EMBO/soda-nel huggingface dataset:
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 1

Snai1 is localised to stem and progenitor cells within mouse intestinal crypts, while Snai2 is restricted to the mesenchyme

A, B Temporal expression of Snail genes in the intestinal tract in (A) embryonic E12.5 to E18.5 and (B) postnatal P0 to P20 measured by qRT–PCR. Shown are means ± SD.
C, E, G, I Immunohistochemical staining for Snai1/2 (Abcam ab85931) (C) E14.5 midgut, (E) E14.5 hind gut, (G) postnatal day 14 SI, (I) postnatal day 14 colon. Snai1 immunohistochemistry peptide competition controls are shown as inserts (C', E', G', I'). Scale bars: 20 mm.
D, F, H, J, K X‐gal staining for β‐galactosidase activity in tissue from Snai2LacZ mice stained in whole mount, sectioned and counterstained with nuclear fast red (D) E14.5 midgut, (F) E14.5 hind gut, (H) postnatal day 14 SI, (J) postnatal day 14 colon, (K) adult SI. Controls for X‐gal staining are shown as inserts (D', F', H', J').
L, M, N Double immunostaining of small intestinal crypts for Snai1/2 (red) and (M) Lgr5‐GFP (green); (N) merge shows Snai1 is present in both CBC stem cells and proliferating transit amplifying cells. Scale bars: 10 mm.
O, P Immunohistochemistry for Snai1 using a different primary antibody (Genetex GTX125918) shows minimal expression of Snai1 in villi. (P) Expression of Snai1 is detected at the base of crypts (P) with strong nuclear expression in CBC stem cells indicated by arrows. Scale bars: 20 mm (O), 10 mm (P).
Q Schematic diagram showing Snai1 expression (red) within different cell types in the crypt. SI = small intestine. See also Supplementary Fig S1.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 2

Loss of Snai1 predisposes differentiation towards secretory enteroendocrine and Paneth cell fate in small intestinal epithelium

To analyse the effect of Snai1 knockout in the mouse small intestine, AhCre Snai1+/+ (control) and AhCre Snai1fl/del (test) adult mice were injected with β‐NF on four consecutive days to induce Cre‐mediated recombination. Tissue was harvested for analysis on day 5.
Periodic Acid Schiff (PAS) staining was used to detect goblet cells in sections of small intestine from control and Snai1 KO mice. Graph shows number of goblet cells per crypt–villus axes (n = 3).
Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells (marked by arrows). Quantification of enteroendocrine cells per crypt–villus axes in control and Snai1 KO tissue revealed a significant increase in the number of enteroendocrine cells following Snai1 loss (n = 3, P = 0.004).
Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cells in control and Snai1 KO indicates a significant increase in Paneth cell numbers following knockout of Snai1 (n = 4, P = 0.002).
Measurement of villus length (μm) (n = 3, P = 0.013) and quantification of enterocytes per crypt–villus unit demonstrate a significant reduction in absorptive enterocytes following Snai1 loss (n = 3, P = 0.036).
Measurement of villus length (μm) (n = 3, P = 0.003) 8 days after β‐NF induction demonstrates a significant shortening of villi. Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.

Source data are available online for this figure.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 3

