Fig. 3: Knockdown of endogenous Δ133p53α inhibits cell proliferation in KGM and CR cultures.

Two independent siRNAs (Δ133-siR#1 and Δ133-siR#2) were designed to target sequences present in Δ133p53α mRNA as 5′-UTRs that are spliced out of full-length p53 mRNA. Transfection was performed in KGM using early-passage HPECs (P3) and HFKs (P4). The control siRNA (Scr-Control) was a scrambled sequence of Δ133-siR#1. a Immunoblot analysis of Δ133p53α and full-length p53 proteins at 3 days post-transfection. GAPDH was used as a loading control. Normalized densitometric values for expression levels are indicated below each lane relative to Scr-Control cells (defined as 1.0). Representative images of the transfected HFKs are shown in Supplementary Information Fig. S9a, b. b Proliferation of the transfected HPECs and c HFKs were monitored for 4 days using IncuCyte. Data are mean ± S.D. from triplicate wells. d Immunoblot analysis for Δ133p53α and full-length p53 proteins at 3 days post-transfection of the HFKs with Scr-Control, Δ133-siR#1 and Δ133-siR#2 siRNA in conditioned media (CM). These HFKs were initially cultured in CRC for eight passages (HFK_CRC/P8) and then cultured in CM for another three passages (CM/P3) before transfection. GAPDH was used as a loading control. Normalized densitometric values are indicated below each lane. e Proliferation of the transfected HFKs in d were monitored for 4 days post-transfection using IncuCyte. Data are the mean ± S.D. from triplicate wells. Representative images of the transfected HFKs are shown in Supplementary Information Fig. S10. f Expression of Δ133p53, full-length p53 and hTERT mRNA in the transfected HFKs in d were measured by qRT-PCR at 3 days post-transfection in conditioned medium (CM). β2-microglobulin mRNA was used for normalization. Data are mean ± S.D. from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Uncropped blot images are shown in Supplementary Information Figs. S29–30