Research Paper Volume 12, Issue 14 pp 14699—14717

Figure 4. K669 acetylation of HSD17B4 promotes its degradation in PCa cells. (A) DHT treatment increases HSD17B4 K669 acetylation levels but decreases endogenous HSD17B4 protein levels in a dose-dependent manner. LNCaP cells were either untreated or treated with DHT at different concentrations as indicated. The K669 acetylation level and the protein steady-state level of HSD17B4 were determined by western blotting and normalized against ACTB (upper panel). The relative K669 protein level compared with the HSD17B4 level (middle panel) and relative HSD17B4 protein level compared with the ACTB level (lower panel) were quantified. ^*denotes P < 0.05, **denotes P<0.01. Error bars represent ±SD for triplicate experiments. (B) DHT treatment increases HSD17B4 K669 acetylation while decreasing its protein in a time-dependent manner. The HSD17B4 acetylation level and protein level were determined by western blotting after treatment with DHT for different lengths of time, as indicated in LNCaP cells (upper panel). The relative K669 protein level compared with the HSD17B4 level (middle panel) and relative HSD17B4 protein compared with the ACTB level (lower panel) were quantified. ^*denotes P < 0.05, ^**denotes P < 0.01, ***denotes P<0.001. Error bars represent ±SD for triplicate experiments. (C) HSD17B4 is not degraded by the ubiquitin proteasome system (UPS). LNCaP cells were treated as indicated, and HSD17B4 protein levels were analyzed by western blotting (upper panel). Relative HSD17B4 protein compared with the ACTB level was quantified. ^*denotes P < 0.05. Error bars represent ±SD for triplicate experiments (lower panel). (D) The lysosome inhibitor chloroquine (CLQ) restores HSD17B4 protein that was reduced by DHT treatment. LNCaP cells were treated with the lysosome inhibitor CLQ, and the HSD17B4 protein level was analyzed by western blotting (upper panel). The relative HSD17B4 protein compared with the ACTB level was quantified. ^**denotes P < 0.01, NS, no significance. Error bars represent ±SD for triplicate experiments (lower panel). Data are shown as the mean ± SD (n = 3) or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05, **p < 0.01, ***p <0.001.