Fig. 4: A quartet of transcriptional regulators maintain pluripotency.

a Left: immunostaining for the pluripotency markers NANOG and POU5F1/OCT4 of KiPS stably expressing an empty vector control (Empty) in presence of DMSO or SB43 and KiPS stably expressing NANOG, KLF7, MYC or ZNF398 in presence of SB43 for 5 days. Representative images of three independent experiments are shown. Right: Violin plots showing fluorescence intensity quantification of NANOG and OCT4. For each condition, at least 1200 nuclei from five randomly selected fields were analysed. Box plot indicates 25th, 50th and 75th percentile; whiskers indicate minimum and maximum. Scale bars 20 µm. See also Supplementary Fig. 3c for results obtained in H9. Source data are provided as a Source Data file. b Diagrams showing an extended set of pluripotency regulators. Gene expression analysis by RNA-seq of KiPS stably expressing an empty vector, NANOG, KLF7, MYC or ZNF398 and treated with SB43 for 5 days. Colours indicate the fold-change relative to Empty DMSO sample, thus yellow indicates the endogenous expression of a given gene in undifferentiated hPSCs. c Box plot showing absolute expression levels (normalised counts, TPM) of 538 genes DOWN-regulated by SB43 treatment (5 days) in KiPS stably expressing an empty vector (see Fig. 5a, blue dots). Shown data refers to KiPS transfected with the empty vector in the presence of DMSO or SB43 (n = 4, 4 independent experiments, respectively) and for KiPS stably expressing NANOG, KLF7, MYC or ZNF398 in presence of SB43 for 5 days (n = 2, 2, 2, 2 independent experiments, respectively). Average fold-change relative to Empty SB43 sample (X) is reported for each condition. Box plots indicate 25th, 50th and 75th percentile; whiskers indicate minimum and maximum. Unpaired two-tailed t-test.