Journal: Nature Communications

Article Title: Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency

doi: 10.1038/s41467-018-08020-0

Figure Lengend Snippet: 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 embryo , naïve, and primed in vitro hESC were plotted, and separated in PC1 axis , , , , , , . c Expression pattern of genes regulated by FLCN KO during exit of naïve state compared to cynomolgus monkey ( Macaca fascicularis ) 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. . 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

Article Snippet: The antibodies used for western blot were β-tubulin III (Promega G7121, 1:1000), β-actin (Cell Signaling 4970, 1:10000), Oct4 (Santa Cruz sc-5279, 1:1000), H3K27me3 (Active Motive 39155, 1:1000), FLCN (Cell Signaling D14G9, 1:2000), NNMT (Abcam 58743, 1:500), TFE3 (Sigma Prestige HPA023881, 1:1000), JARID2 (Cell Signaling D6M9X, 1:1000), LDHA (Cell Signaling 2002, 1:1000), SIN1 (EMD Millipore 05-1044, 1:000), pAkt(S473) (Cell Signaling 9271, 1:1000), pAkt(T308) (Cell Signaling 9275, 1:1000), Akt (Cell Signaling 9272, 1:1000), pS6 (Cell Signaling 2215, 1:10,000), S6 (Cell Signaling 2117, 1:2000), p4EBP1 (Cell Signaling 236B4, 1:1000), ESRRB (ProteinTech, 1:1000), pmTOR (Cell Signaling 2971, 1:1000), mTOR (Cell Signaling 2972, 1:1000), RICTOR (Invitrogen MA5-15681, 1:1000), TFCP2L1 (Novus Biologicals NBP1-85441, 1:1000), and KLF4 (Abcam ab129473, 1:1000).

Techniques: RNA Sequencing, In Vivo, In Vitro, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Construct, Control