Conditional knockout of Snai1 induces loss of CBC stem cells

Sections from control and Snai1 KO small intestine were stained for PCNA to detect proliferating cells (arrows depicting proliferating CBC stem cells at the base of crypts). Quantification of total PCNA‐positive cells per crypt and PCNA‐positive cells in the base of crypts both revealed a significant reduction in the number of proliferating cells after conditional depletion of Snai1 (n = 4, P = 0.049, 0.026). Scale bars: 20 μm.
The expression of 2 CBC stem cell markers Olfm4 and Lgr5 were examined by in situ hybridisation in control and Snai1 KO small intestine. Lgr5 and Olfm4 in situ hybridisation staining shows a loss of CBC stem cell marker expression after loss of Snai1 (high magnification inserts depict a representative crypt demarcated by a dotted line). Loss of Snai1 was confirmed in knockout tissue using immunohistochemistry. Scale bars: 20 μm.
The analysis of sorted and EGFP‐positive cells from Lgr5‐EGFP‐IRES‐creERT2 AhCre Snai1fl/fl and Lgr5‐EGFP‐IRES‐creERT2 AhCre Snai1+/+ mice 5 days after βNF induction show an ˜tenfold reduction in Lgr5 GFP‐positive CBC stem cells.
Immunohistochemistry for active caspase‐3 to detect apoptotic cells revealed a significant increase in the number of apoptotic cells in both crypt (n = 5, P = 0.0001) and villus (n = 5, P = 0.005) regions in Snai1KO tissue (arrows mark active caspase‐3‐positive cells). Scale bars: 20 μm.
A lineage tracing strategy was employed to determine the fate of CBC cells that lack Snai1. Tissue from Lgr5‐EGFP‐IRES‐creERT2 Snai1+/+ (control) and Lgr5‐EGFP‐IRES‐creERT2 Snai1fl/fl (Snai1KO) that also contained the ROSA26LacZ Cre reporter allele was stained with X‐Gal 5 days after tamoxifen injection. Quantification of X‐Gal‐positive foci per cm shows Snai1 knockout tissue failed to lineage trace indicating that no progeny are produced from Snai1 knockout cells. Bars represent mean ± SD (n = 3, P = 0.0001). Scale bars: 0.50 mm. Two‐tailed Student's t‐test was used to assess significance. See also Supplementary Figure S2.

Source data are available online for this figure.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 4

Snai1 function is essential for in vitro organoid formation and survival

VillinCreERT2 Snai1fl/fl organoids treated with tamoxifen show arrested growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.
Quantification of viable cell growth in VillinCreERT2 Snai1fl/fl organoids treated with tamoxifen compared to controls over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.
Assessment of apoptosis in control and VillinCreERT2 Snai1fl/fl organoids after 4 days of growth. Photographs show an overlay of bright‐field and fluorescent images with propidium iodide and Hoechst staining. Quantification of apoptotic organoids. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.00001. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 50 μm. See also Supplementary Figure S3.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 5

Snai1 function is essential for in vivo intestinal regeneration after irradiation treatment

The consequences of Snai1 knockout were analysed over a time course. Immunohistochemistry for Snai1 revealed significant loss of expression at day 5 following induction compared to control. The epithelium is re‐populated by wild‐type cells as shown on day 60. Scale bars: 20 μm.
To analyse the effect of Snai1 knockout after radiation treatment in the mouse small intestine, VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl (test) adult mice were irradiated on day 1 and injected with tamoxifen on 4 consecutive days to induce Cre‐mediated recombination. Tissue was harvested for analysis on day 5.
Sections from irradiated control and Snai1 KO small intestine were stained for BrdU to detect proliferating cells. Quantification of BrdU‐positive cells per crypt and total number of cells per crypt revealed a significant reduction in both number of proliferating cells and total cells after irradiation and the conditional depletion of Snai1 (n = 4, P = 0.00019, 0.014). Bars represent mean ± SD. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 20 μm.

Source data are available online for this figure.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 6

Elevated levels of Snai1 result in reduction of secretory enteroendocrine and Paneth cells and an increase of CBC intestinal stem cells

The effect of elevated levels of Snai1 was examined using a conditional transgenic mouse model (Rosa26Snai1) that permits expression of Snai1 when exposed to Cre recombinase. AhCre (control) and AhCre Rosa26Snai1 (RS) mice were injected with β‐NF for 4 days and tissue harvested for analysis on day 5.

