Fig. 2

Trincr1 represses FGF/ERK signaling in ESCs. a Gene structure of Trincr1_L and S. Exon 1 (L): 87,472,809–87,472,960; Exon 1 (S): 87,472,871–87,472,960; Exon 2: 87,521,736–87,521,813; Exon 3: 87,524,296–87,524,427; Exon 4: 87,525,415–87,525,554; Cleavage and polyadenylation signal sequence “AAUAAA”: 87,525,535–87,525,540. Scissors indicate the position of two gRNA sequences used to knockout Trincr1 in this study. b Fraction of Trincr1_L and S in ESCs. c RT-qPCR analysis of Trincr1 and Trim71 in differentiated ESCs induced by 100 nM All-Trans-Retinoic Acid for 4 days. The Gapdh gene was used as a control. For each gene, data were normalized to the RNA level of undifferentiated ESCs (day 0). n = 3 biological replicates. Appropriate differentiation is supported by the expression change of Oct4 and Rex1. d RT-qPCR analysis of Trincr1 in ESCs with Trincr1 knocked out by Cas9. Four sets of primers corresponding to four exons were used to confirm the loss of Trincr1 in Trincr1−/− ESCs. The β-actin gene was used as a control. Data were normalized to wild-type ESCs. n = 2 biological replicates. e Western blotting analysis of phosphorylated ERK in wild type and Trincr1−/− ESCs in 2i + LIF and PD + LIF. For quantification of pERK/ERK, data were normalized to GAPDH and then to wild-type ESCs cultured in 2i + LIF. n = 3 independent experiments. f Western blotting analysis of phosphorylated ERK in control and Trincr1 overexpressing ESCs induced by bFGF. For quantification of pERK/ERK, data were normalized to GAPDH and then to control overexpression vector transfected ESCs cultured in CHIR and induced by 12 ng per ml bFGF. n = 3 independent experiments. Shown are mean ± SD. For c and f, P values were determined by unpaired two-way and one-way ANOVA with two-sided Dunnett’s test, respectively. For e, P values were determined by paired two-sided Student’s t-test