Reduced USP13 expression and increased Siah2 activity in melanoma cells under hypoxia. A, quantitative real time PCR detection of relative transcription levels of USP13 mRNA in different melanoma cell lines in normoxia and after 6 and 18 h in hypoxia (2% O2). All of the values were calculated using the ΔΔCt method as relative quantity value, using histone 3.3A as internal control. The results are shown as the means ± S.E. of three independent experiments. B, melanoma cell lines 501, UACC-903, and Lu1205 were kept in normoxic or subjected to hypoxic (2% O2) conditions for 24 h. The cells were harvested, and cell lysates were subjected to Western blot analysis to detect USP13, Siah2, Spry2, and HIF-1α. β-Actin served as the loading control. C, 293T and HeLa cells were kept in normoxic or subjected to hypoxic (2% O2) conditions for 24 h. The cells were harvested, and cell lysates were subjected to Western blot analysis to detect USP13 and HIF1α. β-Actin served as the loading control.