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in Group 3 MB cell lines and PDXs. The level of total Smad2 (Smad2) was assessed and β-actin was used as a loading control. Quantification of P-Smad2 (P-S2) to β-actin are shown on right panel. (B) Expression of INHBB in Group 3 MB cell lines and PDXs relative to HDMB03 (set at 1) by RT-qPCR. (C) Immunoblot of phosphorylated Smad2 (P-Smad2), total Smad2 and β-actin upon ActivinB or TGFβ stimulation for 1 hour. (D) Conditioned media experiments were performed on HDMB03 MB cell line. Phosphorylation of Smad2 (P-Smad2) was analyzed by immunoblot upon treatment with either non-conditioned media (NCM), media conditioned with HDMB03 cells (CM-HDMB03) or media conditioned on PDX4 cells (CM-PDX4). Pre-incubation with blocking antibody against ActivinB (Ab α-ActB) or with vehicle was performed before HDMB03 cell-line treatment as indicated. (A, C, D) Right (A) and lower (C-D) panels represent the relative quantification of P-Smad2 normalized to β-actin. (E) Immunoblots of phosphorylated Smad2 (P-Smad2), total Smad2, MYC, PMEPA1, OTX2 and β-actin were performed on extracts from cell cultures of PDX4, PDX3 and PDX7 treated by either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α-ActB), Follistatin, PBS or ActivinB for 24 hours. WB quantification is depicted in Appendix Figure S4C. P-Smad2 to total Smad normalization is provided on Appendix Figure S5. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed statistics are presented in Appendix Table S2 for B. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.
An autocrine ActivinB mechanism drives TGFβ/Activin signaling in Group 3 medulloblastoma (2019)
Figure 7

Figure 7: ActivinB signaling is a potential therapeutic target for patients of group 3 MB. (A) Kaplan-Meier representing survival of mice treated with either vehicle (black) or Galunisertib (LY2157299, red) or Cisplatin (blue) or a combination of Galunisertib and cisplatin (purple) after orthotopic grafting of PDX4 cells into the cerebellum. The pink rectangle represents Galunisertib treatment duration, while the blue dotted lines represent the 3 cisplatin administrations. (B) Boxplot of tumor area after 25 days of treatment. On the right, boxplots represent quantification of P-Smad2 staining on tumor (IHC). The code color is similar to panel A. (C) P-Smad2 staining by IHC in 3 representative tumors per group after 25 days of treatment. The scale represents 500μm and 100μm on the left and right panels, respectively. (D) Boxplots representing the expression level of INHBB, PMEPA1, MYC and NRL in the different MB subtypes, as defined in Cavalli et al. (Cavalli et al., 2017). Only p-values corresponding to comparisons between Group 3 subtypes are indicated. Detailed statistics are presented in appendix Table S3. Patient samples are colored by subtypes as indicated. (E) Scatter plot of INHBB and PMEPA1, expression levels in Group 3 patient samples. Colored dots represent each patient sample and colors represent the group 3 MB subtypes (α in yellow, β in brown and δ in orange). Note that this panel is identical to that shown in Appendix Figure S3A (yellow) except for the color code. The p-values were determined by Log-Rank (Mantel-Cox) test on panel A and unpaired t-test on panel B. Wilcoxon rank-sum tests were performed for panel D. Spearman's rank correlation coefficient ρ and p-value are indicated on panel E. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3. The exact p-values and number of replicates are indicated in Appendix Table S5.

Expanded View figure Legends
RADX interacts with single-stranded DNA to promote replication fork stability (2017)
Figure 1

Figure 1.

RADX (CXorf57) is an ssDNA-binding protein

A. Domain organization of human RADX and RPA1, showing location of OB folds, including three DNA-binding domains (DBDs) in RPA1, and RADX mutants used in this study (see also Fig EV1A). B. Predicted in silico folding of OB folds 2 and 3 of human RADX, modeled by Phyre2, showing similarity to the structure of the region encompassing DBD-A and DBD-B in human RPA1 (Fig 1A). C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies. D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting. E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies. F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B. G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and U2OS/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm. H. Quantification of data in (F) by quantitative image-based cytometry (QIBC) (n≥3000 cells per condition; data from a representative experiment are shown). See also Fig EV1G. I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.
RADX interacts with single-stranded DNA to promote replication fork stability (2017)
Figure 2

Figure 2.

