C3G null ES cells exhibit enhanced self-renewal. a Schematic showing the 5′ region of mouse C3G gene indicating exons and guide RNA positions. EF1α-Puromycin cassette was knocked-in to replace exons 2–4 of the C3G gene. b RT-PCR analysis of C3G transcripts in three independent knockout clones (B5, D1 & D3), WT, and vector control (VC) using N-terminal primers. c Schematic representation of C3G protein domains and western blot showing C3G expression in WT and knockout clones. Actin was used as a loading control. d Bright-field images of representative colony morphology of WT cells and C3G KO clones. Scale bar, 40 μm. e Clonogenic assay of WT and KO cells grown for 7 days. The bar diagram shows the quantification of colonies with the indicated morphology. Data were averaged from three independent experiments carried out in duplicates, ***P < 0.001. f Clonogenic assay of WT and C3G KO clone D1 grown in the absence and presence of indicated LIF concentrations. Quantification of colony type is shown in the bar diagram. ***P < 0.001; n = 3.