EMBO/SourceData huggingface dataset:
Figure 2(A-F) Human skin immunofluorescence images of mouse basal non-LRC/LRC preferred-factor staining in rete ridges (RR) versus inter-ridges (IR). Representative images from 30 samples of different body regions and ages Scale bars are indicated on the panels(G) LRC/non-LRC preferred gene expression in RR and IR samples from two 30-year-old breast samples, quantified using background subtraction P-values from paired Student's t-test. Error bars are SDs. 8-10 images were quantified each from two females.
Figure 5(A) Schematic showing the tamoxifen injection paradigm for tdTomato-labelling of all IFE lineages in tail and back. Co-expression of markers with the lineage-traced cells at 1-month post-tamoxifen. Dlx1-CrER is shown as example in tail; Scale bar =50 µm (B) Quantification of images like those in (A) showing % tdTomato+ cells expressing indicated markers. Sox6 (black bar) was not analyzed in K14-Cre/EYFP lineage. Error bars are SDs. P-values calculated by Student's t-test (mixed effects model) from n=2-3 mice and 5-6 images per replicate.(G) Immunofluorescence images of Sox6 staining in noUVB (0hr) or UVB-exposed (6hrs- or 24hrs-post) mouse back skin. (H) Boxplot shows % Sox6 expressing basal cells quantified from images like those in (G). Central band is the mean value, the top of box indicates 25th percentile while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.
Figure 6(B-G) (B,D,F) Images of tail skin immune-stained for markers indicated and (C,E,G) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 total images from 3 biological replicates.(I-N) (I,K,M) Images of skin immune-stained for markers indicated and (J,L,N) corresponding box plots. Central band is the mean value, the top of box indicates 25th percentile, while the bottom indicates 75th percentile. Whiskers indicate minimum and maximum values in the data, with dots indicating potential outliers. Linear mixed model statistical test was used with data from 18 images from 3 biological replicates.
Figure 1Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B).Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C).Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D).Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F).Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I).Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.
Figure 2Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E).Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I).Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L).Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.
Figure 3p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells. Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2, capsid or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid (A,B).Cells were transfected with Ub‐HA and capsid‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐HA antibody (C).Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to p62, FK2 or capsid (D,E).Cells were infected for 3 h and then transfected with empty plasmid (CTRL) or p62WT‐3XFLAG or p62deltaUBA‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. with anti‐FLAG antibody. Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid (F).Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using anti‐capsid antibody (G).Cells were infected for 15 h and labeled with antibodies to p62, LAMP‐1 or capsid (H).Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I).HeLa cells were transfected with empty plasmid (CTRL), control protein (cherry‐3XFLAG) or capsid‐3XFLAG at fixed (750 ng) or at the indicated concentrations. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (J,K).Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (L).Cells were infected with CHIKV for 3 h and transfected with empty plasmid (CTRL), p62WT‐3XFLAG, p62deltaUBA‐3XFLAG or p62deltaLIR‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 24 h post transfection (M).
Figure 4Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A).Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D).Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E).Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F).Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G).Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H).
Figure 5NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C).MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D).MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I).MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).
Figure 1TRIP6 promotes YAP activity by inhibiting LATS1/2 (A) Full length (WT), the amino-terminal half (1-277), or the carboxy-terminal half (278-476) of TRIP6 were tested for binding to LATS2 by immunoprecipitation. FLAG-TRIP6 variants were co-expressed with LATS2-GFP in HEK293 cells, anti-FLAG or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS2-GFP and FLAG-TRIP6. Schematic diagram depicts TRIP6 domains (NES: Nuclear Export Signal; LIM: LIM domain; PDZ: PDZ domain binding motif).(B) Lysates from MCF10A cells were subjected to immunoprecipitation using anti-TRIP6 or control (IgG) antibodies, and immune complexes and lysates were probed for TRIP6 and LATS1.(C) FLAG-TRIP6 was tested for binding to various LATS2-GFP deletion mutants as described in part (A). Schematic diagram of LATS2 shows MOB1 binding domain, and the autophosphorylation (S872) and MST1/2 phosphorylation sites (T1041) in the kinase domain.The regions marked in green depict TRIP6 binding sites on LATS2 (D) Lysates from HEK293A cells transfected with control or FLAG-TRIP6 plasmid were analyzed by western blotting using the indicated antibodies (quantification is shown in (F)).(E) Lysates from control (WT) or CRISPR generated TRIP6 null (TRIP6-KO) HEK293A cells were analyzed by western blotting using the indicated antibodies (quantification shown in (G)).(F) The relative levels of LATS1 activating phosphorylation (pLATS1-1079, 909) and YAP S127 inhibitory phosphorylation from (D) were measured relative to LATS1 and YAP levels respectively. (Mean ± SD; n=3; **P≤0.01, ***P≤0.001, T-test)(G) The levels of LATS1 activating phosphorylation and YAP inactivating phosphorylation from part (E) were quantified (Mean ± SD; n=3; *P≤0.05, T-test).(H) GFP-MST2 was expressed with or without FLAG-TRIP6 in HEK293 cells and the levels of MST2, MST2 activating phosphorylation (pMST2-T180), and FLAG-TRIP6 were measured by western blotting with the indicated antibodies. (Mean ± SD; n=3; n.s.≥0.05, T-test).(I) TRIP6 was overexpressed in HEK293A cells and the levels of TRIP6, and YAP target gene expression was analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ****P≤0.0001, T-test).(J) The levels of YAP target gene expression was analyzed using RT-qPCR in control (WT) and TRIP6-KO HEK293A cells. (Mean ± SD; n=3; ***P≤0.001, T-test)(K) Control (WT) and TRIP6-KO HEK293A cells were stained for YAP and TRIP6. Merged image shows YAP (green), TRIP6 (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ****P≤0.0001, Fisher's test). Scale bar=20µm.
