Fig. 3

FLCN is essential for the exit from the naïve pluripotency. a Schematic representation of experimental procedures used to exit naïve pluripotent state. b Principal component analysis after RNA-seq analysis reveals that FLCN KO hESC do not fully exit naïve state. RNA-seq from various pre-implantation in vivo human embryo⁶⁷, naïve, and primed in vitro hESC were plotted, and separated in PC1 axis^{1,6,7,11,12,67,68}. c Expression pattern of genes regulated by FLCN KO during exit of naïve state compared to cynomolgus monkey (Macaca fascicularis³⁴) pre- vs. post-implantation stage. Red: genes up-regulated in 7D TeSR FLCN KO and pre-implantation; green: genes up-regulated in 7D TeSR WT and post-implantation. d, e RT-qPCR analysis of naïve (DNMT3L, d) and primed (IDO1, e) markers after exit of 2iL-I-F naïve state (7 day TeSR) in WT, FLCN KO, and rescue line (FLCN KO + OE FLCN-GFP). S.e.m.; *p < 0.05, **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–6 biological replicates. Presented are the fold changes compared to naïve 2iL-I-F. f Western blot analysis of primed markers JARID2, LDHA, and H3K27me3 marks in naïve 2iL-I-F hESC and 10D TeSR hESC. g RT-qPCR analysis of NNMT expression. S.e.m.; **p < 0.005; two-tailed t-test, n = 3–6 biological replicates. h Model of doxycycline inducible- NNMT-GFP fusion construct inserted into AAVS1 locus of 2iL-I-F FLCN KO hESC. i Western blot analysis of NNMT, GFP, H3K27me3, JARID2, and OCT4 after NNMT OE in FLCN KO 7D TeSR hESC. j, k FLCN KO cells retain naïve 5iLA morphology (j) and naïve markers DNMT3L, TFCP2L1, and KLF4 (k) when pushed to exit the naïve state (Exit assay: FGF 4 or 7 days; relative mRNA expression: fold changes of expression in FLCN KO vs. control (WT FGF: 4D or 6D, red bar). Complete data presented in Supplementary Fig. 3B. S.e.m.; *p < 0.05, **p < 0.005, ***p < 0.001; two-tailed t-test, n = 3–6 biological replicates. Scale bars represent 100 μm