A PAS staining was used to detect goblet cells in the small intestine, and no differences in cell numbers were observed in test versus control mice (n = 4, NS).
B Immunohistochemistry for synaptophysin was used to detect enteroendocrine cells. Quantification of the number of enteroendocrine cells per crypt–villus axes revealed a significant decrease in enteroendocrine cell numbers when Snai1 levels are elevated (n = 4, P = 0.003).
C Immunohistochemistry for lysozyme was used to detect Paneth cells. Quantification of Paneth cell numbers indicated a significant decrease in Paneth cells in the base of crypts in mice with elevated Snai1 levels (n = 5, P = 0.027).
D, E Quantification of total PCNA‐positive cells per crypt revealed a significant increase when Snai1 levels were elevated (n = 4, P = 0.009). This was confirmed in VillinCreERT2 ROSA26Snai1 mice 5 days after induction with tamoxifen where analysis of (E) PCNA‐positive cells in the base of crypts revealed a significant increase in proliferating cells in crypts with elevated Snai1 levels compared to control tissue (n = 4, P = 0.0005). Bars represent mean ± SD.
F PCNA immunostaining (green) and DAPI staining (blue) of VillinCreERT2 ROSA26Snai1 compared to control VillinCreERT2 littermates. An expansion of proliferating CBC cells (indicated by arrows) is observed at the base of crypts. Scale bars: 10 μm.
G qRT–PCR analysis of stem cell marker expression in the small intestine from VillinCreERT2 ROSA26Snai1 compared to control VillinCreER2T littermates. Bars represent mean ± SD. n = 3 experiments per group, Olfm4 P = 0.013, Cdh1/E‐cadherin P = 0.003, Lgr5 P = 0.013.
H CD44v6 immunostaining of tissue from AhCre control compared to AhCre RosaSnai1 tissue. A significant increase in CD44v6‐positive cells localised at the base of crypts (depicted by arrows) was observed. Bars represent mean ± SD. n = 3 mice per group, *P = 0.00023. Two‐tailed Student's t‐test was used to assess significance. Scale bars: 10 μm.

Source data are available online for this figure.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 7

Elevated levels of Snai1 in in vitro organoids result in reduction of organoid growth and increase in cell death

VillinCreERT2 RosaSnai1 organoids treated with tamoxifen show slower growth after 2 and 4 days of treatment compared to controls. Scale bars: 50 μm.
Quantification of viable cells in VillinCreERT RosaSnai1 organoids treated with tamoxifen compared to control growth over 4 days. Bars represent mean ± SD. n = 3 experiments per group, **P = 0.002.
Assessment of apoptosis in control and VillinCreERT2 RosaSnai1 organoids after 4 days of growth. Photographs show an overlay of bright‐field, propidium iodide and Hoechst fluorescence. Scale bars: 50 μm.
Quantification of apoptotic organoids with and without Wnt3A treatment shown as mean ± SD. n = 3 experiments per group, **P = 0.00003. Two‐tailed Student's t‐test was used to assess significance.
Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium (2015)
Figure 8

Snai1 does not regulate BH3‐only protein expression in the intestinal epithelium but regulates Serinc3 expression

A–C Droplet digital PCR analysis of Bmf, Bbc3/Puma and Bcl2l 11/Bim after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 h with tamoxifen. No differences in gene expression were observed (n = 3).
D qRT–PCR analysis of Serinc3 downregulation after conditional Snai1 loss in in vitro organoid culture. VillinCreERT2 Snai1+/+ (control) and VillinCreERT2 Snai1fl/fl SI organoids were treated for 24 (P = 0.0001) and 72 h (P = 0.006) with tamoxifen. Bars represent mean ± SD. n = 3 mice per group.
E SW480 cells, which transiently express GFP‐SNAI1, were used to detect direct binding of SNAI1 to the promoters of known target genes CDH1, IL8 and a novel target SERINC3 revealed by microarray analysis. An unrelated DNA region was used as a negative control. Anti‐GFP antibody was used to immunoprecipitate GFP‐tagged SNAI1, while IgG control antibody was used as a negative control. The results are given as relative enrichment compared to input material and are representative of three independent experiments.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 1

Figure 1. SMC5 dysfunction causes slower proliferation.

A. WB analysis of smc5-AID clones in the absence or presence of Auxin using FLAG. Tubulin is used as loading control and wt cells is used as a negative control for the FLAG tag.

B. Proliferation curve of wt and smc5-AID cells in the presence or absence of Auxin. The data represents means ± SD of 3 independent experiments.

C. Proliferation curves of wt and 2 independent smc5 clones grown at 39.5ºC, with the estimated doubling time on the right. Data represents means ± SD of 4 experiments. Asterisks indicate p value ≤ 0.05, as derived from unpaired t-test type 3, two-sample unequal variance, carried out to check statistical significance between smc5 clones and wt. smc5 clone 1 versus wt, p=0.016, smc5 clone 2 versus wt, p=0.04.