Figure 2(A) LATS2-GFP and Myc-MOB1A were overexpressed in HEK293 cells with or without co-overexpression of full length FLAG-TRIP6 and FLAG-TRIP6 1-277. Myc-MOB1A was immunoprecipitated using anti-Myc antibodies and immune complexes were assayed for Myc-MOB1A and LATS2-GFP levels. Levels of FLAG-TRIP6, FLAG-TRIP6 1-277, Myc-MOB1A, and LATS2-GFP in the lysate are also shown. The levels of LATS2-GFP in immune complexes relative to the level of Myc-MOB1A are shown in the graph (Mean ± SD; n=3; **P≤0.01, T-test)(B) Competitive binding experiments were done using purified recombinant MBP-LATS2, GST-TRIP6, and 6His-MOB1A. MBP-LATS2 bound to maltose beads was incubated with GST-TRIP6 with or without increasing amounts of 6His-MOB1A, and the levels of each protein bound to MBP-LATS2 on the beads at the end of the experiment was determined by western blotting. The levels of input proteins are shown (lysate). The binding of 6His-MOB1A and GST-TRIP6 to MBP alone, and the use BSA as a competitor instead of 6His-MOB1A are shown as controls. The numbers at the bottom are referred to in the text.
Figure 3(A) MCF10A cells were infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4) and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue) (quantification of LATS1 localization at cell-cell junctions is shown in Figure EV3D). Scale bar=20µm.(B) LATS1 and LATS2 were knocked down using siLATS1 and siLATS2 SMARTPools in MCF10A cells. MCF10A control cells were treated with control siRNA (siControl) against fire fly luciferase. Cells were stained for TRIP6 and LATS1 as in (A) (quantification of TRIP6 localization at cell-cell junctions is shown in Figure EV3E). Scale bar=20µm.(C) MCF10A cells were grown to high density, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(D) Cells were grown as in (C), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).(E) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and lysed while under tension. Anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6. Quantification is shown. (Mean ± SD; n=3; *P≤0.05, T-test).(F) Cells were treated as in (E), fixed while under tension, and stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(G) MCF10A cells infected with lentivirus carrying control shRNA (shEGFP), or a mix of two different shRNA against TRIP6 (shTRIP6-1 and shTRIP6-4), were grown at high density on PDMS membranes and were stretched or not (only stretched cells shown) at 17% elongation for 2 hours, fixed while under tension, and were stained for TRIP6 and LATS1 as in (A). Scale bar=20µm.(H) Cells were treated as in (G) and YAP target gene (CTGF and Cyr61) and TRIP6 expression were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, T-test).(I) TRIP6 expression levels in (H) in control (shEGFP) and TRIP6 (shTRIP6) knockdown cells just prior to stretch were analyzed using RT-qPCR. (Mean ± SD; n=3; *P≤0.05, T-test).
Figure 4(A) MCF10A cells were either not treated or treated separately with Latrunculin B, Blebbistatin, EGTA, serum starvation, and Y27632, and were stained for TRIP6 and LATS1. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.(B) Cells were treated as in (A), then lysed and anti-TRIP6 antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for LATS1 and TRIP6.
Figure 5(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.(B) MCF10A cells were either not treated (control) or treated separately by growth to high density, serum starvation, Latrunculin B, Blebbistatin, EGTA, or Y27632 treatment, and stained using anti-vinculin antibodies by immunofluorescence. Scale bar=20µm.(C) MCF10A cells grown at high density on PDMS membranes and were stretched (or not) at 17% elongation for 2 hours and fixed while under tension and stained for vinculin. Scale bar=20µm.(D) Vinculin was knocked down using two different siRNAs or control siRNA in MCF10A cells and the cell lysates were probed by western blotting for phospho-LATS1 (T1079 and S909), phospho-YAP (S127), LATS1, YAP, vinculin, and tubulin antibodies and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).(E) Vinculin was knocked down as described in (D) and cells were stained for YAP and vinculin. Merged image shows YAP (green), vinculin (red), and DNA (blue). Quantification of at least 100 cells is shown (Mean ± SD; n=3; ***P≤0.001, Fisher's test). Scale bar=20µm.(F) Vinculin was knocked down as described in (D) and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).(G) Vinculin was knocked down in MCF10A cells as described in (D), grown at high density on PDMS membranes and cells were stretched (or not) at 17% elongation for 2 hours and the levels of vinculin and YAP target gene expression was analyzed using RT-qPCR (Mean ± SD; n=3; ***P≤0.001, ****P≤0.0001, T-test).
