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. 2018 Nov 8;11(6):1523–1538. doi: 10.1016/j.stemcr.2018.10.009

Figure 7.

Figure 7

p53 Knockdown in Rpt3-Deficient Satellite Cells Rescues the Defect in Proliferation

(A) DAVID v6.7 functional annotation bioinformatics microarray analysis software was used to obtain KEGG pathway functional classification. Only KEGG pathway terms for classification that showed statistically significant differences, in terms of the number of differentially regulated genes (satellite cells from satellite cell-specific Rpt3 mice versus satellite cells from control mice), are shown (p < 0.05). The 1,254 significantly differentially regulated genes are shown in Table S2. The red dash line denotes the significance level of 0.05.

(B) Time course of tamoxifen (Tmx) treatment and tissue harvesting. Con indicates Rpt3f/f mice and scKO indicates satellite cell-specific Rpt3 knockout mice (Pax7CreERT2/+; Rpt3f/f).

(C) Real-time RT-PCR was used to measure the mRNA expression of p53-related genes (p53, p21, Cyclin D1, and Cdk4) in Pax7CreERT2/+; Rpt3f/f satellite cells treated with vehicle or 4OH-Tmx. Data represent means ± SEM (t test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n = 4–5 per group). AU, arbitrary units.

(D) Immunoblotting analysis of relative protein levels of p53 targets in Pax7CreERT2/+; Rpt3f/f satellite cells treated with vehicle or 4OH-Tmx.

(E) Immunostaining for p53 (green) and DAPI (blue) in Pax7CreERT2/+; Rpt3f/f satellite cells treated with vehicle or 4OH-Tmx. Arrows indicate p53-positive cells. Scale bar, 10  μm. The y axis shows the relative ratio of p53-positive cells. Data represent means ± SEM (t test: ∗∗∗p < 0.001; n = 4 per group).

(F) Time course analysis of p53 siRNA knockdown and Tmx treatment.

(G) Immunostaining for EdU (green) and DAPI (blue) in satellite cells treated with vehicle or 4OH-Tmx. Scale bar, 50  μm. The y axis shows the relative ratio of EdU-positive cells. Data represent means ± SEM (one-way ANOVA followed by the Bonferroni post hoc test: ∗∗p < 0.01; NS, no significance; n = 4 per group).

(H) Relative ratio of DAPI-positive cells. Data represent means ± SEM (one-way ANOVA followed by the Bonferroni post hoc test: ∗∗p < 0.01, ∗∗∗p < 0.01; NS, no significance; n = 4–6 per group).

See also Figure S7.