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. 2018 Oct 12;7:e38279. doi: 10.7554/eLife.38279

Figure 6. WNT priming unveils ACTIVIN-dependent mesendoderm differentiation.

(A) Schematic outlining the 2 day experimental protocol. (B–C) Cells were cultured for one day with or without WNT3A (100 ng/mL, top bar). On the second day they were washed to remove WNT and treated with or without ACTIVIN (10 ng/mL, bottom bar). After the second day cells were fixed and analyzed by immunofluorescence (IF). (B) Images: NANOG (red), OCT4 (green), SOX2 (cyan), DAPI (gray). Scale bar, 50 µM. (C) Histograms showing the nuclear IF signal quantified in single cells (n > 5,000 cells per condition). (D–E) Cells were cultured for one day with or without WNT3A (100 ng/mL, top bar). On the second day they were washed to remove WNT and treated with or without ACTIVIN (10 ng/mL, bottom bar). After the second day cells were fixed and analyzed by immunofluorescence (IF). (D) Images: BRA (red), EOMES (green), GSC (cyan), DAPI (gray). Scale bar, 50 µM. (E) Histograms showing the nuclear IF signal quantified in single cells (n > 5,000 cells per condition). (F–G) Kolmogorov-Smirnov (KS) distance of the cumulative probability distribution (CDF) of each marker to the reference CDF (-/ACT) for independent experiments in RUES2 and in RUES1 for the (F) pluripotency and (G) mesendoderm marker sets. n.s. (or comparison not shown), not significant, *p<0.05, **p<0.01, ***p<0.001, ANOVA.

Figure 6.

Figure 6—figure supplement 1. ACTIVIN dose-dependent mesendoderm differentiation.

Figure 6—figure supplement 1.

(A) RUES2 and RUES2-SMAD3-/- cells were cultured for one day with WNT priming. On the second day they were washed to remove WNT and treated with or without ACTIVIN (10 ng/mL) for an additional day. Cells were fixed and analyzed by immunofluorescence (IF) for BRA, EOMES, and GSC expression. Histograms show the nuclear IF signal quantified in single cells (n > 5,000 cells per condition). (B) Histograms showing the immunofluorescence data from samples treated with different doses of ACTIVIN (1, 10, 100 ng/mL) for 12 hr following WNT priming. The data for WNT (24 hr) shows that all markers were poorly expressed prior to ACTIVIN addition and that their distributions are identical to cells in pluripotency conditions (-/ACT10, 36 hr) or cells that were left untreated with ACTIVIN for the additional 12 hr (WNT/-). Quantification for n > 5,000 cells per condition. (C) Scatter plot of the single-cell EOMES and GSC nuclear signal from the data shown in B. EOMES and GSC expression increases in WNT/ACT10 condition and expression is positively correlated. (D) Scatter plot of the single-cell BRA and GSC nuclear signal from the data shown in B. Expression is not well correlated. Similar numbers of cells were analyzed in all conditions in C and D, but the data points cluster on top of one another in the WNT/- and WNT/ACT1 conditions. r, Pearson correlation coefficient with the 95% confidence interval shown in brackets.