VFL is involved in Hakai degradation via proteasome-mediated in 5637 cell line. A, the levels of Hakai mRNA in 5637 (upper panel, left), HT1376 (upper panel, right), and UMUC3 (lower panel) cells following treatment with 5 μM VFL for 48 hours were determined by RT-qPCR analysis. HPRT mRNA levels were measured for normalization. The means ± SEM are represented from three independent experiments (*p < 0.05, n = 3). B, effect of proteasome inhibitor, MG132, on Hakai expression in 5637. Cells following treatment with 5 μM VFL for 48 hours were incubated for 2 h in the absence or presence of proteasome inhibitor (20 μM MG132) and cell lysates were prepared for Western blot analysis to detect Hakai, E-cadherin and normalization control GAPDH. Western blot data are representative of three independent experiments and quantification by densitometry was represented (lower panel, *p < 0.05, **p < 0.01).