Extended Data Fig. 5: Characterisation of ZNF398 genetic perturbations in both 2D and 3D hPSC models.
a, Gene expression analysis by qPCR of KiPS stably expressing the empty vector control (Empty) without (blue) or with SB43 (yellow), or expressing ZNF398 without SB43 (lilac) or with SB43 (orange) after 4 days of self-organisation. Bars indicate the mean +/- s.e.m. of 4 experiments, shown as dots. Expression was normalised to the mean of Empty DMSO samples. Unpaired two-tailed Welch’s t-test. b, Heatmap showing gene expression analysis by qPCR for markers of pluripotency. Human PSCs, either expressing ZNF398 or an empty vector, were analysed when cultured under conventional conditions (2D) and after 4 days of self-organisation (3D) in presence of SB43 or DMSO. Values are expressed as fold-change relative to the mean of Empty 2D samples from 4 experiments and are scaled by rows (genes). Red and purple indicate high and low expression, respectively. c, Representative chromatograms of the ZNF398 locus in a WT (top panel) and a heterozygous clone. The matching part of the guide (before the cut) is highlighted in light blue. Table summarizing the ZNF398 sequence analyses in 365 clones. d, Representative brightfield and fluorescence images of 2D ZNF398_CRISPRi and parental cell lines cultured without and with DOX/TMP for 7 days. Scale bar: 200 µm. e, Bar plots showing gene expression analysis by qPCR of pluripotency and epithelial genes on 2D ZNF398_CRISPRi cell line cultured without and with DOX/TMP for 7 days. Bars indicate the mean of 2 experiments, shown as dots. f, Bar plots showing the fraction of organised (red), non-organised (gray), and no-cavity (orange) ZNF398_CRISPRi-derived structures, with or without DOX and TMP. Each bar represents the average of 2 experiments. g, Bar plots showing gene expression analysis by qPCR of pluripotency and epithelial genes on ZNF398_CRISPRi 3D-hE-Gastruloids without and with DOX/TMP after 4 days of self-organisation. N = 2 independent experiments. h, PCA of the ZNF398_CRISPRi 3D-hE-Gastruloids RNA-seq experiment, computed using the 2000 most variable genes. Colors indicate the analyzed conditions, while shape indicates whether the culture is 2D or 3D.