D. Bidimensional FACS analysis shows significant accumulation in G2/M and decrease in the S phase in smc5 mutants. The data represents means ± SD of 3 independent experiments for smc5 clones 2 and 3, and 4 independent experiments for wt and smc5 clone 1. Asterisks indicate p value ≤ 0.05, detailed below, using paired t-test. S-phase (smc5 clones 1, 2, 3 versus wt: p= 0.0031; p=0.0002; p=0.004, respectively). G2/M (smc5 clones 1, 2, 3 versus wt: p= 0.029; p=0.0014; p=0.02, respectively).
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 2

Figure 2. SMC5 contributes to the repair of intra- and inter-strand crosslinks, independently of KU70 and jointly with FANCC

A. Survival curve of smc5 cells treated with different concentrations of cisplatin (CDDP). Data represents means ± SD of 3 experiments.

B. Survival curve of smc5 cells complemented or not with chicken SMC5 cDNA and treated with different concentrations of cisplatin. The experiment is carried out at 39.5ºC. Data represents means ± SD of at least 3 experiments.

C. Survival curve of smc5 ku70 cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.

D. Survival curve of smc5 fancc cells treated with different concentrations of cisplatin. Data represents means ± SD of 3 experiments.

E. WB analysis of CHK1-P levels at steady state or after treatment with 1 μM cisplatin for 3 hours. CHK1 total protein is used as loading control. Quantitative analysis of CHK1-P levels at steady state versus total CHK1 is shown in the bottom panel. Data represents means ± SD of 4 experiments.

F. WB analysis of FANCD2 ubiquitination levels at steady state after treatment with 1 μM cisplatin for 3 hours. Analysis of the ratio between FANCD2 ubiquitinated/non-ubiquitinated forms is shown on the bottom panel. Data represents means ± SD of 3 experiments.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 3

Figure 3. SMC5 acts jointly with FANCM and the FA-like pathway involving the RAD17 checkpoint clamp loader and the DDX11 DNA helicase

A. Left panel: Growth curve was carried out to test smc5 fancm cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 fancm cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.

B. Clonogenic assay to test cisplatin sensitivity of smc5 fancm cells in long term assays. Data represents means ± SD of 2 independent experiments with two independent clones analyzed for smc5 fancm.

C, D. Left panel: Growth curve was carried out to test smc5 rad17 (C) and smc5 ddx11 (D) cell proliferation. Data represents means ± SD of 3 experiments. Right panel: Survival curve of smc5 rad17 (C) and smc5 ddx11 (D) cells treated with different concentrations of cisplatin. Data represents means ± SD of the 3 experiments.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 4

Figure 4. SMC5/6 and FANCD2 jointly promote DNA repair of MMC-induced lesions.

A. Left panel: Representative Western blots illustrating the expression of SMC6, SMC5 and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Right panel: Relative expression of SMC5, SMC6 and FANCD2. We performed individual experiments for each cell line with technical replicates. In each individual experiment, we first normalized SMC5, SMC6 or FANCD2 expression to that of Vinculin (internal control) and calculated its relative level in each cell line in relation to the target protein/Vinculin ratio of the siLacZ transfected cells, which was set as one in each experiment. Data present the mean+/-S.E.M. and were analyzed by unpaired t-test. SMC5 in siSMC6: n=7 and p=0.0002; SMC5 in siFANCD2: n=7 and p=0.0016; SMC6 in siSMC5: n=8 and p<0.0001; SMC6 in siFANCD2: n=24 and p<0.0001; FANCD2 in siSMC6: n=30 and p<0.0001; FANCD2 in siSMC5: n=10 and p=0.0008.

B. Top. Representative Western blots illustrating the expression of SMC6, FANCJ and FANCD2 in protein extracts from HeLa cells 48 hours after transfection with the indicated siRNAs. Bottom. Survival curves of cells transfected with indicated siRNA and treated with different concentrations of mitomycin C (MMC). Data represents the means and SEM (standard error of the mean) of 3 independent experiments.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 5

Figure 5. SMC6 is not critical for efficient FANCD2 ubiquitylation or RAD51 focus formation.

A. Representative Western blots illustrating the expression of SMC6 and of monoubiquitylated FANCD2 in protein extracts from HeLa cells transfected with the indicated siRNAs and treated 48 hours after transfection with HU (5mM, 18 hours) or Mitomycin C (200 ng/ml, 18 hours). Vinculin is used as loading control.