Recruitment of RADX to sites of replication stress

A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU. B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A. C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody. D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody. E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation. F. Quantification of data in (E) by QIBC (n≥3000 cells per condition; data from a representative experiment are shown). See also Fig EV2D. All scale bars, 10 μm.
RADX interacts with single-stranded DNA to promote replication fork stability (2017)
Figure 3

Figure 3.

RADX promotes DNA replication integrity

A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ). B. Schematic for DNA fiber assays in (C-E). C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments). G. Schematic for DNA fiber assays in (H) and (I). H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).
RADX interacts with single-stranded DNA to promote replication fork stability (2017)
Figure 4

Figure 4.

The balance between RADX and RPA ssDNA-binding activities is critical for DNA replication integrity

A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments). D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments). E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition). G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition). H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).
RADX interacts with single-stranded DNA to promote replication fork stability (2017)
Figure 5

Figure 5.

RADX promotes replication fork stability and cell survival upon replication stress

A. Schematic for replication fork protection assays in (B) and (C). B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition). C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines. D. U2OS or U2OS/GFP-RADX cells transfected with indicated siRNAs and exposed to HU for 4 h were co-immunostained with RPA1 and γ-H2AX antibodies and analyzed by QIBC. Proportion of cells displaying maximal RPA chromatin loading accompanied by H2AX hyperphosphorylation (Fig EV5A), reflecting replication fork catastrophe [17], is indicated (bars, mean; n=5 independent experiments (≥3000 cells analyzed per condition); *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001, unpaired t-test). E. As in (D), but using U2OS or U2OS/Super-RPA cells exposed or not to HU (n=6 independent experiments). See also Fig EV5B. F. Clonogenic survival of U2OS cells transfected with indicated siRNAs and subjected to different CPT doses for 24 h (mean±SEM;n=3 independent experiments). LQ (Linear Quadratic) model was fitted to the fractional survival data, using non-linear least square method. Overlap between confidence intervals of the fitting coefficients was used to evaluate the statistical difference between cell survival after siRADX and siCTRL treatment (*No overlap of 95% confidence interval with siCTRL). G. As in (F), except cells were treated with indicated doses of HU (mean±SEM;n=3 independent experiments). H. As in (F), using U2OS and U2OS/Super-RPA cells (mean±SEM;n=3 independent experiments).

Expanded View Figure legends
Identification of TOR-responsive slow cycling neoblasts in planarians (2020)
Figure 1

Figure 1: Planarian neoblasts have variable rates of division.

A Whole mount FISH for piwi-1 and immunostaining for BrdU at 1-day, 1-week and 5-weeks post-BrdU administration. White boxes denote the magnified regions shown in the bottom panels. Scale bars, 30 µm.

B Quantification of the percentage of BrdU label-retaining neoblasts. Data are presented as mean ± s.d. (n = 4-6 animals per time point).

C Double pulse experiment showing colocalization of BrdU after a 2-week chase period with EdU after a 1-day chase period. White boxes denote the magnified region shown on the left. Dashed lines mark the edges of the animal and white arrows mark colocalization. Scale bar, 50 µm.

Data information: All images are single confocal planes taken from the prepharyngeal region as indicated in the diagrams.
Identification of TOR-responsive slow cycling neoblasts in planarians (2020)
Figure 5

Figure 5: RNAlow neoblasts exhibit TOR-dependent cell cycle entry during regeneration.

A Schematic of the experimental design.

B FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and 48 hpa regenerating animals following 1 day of NDZ treatment.

C RNAlow neoblast size in intact animals and at 48 hpa in control or TOR KD animals (n ≥ 55).

D Quantification of H3P immunostaining in intact animals and at 48 hpa in control or TOR KD animals (n = 5-10).

E RNAlow neoblast size in intact animals and at 48 hpa in control, raptor or rictor KD animals (n ≥ 54).

F FACS plots of Hoechst and Pyronin Y stained cells isolated from intact and regenerating animals following 1 day of nocodazole treatment in control or TOR KD animals. Percentages indicate the proportion of events within each gate.

Data information: Data in C-E are presented as mean ± s.d. Statistical significance was assessed using Welch's t-tests (p values are indicated; ns, not significant (p > 0.05)). In C and D, animals were amputated at 5fd3. In E, Animals were amputated at 7fd3. Data are representative of 3 (B) or 2 (C-F) independent experiments.