Fig. 5

Shp2 chemical inhibitor as an iMek1 replacement. A Chemical structure of Shp2 inhibitor (iShp2: RMC-4550). B Immunoblotting analysis of WT ESCs at 30 min after indicative dose of iShp2 treatment, α-tubulin was used as a loading control. C Immunoblotting analysis of WT ESCs at indicted time after 1000 units of LIF stimulation, pretreated with either DMSO (Mock) or 5 μm of iShp2 (+ iShp2) for 1 h while LIF starvation (L/2i: LIF + 2i control). D Graphical presentation of luciferase reporter activity of Stat3 at 8 h after LIF deprivation with either DMSO (Mock) or 5 μm of iShp2 (+ iShp2) for 1 h prior to LIF deprivation. White column (LIF) indicates reporter activity of Stat3 without LIF deprivation as a control (***p < 0.001, n = 3). E Immunoblotting analysis for pStat3 at 30 min after indicative dose of LIF stimulation in WT ESCs pretreated with either DMSO (Mock) or 5 μm of iShp2 (+ iShp2) for 1 h while LIF starvation. F Light microscopic images of WT cultured under indicative dose of LIF for 3 days supplemented with DMSO (Mock) or 5 μm of iShp2 (+ iShp2) (scale bars, 500 μm). G Light microscopic images of WT ESCs under control (LIF + 2i), deprivation of iMek1 [(-)iMek1], iGsk3β [(-)iGsk3β] or iMek1/iGsk3β[(-2i)] with DMSO (Mock) or 5 μm of iShp2 (+ iShp2) treatment for 2 days (scale bars, 200 μm) H Relative gene expression of Rex1 (left) and Essrb (right) at indicated culture condition with DMSO (Mock: blue) or 5 µM of iShp2 (+ iShp2: red) for 2 days (*p < 0.05, **p < 0.01, ***p < 0.001, n.s. for not significant). I Flow cytometry of GFP intensity of WT ESCs with either LIF + 2i control (LIF + 2i) or iMek1 depletion [(-)iMek1] for 2 days, pretreated with DMSO (Mock) or 5 μM of iShp2 (+ iShp2) (****p < 0.0001, n = 6). J Graphical illustration of endogenous Oct4 with distal enhancer (DE) and proximal enhancer (PE)(top), Oct4-ΔPE-GFP (middle) and Oct4-ΔDE-RFP (bottom), activation of DE and PE at naïve and inner cell mass (ICM) and at primed and epiblast stem cells (EpiSCs) respectively (left), ESCs with GFP + /RFP- (Green), GFP + /RFP + (Yellow) and GFP-/RFP + (red) at naïve, intermediate and primed status respectively (top), expected growth of GFP + or RFP + ESCs under each indicative condition was shown (bottom). K Cell growth ratio at indicated time of GFP + or RFP + ESCs under LIF + 2i/LIF + 2i’ (iShp2 instead of iMek1) and bFGF/Activin with DMSO (F.A) or iShp2 (F.A + iShp2) (top panels), Table of slope and p value of linear regression of GFP + or RFP + at indicated culture condition, effect of iShp2 on slope was highlighted in red