Figure 3: Endogenous STUB1 regulates Δ133p53α expression and senescence. | Nature Communications

Figure 3: Endogenous STUB1 regulates Δ133p53α expression and senescence.

From: Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence

Figure 3

(a) Endogenous STUB1 protein is downregulated at replicative senescence. Immunoblot was performed using early-passage (Y) and replicatively senescent (S) fibroblasts (the same set of cells without treatment as in Fig. 1c). (b) STUB1 mRNA is downregulated at replicative senescence. The same set of cells as in a were examined by qRT–PCR. Data were from triplicate experiments (mean±s.d.). *P<0.01; **P<0.001 (Student’s t-test). (c) Knockdown of STUB1 represses Δ133p53α. MRC-5 fibroblasts (30 PDLs) were transfected with two siRNAs against STUB1 (no. 1 and no. 2) and control siRNA. At 4 days after transfection, immunoblots were performed using anti-STUB1, anti-Δ133p53α, DO-1 (detecting full-length p53), CM1 (detecting both Δ133p53α and full-length p53), anti-p62/SQSTM1 and anti-β-actin antibodies. (d) STUB1 knockdown inhibits cell proliferation. MRC-5 fibroblasts (30 PDLs) were seeded on six-well plates on day 0 (4 × 104 cells per well in 6-well plate; nine wells for each siRNA). Cells were counted on days 1, 4 and 7 (three wells each; mean±s.d.), while siRNA transfection was performed on days 1 and 4. *P<0.01 (Student’s t-test). (e) STUB1 knockdown induces cellular senescence. The siRNA transfection was performed as in c and repeated 4 days later, followed by incubation for 3 days and SA-β-Gal staining. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P<0.001 (Student’s t-test). (f,g) STUB1 knockdown upregulates p21WAF1 and IL-8. The same set of cells as in e were examined in qRT–PCR for p21WAF1 mRNA (f) and IL-8 mRNA (g). Data were from triplicate experiments (mean±s.d.). **P<0.001 (Student’s t-test). (h) Δ133p53α rescues STUB1 knockdown-induced senescence. MRC-5 fibroblasts were transfected with STUB1 siRNA (no. 1) as in e. At the next day after the second transfection (on day 5), the cells were transduced with a lentiviral vector driving Δ133p53α or its vector control. At 5 days after the lentiviral transduction, the cells were stained for SA-β-Gal activity. Representative pictures (left) and quantitative data (right) are shown. Percentages of SA-β-Gal-positive cells were from triplicate experiments (mean±s.d.). **P<0.001 (Student’s t-test).

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