Fig. 2: MPP8 is essential for stem cell self-renewal of ground-state mESCs.

a Schematic representation of the auxin-inducible degradation system for the MPP8 protein. b Kinetic evaluation of Mpp8^mAID degradation in OsTIR1-expressing mESCs grown in serum/LIF after 1 or 2 h of auxin treatment (+ IAA) in Mpp8^mAID; OsTIR1 or Mpp8^mAID (control) cells (n = 1). c, d Cell proliferation assay in 2i/LIF (c) or serum/LIF (d) culture conditions ± IAA (500 µM) (mean ± s.d., n = 3 biological replicates). e Cell cycle analysis in 2i/LIF culture conditions ± IAA (500 µM) (mean ± s.d., n = 3 independent experiments for untreated, 6 h, 72 h; mean, n = 2 independent experiments for 24 h, 48 h). p = 0.0136 (S), p = 0.0169 (G1), p = 0.7009 (G2M), p = 0.1793 (SubG1) comparing each IAA-treated (72 h) cell population to the respective untreated cell population of Mpp8^mAID; OsTIR1 cells and p = 0.0209 (S), p = 0.0014 (G1), p = 0.2478 (G2M), p = 0.6382 (SubG1) comparing each IAA-treated (72 h) cell population to the respective untreated cell population of Mpp8^mAID cells (two-tailed paired Student’s t test). f Quantification of the Alkaline Phosphatase (AP) staining assay in serum/LIF culture conditions. Stained colonies were scored based on AP+ (undifferentiated), mixed or AP- (differentiated) morphology (mean ± s.d., n = 3 independent experiments). p = 2.0036e-06 (AP+), p = 0.6763 (mixed) and p = 1.8854e-06 (AP-) comparing each IAA-treated subgroup to the respective untreated subgroup. (We report the maximum p value of pairwise two-sample t-tests (two-tailed), that is each replicate of untreated against each replicate of auxin-treated cells). g Quantification of colony formation. Differentiation was induced by removal of LIF under serum-containing culture conditions (mean ± s.d., n = 3 independent experiments). p = 2.8985e-05 for Mpp8^mAID; OsTIR1 and p = 0.3808 for Mpp8^mAID (two-tailed unpaired Student’s t test). h Experimental setup for monolayer differentiation of naïve mESCs in N2B27 by withdrawal of 2i. i, j qRT-PCR analysis of selected pluripotency genes (i) or early post-implantation epiblast markers (j) (n = 2 biologically independent samples). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Source data are provided as a Source Data file.