Fig. 4: SUMOylation stabilizes p14ARF.

A PC3 cells were transfected with SUMO2 or the empty vector pcDNA. At 48 h after transfection, p14ARF levels were analyzed by western-blot (upper panel). B PC3 cells were treated with the SUMOylation inhibitor ML-792 or DMSO and 16 h after treatment, the levels of p14ARF were analyzed by western-blot (upper panel). C PC3 cells were transfected with siRNA against UBC9 (siUBC9) or siRNA control (siC). At 72 h after transfection, UBC9 and p14ARF protein levels were analyzed by western-blot (upper panel). The intensity of the bands in (A–C) was quantified using ImageJ software. Intensity of p14ARF bands were normalized to GAPDH or actin bands and plotted (lower panels). Columns are representative of the mean and error bars represent the standard deviation of three biological replicas. Statistical analysis was assessed by a Student’s t-test. *P < 0.05; **P < 0.005; ****P < 0.0001. D PC3 cells were treated as indicated in (B) or transfected as indicated in (C). RNA was extracted and expression of mRNAs encoding p14ARF was analyzed by QRT-PCR. Values were normalized to GAPDH expression. Columns are representative of the mean and error bars represent the standard deviation of three biological replicas. E PC3 cells were transfected with siUBC9 or siC and 48 h after transfection (hpt), cells were treated with cycloheximide (CHX) and, at different times after treatment, levels of p14ARF were evaluated by western-blot analysis using anti-p14ARF antibody. p14ARF bands intensity were normalized to actin bands for each respective time and plotted (lower panel). Data are representative of the mean and error bars represent the standard deviation of three biological replicas. Statistical analysis was assessed by a Student’s t-test. *P < 0.05. F HEK-293 cells were transfected with p14ARF-HA and 48 h after transfection, cells were treated with DMSO or ML-792 for 4 h. Then, cells were treated with cycloheximide (CHX) in the presence or absence of ML-792 and, at different times after treatment, levels of p14ARF-HA protein were evaluated by western-blot with anti-HA antibody. p14ARF-HA bands were normalized to GAPDH bands for each respective time and plotted (lower panel). Data are representative of the mean and error bars of three biological replicas. Statistical analysis was assessed by a Student’s t-test. *P < 0.05.