B. Top. Representative images of nuclei (DAPI stained, blue) with RAD51 (green) foci in HeLa cells transfected with the indicated siRNAs and treated with HU or MMC, as indicated in panel A. The scale bar represents 10 μm. Bottom. Histograms presenting the percentage of RAD51 foci-positive cells as evaluated in the different conditions of above. Data represent the means+/-S.E.M. and were analyzed by unpaired t-test. siSMC6 vs siLacZ: n=3, NT, MMC or HU p=n.s.; siFANCD2 vs siLacZ: n=4, NT p=n.s., MMC p=0.0003, HU p=0.0479; siSMC6+siFANCD2 vs siLacZ: n=3, NT p=n.s., MMC p=0.0439, HU p=0.0458.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 6

Figure 6. SMC6 acts jointly with FANCD2 to prevent micronuclei and mitotic abnormalities.

A. Left panel: Representative images of anaphase HeLa cells with micronuclei (white arrows). 48 hours after transfection with indicated siRNAs cells were incubated 18 hours with 2 μg/ml cytochalasyn B to block cytokinesis. The scale bar represents 20 μm. Right panel: Histograms presenting the percentage of binucleate cells with micronuclei. Data represent the mean +/- SEM of 3 independent experiments, t-test, unpaired. siSMC6 vs siLacZ, p=0.0085; siFANCD2 vs siLacZ, p=0.0057; siSMC6+siFANCD2 vs siLacZ, p=0.0019.

B. Top: Examples of HeLa mitotic catastrophes and anaphase bridges as observed 48 hours after siRNA transfection. The scale bar represents 20 μm. Bottom: Histograms presenting the percentage of cells with mitotic abnormalities. Data represent the mean +/- S.E.M. of 3 independent experiments. t-test, unpaired. siSMC6 vs siLacZ: Aberrant mitosis p=0.0125, Nuclear bridges p=0.0198, Mitotic catastrophes p=0.0145; siFANCD2 vs siLacZ: Aberrant mitosis p=0.0005, Nuclear bridges p=0.0002, Mitotic catastrophes p=0.0011; siSMC6+siFANCD2 vs siLacZ: Aberrant mitosis p=0.0012, Nuclear bridges p=0.0116, Mitotic catastrophes p=0.0033.
SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability (2019)
Figure 7

Figure 7. SMC5/6 physically interacts with FANCD2-I in human cells.

A. Immunoprecipitation of FLAG-SMC6 and FLAG-NSMCE2 with FANCI in HEK293T cells overexpressing the SMC6 or NSMCE2, respectively, in the presence or absence of Ethidium bromide (EtBr). The experiment was performed 3 times, and the blot shows a representative experiment. The band under the one indicated as FANCI may represent a degradation product. B-C. Co-immunoprecipitation of endogenous SMC5-mAID-EGFP and FANCD2 in TK6 cells in the presence of benzonase. Both experiments were performed twice independently, with the note that the cisplatin condition was performed only in one of the experiments, and the FANCD2 pull down in the experiment not shown was performed without benzonase. D. Model schematizing the proposed view of SMC5/6 action in genome integrity and DNA repair. SMC5/6 acts differently from non-homologous end joining and downstream of RAD51 filament formation and of homologous recombination branches mediated by DDX11, RAD17 and FANCD2, RAD17.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 1

Figure 1. Spata2 is a negative regulator of inflammation.

(A) Survival of age- and sex-matched (8-10 week old) Spata2+/+ (WT) and Spata2-/- (KO) mice injected intraperitoneally (i.p.) with LPS (20 mg/kg bodyweight). Data pooled from three independent experiments are shown. Statistical analysis was performed using long-rank test.

(B) ELISA of serum IL-1β, IL-18, TNFα and IL-6 at 12 h after injection of WT or Spata2-KO mice with 20 mg/kg LPS. Circles and triangles represent individual mice. Horizontal bars indicate mean values. **P < 0.01; ****P < 0.0001. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(C) Immunoblot analysis of the indicated phosphorylated (P) and total proteins in lysates of LPS-stimulated BMDMs derived from WT or Spata2-KO mice. Molecular weights in kDa are indicated to the right.

(D) qRT-PCR analysis of the indicated mRNAs in untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(E) ELISA of TNFα and IL-6 in the supernatants from untreated or LPS-stimulated WT or Spata2-KO BMDMs. Bars and error bars represent the mean ± S.D. of triplicate experiments. Statistical analysis was performed using unpaired two-tailed Student's t-test.