Figure 6(A) Vinculin was knocked down as described (Figure 5D) in MCF10A cells and cells were stained for LATS1 and TRIP6. Merged images show LATS1 (green), TRIP6 (red) and DNA (blue). Scale bar=20µm.(B) Vinculin was knocked down as described (Figure 5D) and TRIP6 immune complexes and lysates were probed by western blotting for LATS1, TRIP6, and vinculin and the relative levels quantified (Mean ± SD; n=3; **P≤0.01, T-test).(C) MCF10A cells were subjected to different treatments as described (Figure 4A) and TRIP6 immune complexes and lysates were probed by western blotting for vinculin and TRIP6.(D) Quantification of TRIP6-vinculin binding from part (C) is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(E, F) MCF10A cells were treated separately (or not) with Latrunculin B (E) and by serum starvation (F) and LATS1 immune complexes and lysates were probed by western blotting for vinculin, LATS1, and TRIP6. Quantification of LATS1-TRIP6-vinculin binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(G) Wild type and T12 mutant of vinculin were co-expressed with LATS2 and FLAG-TRIP6 and anti-FLAG antibody was used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin, LATS2 and FLAG-TRIP6. Quantification of LATS2-TRIP6-vinculin (WT or T12) binding is shown. (Mean ± SD; n=3; *P≤0.05, **P≤0.01, ***P≤0.001, T-test).(H) Vinculin was knocked down as described (Figure 5D) in MCF10A cells grown at high density on PDMS membranes. The membranes were stretched at 17% elongation for 2 hours and fixed while under tension and stained for TRIP6. Merged images show TRIP6 (red) and DNA (blue). Scale bar=20µm.
Figure 1a, Survival curves of wild type (n=27) and Cdc14 DKO (n=51) mice during the first 6 weeks of life. (***, p<0.001; Log-rank, Mantel-Cox test). b, Representative images of postnatal day 9 Cdc14 DKO and wild-type littermates. c, Weight of Cdc14 DKO and wild-type littermates during the first 3 weeks of life (n=4 per genotype). Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.d, Representative macroscopic pictures of brain dorsal view of wild type (WT) and Cdc14 KO mice at postnatal 0, 5 and 9 days (P0, P5 and P9). Scale bar, 2 mm. The bar plots show the quantification of brain width, length and area, respectively. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.e, Representative cresyl violet staining images of midsagittal sections of cerebellar vermis of WT and Cdc14 KO mice at P0, P5 and P9. Fissures underdeveloped in mutant samples are labeled with arrowheads. Scale bar, 700 μm. The bar plots show the quantification of the cerebellar vermis area, the number of folia and the average length of the fissures, respectively. Data information: Bars indicate mean ± SEM. p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice.f, Detailed cresyl violet staining images of midsagittal sections of cerebellar vermis of the same samples. The molecular layer (ML), internal granule cell layer (IGL) and external granule cell layer (EGL) are indicated. The width of these layers is shown in the bar plots. Data information: Bars indicate mean ± SEM. *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction. Non-significant data (p>0.05) are not labeled.In data show the individual data, average and SEM from 3 WT and 3 DKO mice. In f, each point represents the average value of 9 different random measurements across distinctive folia for each mouse.g, Representative images of misdigital sections stained with DAPI (blue), Calbindin (green) and GFAP (red) of cerebellar vermis of wild-type ad Cdc14 DKO mice at 9 days after birth. Scale bar, 50 μm. Plots show the quantification of Purkinje cell dendritic length (average distance of 30 random measurements) and the number of cells per 100 μm from 3 different fields from 3 different mice in each plot. Data information: Bars indicate mean ± SEM. , *, p>0.05; **, p<0.01; ***, p<0.001, Student's t-test with Welch's correction.
Figure 2a. Confocal microscopy analysis of wild-type and Cdc14 DKO embryonic stem cells (ESCs) subjected to neural differentiation. Neuronal marker TUJ1 (green), glial marker GFAP (red) and nuclear marker DAPI (blue) are stained and quantified after 18 days in differentiation media. Each dot represents the ratio between TUJ or GFAP and DAPI area in an image containing at least 100 cells from 4 independent experiments with two different clones. **** p-value<0.0001. Student's t-test. Scale bar 50 μm.c, Confocal microscopy analysis of WT and Cdc14 DKO ESC subjected to neural differentiation. Neural progenitor marker Nestin was stained and quantified after 6 days in differentiation media. Each dot represents the ratio between Nestin and DAPI area in an image containing at least 100 cells. ****, p<0.0001; Students' t-test.