Fig. 3: VGLL1 is required for human TELC induction from naive PSCs.

a RT-qPCR showing the VGLL1 knockdown efficiency for 4CL H9 ESCs transduced with shVGLL1−2 or shVGLL1−4 compared to the shLuc control at day 5 of TELC differentiation. Data are presented as the mean ± SEM, n = 3 biological replicates. P value was calculated using a two-tailed unpaired Student’s t-test, ***P < 0.001. b Western blotting analysis of the indicated proteins for 4CL H9 ESCs transduced with shVGLL1−2 or shVGLL1−4 compared to the shLuc control at day 5 of TELC differentiation. Representative of three independent experiments. c Representative phase contrast images of 4CL H9 ESCs transduced with shLuc (control) or shVGLL1 (2 and 4) at day 5 of TELC differentiation. Scale bar, 100 µm. Representative of three independent experiments. d Analysis of cell numbers for 4CL H9 ESCs transduced with shLuc, shVGLL1−2 or shVGLL1−4 throughout the TELC induction time course. Data are presented as the mean ± SEM. n = 3 biological replicates. P value was calculated using a two-tailed unpaired Student’s t-test, ***P < 0.001, **P < 0.01. e Heatmap showing the expression of pluripotency, cell cycle and TE genes in 4CL H9 ESC-derived cells from the indicated conditions. Example genes are shown for each cluster in the boxes. n = 2 biological replicates. f RT-qPCR showing the expression of TE-related genes for 4CL H9 ESCs transduced with shVGLL1−2 and shVGLL1−4 compared to the shLuc control at day 5 of TELC differentiation. Data are presented as the mean ± SEM. n = 3 biological replicates. P value was calculated using a two-tailed unpaired Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05. g Enriched GO terms for downregulated genes in 4CL H9 ESCs transduced with shVGLL1 compared to shLuc control at day 5 of TELC differentiation. P value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. h Enriched GO terms for upregulated genes in 4CL H9 ESCs transduced with shVGLL1 compared to shLuc control at day 5 of TELC differentiation. P value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction. i TA cloning followed by Sanger sequencing results showing homozygous deletion for VGLL1-knockout clones [clone 45 (C45) and clone 68 (C68)]. WT: wild-type. j Western blotting analysis of VGLL1 expression in H9 WT and VGLL1-knockout clones at day 5 of TELC induction. Representative of three independent experiments. k Representative phase contrast images of H9 WT and VGLL1-knockout clones at day 5 of TELC induction. Scale bar, 100 µm. Representative of three independent experiments. l Analysis of cell numbers for H9 WT and VGLL1-knockout clones at day 5 of TELC induction. Data are presented as the mean ± SEM. n = 3 biological replicates. P value was calculated using a two-tailed unpaired Student’s t-test, ***P < 0.001. m Heatmap showing the expression of pluripotency, cell cycle and TE genes in bulk RNA-seq of H9 VGLL1-knockout clones compared to WT at day 5 of TELC induction. Example genes are shown for each cluster in the boxes. n = 2 biological replicates. n Enriched GO terms for downregulated genes in H9 VGLL1-knockout clones compared to WT at day 5 of TELC induction. P value was calculated using a hypergeometric test (one-sided) and adjusted for multiple testing using the Benjamini-Hochberg correction.