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. 2018 Jul 3;9(7):747. doi: 10.1038/s41419-018-0774-8

Fig. 3. PP2A-mediated T308 dephosphorylation was responsible for YY1 knockdown-induced enhancement of anti-proliferative effect of cisplatin.

Fig. 3

a BX-795 enhanced cisplatin-induced cell proliferation inhibition. CAL27 cells were treated with BX-795 (10 μM) or cisplatin (10 μM) or both for 48 h and subjected to CCK-8 assay, (n = 4). One-way ANOVA: *P < 0.05 vs. control group; #P < 0.05 vs. BX-795 or cisplatin group. b Forskolin enhanced cisplatin-induced cell proliferation inhibition. CAL27 cells were treated with forskolin (20 μM) or cisplatin (10 μM) or both for 48 h and subjected to CCK-8 assay, (n = 4). One-way ANOVA: *P < 0.05 vs. control group; #P < 0.05 vs. forskolin or cisplatin group. c YY1 overexpression partially rescued LY294002-induced AKT phosphorylation inhibition. CAL27 cells infected with lentivirus carrying GFP empty vector or PLVX-GFP-YY1 vector and exposed to LY294002 for 48 h. Total protein was extracted and subjected to Western blot analysis. d YY1 overexpression inhibited PP2A activity and PPP2CA protein expression but upregulated AKT T308 phosphorylaiton. CAL27 cells infected with lentivirus carrying GFP empty vector or PLVX-GFP-YY1 vector. Whole-cell lysates were immunoprecipitated with PPP2CA antibody and subjected to PP2A immunoprecipitation phosphatase assay. Protein was extracted from the same samples and subjected to Western blot. t-test, *P < 0.05 (n = 4). e Knockdown of YY1 upregulated PP2A activity and PPP2CA protein expression but inhibited AKT T308 phosphorylation. CAL27 cells stably transfected with scramble shRNA or YY1 shRNA lentivirus. Cells treated with forskolin as a positive control. The experiment was performed similar to that in (d), one-way ANOVA: *P < 0.05 (n = 4). f Okadaic acid rescued YY1 knockdown-induced AKT T308 phosphorylation inhibition but failed to rescued YY1 knockdown-induced AKT S473 phosphorylation inhibition. CAL27 cells stably transfected with scramble shRNA or YY1 shRNA and treated with okadaic acid (15 nM) or not for 48 h. Total proteins were extracted and subjected to Western blot analysis. g Okadaic acid abolished YY1 knockdown-induced or cisplatin-induced or their combination-induced inhibition of cell proliferation. CAL27 cells stably transfected with Tet-on YY1 shRNA were exposed to either doxycycline (100 nM) or okadaic acid (15 nM) or cisplatin (10 μM) or both for 48 h and subjected to CCK-8 assay (n = 4), one-way ANOVA: *P < 0.05 vs. control group; #P < 0.05 vs. YY1 knockdown or cisplatin group; &P < 0.05 vs. cisplatin group; $P < 0.05 vs. YY1 knockdown group; %P < 0.05 vs. the combination of cisplatin and YY1 knockdown group. h Forskolin blocked YY1 overexpression-induced upregulation of AKT T308 phosphorylation but failed to blocked YY1 overexpression-induced upregulation of AKT S473 phosphorylation. CAL27 cells infected with GFP empty or PLVX-GFP-YY1 lentivirus and treated with forskolin (20 μM) or not for 48 h. Total proteins were extracted and subjected to Western blot analysis. c, d, e, f, h The target bands were exposed, and densitometry was performed as fold change ratio from the mean of three independent experiments, one-way ANOVA (c, e, f, h) or t-test (d), *P < 0.05 vs. control group. (Bars: mean ± SD)