Fig. 3: Deubiquitinase USP14 stabilizes PSAT1 in lung adenocarcinoma.

a, b H1299 cells were co-transfected with His-USP14 and Flag-PSAT1. The immunoprecipitations were blotted with anti-His or anti-Flag antibodies. c, d In H1299 cells, endogenous interaction between PSAT1 and USP14 was tested using anti-Flag antibodies for Co-IP. The immunoprecipitations were blotted with anti-PSAT1 or anti-USP14 antibodies. e The protein levels of USP14 and PSAT1 were detected in lung cancer cell lines and normal bronchial epithelial cell BEAS-2B. f In H1299 cells, USP14 was overexpressed to detect PSAT1 protein by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), ***P < 0.001. g H1299 cells were experimented with different concentrations of IU1. The PSAT1 protein expressions were detected by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), *P < 0.05, **P < 0.01, ***P < 0.001. h H1299 cells were transiently transfected with CTL siRNA or USP14 selective siRNAs. The PSAT1 protein expressions were detected by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), **P < 0.01, ***P < 0.001. i H1299 cells were transfected with USP14 selective siRNAs and MG132 (20 μM) was added 12 h before sample collection. The PSAT1 protein expressions were detected by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), ns: P > 0.05, **P < 0.01. j H1299 cells were transfected with control Vector or His-USP14 plasmid or transiently transfected with CTL siRNA or USP14 selective siRNAs or experimented with different concentrations of IU1. The mRNA levels of PSAT1 and USP14 were detected by qPCR. Data represented as the average of three independent experiments (mean ± SD), ns: P > 0.05, *P < 0.05, ***P < 0.001. k–m H1299 cells were experimented with CHX (100 μg/mL) for various time points and were transfected with His-USP14 or USP14 selective siRNA or treated with IU1. The PSAT1 protein expressions were detected by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), *P < 0.05, **P < 0.01, ***P < 0.001.