Fig. 4: CYH33 accumulated p21 via blocking SKP2-mediated ubiquitination.

a ESCC cells were treated with CYH33 at indicated concentrations for 24 h and mRNA level of CDKN1A was measured by quantitative real time PCR (n = 3). b, c KYSE410 (b, n = 3) and KYSE450 (c, n = 2) cells were pretreated with CYH33 (1 μM) or vehicle for 12 h, and then treated with CHX (25 μM) for indicated time. Cell lysates were subjected to Western blot with the indicated antibodies. The intensity of protein bands was quantified by Imagelab and the half-life of p21 was calculated after linear fitting. d, e KYSE410 cells were treated with NH₄Cl (d) or MG132 (e) at indicated concentrations for 24 h. Cell lysates were subjected to Western blot with the indicated antibodies. f KYSE410 cells were treated with CYH33 (1 μM) for 24 h in the presence of MG132 (20 μM). Cell lysate was immunoprecipitated with the antibody against p21 and followed by Western blot with an anti-Ubiquitin antibody. g KYSE410 cells were treated with CYH33 (1 μM) for 24 h, and immunoprecipitation with the antibody against p21 was performed followed by Western blot for the indicated proteins. The bands were quantified by Imagelab (n = 2). h Ubiquitination of p21 was determined in KYSE450 cells transfected with plasmid expressing SKP2 (S) or vehicle (V) by immunoprecipitation with anti-p21 antibody followed by Western blot with anti-Ubiquitin antibody. i KYSE410 cells transfected with plasmid expressing SKP2 or vehicle were treated with CYH33 for 24 h and cell cycle distribution was analyzed with flow cytometry (n = 3). j SKP2 gene was knocked out in KYSE450 cells by CRISPR. Monoclonal SKP2-KO cells (S2-16) and parental cells (Vehicle) were treated with CYH33 for 24 h and cell cycle distribution was analyzed with flow cytometry (n = 3). Data were presented as mean ± SD. p values were determined by two-tailed one-way ANOVA with Tukey multiple group comparison test. ns, p > 0.05; *p < 0.05; **p < 0.01