Research Paper Volume 14, Issue 19 pp 8032—8045

Figure 2. SENP3 promotes proliferation and deSUMOylation of STAT3. T24 and EJ cells were transfected plasmids with overexpression of SENP3 [STAT3(OE)] and knock-down of SENP3 [STAT3(KD)]. (A) Cell proliferation assay in vitro in NC, SENP3(OE), SENP3(KD) T24 and EJ cells. [mean ± S.D. (error bars), n = 3. ^**p ≤ 0.01; ^***p ≤ 0.001; ^****p ≤ 0.0001, compared with NC group; ^##p ≤ 0.01; ^####p ≤ 0.0001, compared with NC group, two-way analysis of variance]. (B) Left: Cell invasion determined by trans well staining in SENP3 overexpression or knockdown T24 and EJ cells. [scale bar, 25 μm]. right: quantitative analysis of immunohistochemistry for positive trans well staining. [mean ± S.D. (error bars), n = 3. ^*p ≤ 0.05, compared in T24 cells; ^#p ≤ 0.05, compared in EJ cells, two-way analysis of variance] (C) EDU positive cells in NC, SENP3(OE), SENP3(KD) T24 and EJ cells determined by Confocal immunofluorescence. [scale bar, 50 μm]. (D) SENP3, STAT3 and p-STAT3 protein levels in NC, SENP3(OE), SENP3(KD) T24 and EJ cells, as measured by western blot. (E) The mRNA level of SENP3 and STAT3 measured by qPCR. (F) Co-immunoprecipitation (co-IP) of endogenous SENP3 with STAT3 and its SUMO2. (G) Abundance of p-STAT3 protein in in NC, SENP3(OE) T24 and EJ cells. (H) T24 cells were transfected with the indicated constructs and treated for the indicated times with CHX and MG132, whole cells were collected and STAT3 protein level was determined by western blot. All experiments were performed in triplicates.