Research Paper Volume 12, Issue 14 pp 14699—14717

Figure 7. CREBBP increases K669 acetylation levels and promotes the degradation of HSD17B4. (A) CREBBP overexpression increases the K669 acetylation level but decreases the HSD17B4 protein level. LNCaP cells were transfected as indicated. Cell lysates were subjected to western blotting. The K669 acetylation level and HSD17B4 levels were normalized against ACTB. (B) CLQ blocks the decrease in HSD17B4 protein induced by CREBBP overexpression. FLAG-CREBBP was transfected into LNCaP cells with or without CLQ treatment and then subjected to western blotting. (C) The knockdown of CREBBP decreases HSD17B4 K669 acetylation while increasing its protein level. LNCaP cells were transfected with siCREBBP or control. The acetylation and protein levels of HSD17B4 and the protein level of CREBBP were determined by western blotting and were normalized against ACTB. (D) Endogenous HSD17B4 binds CREBBP in LNCaP cells. LNCaP cells were cultured and harvested until the cell density reached 90%. The interaction between endogenous HSD17B4 and CREBBP was determined by co-IP and western blotting. (E) CREBBP is negatively correlated with HSD17B4 in PCa. The correlation between the mRNA expression levels of CREBBP and HSD17B4 in patients with PCa was analyzed using the public dataset GSE70770. (F) CREBBP knockdown promotes LNCaP cell growth, while CREBBP overexpression inhibits cell growth. The CCK-8 assay showed that the proliferation rate of LNCaP cells was affected by CREBBP knockdown or overexpression. The data shown are representative of three independent experiments. (G) CREBBP knockdown promotes the migration of LNCaP cells, while CREBBP overexpression acts the opposite way. LNCaP cells were transfected with siCREBBP or Flag-CREBBP as indicated, followed by a migration assay in 24-well chambers without Matrigel. Quantitative analysis of cell migration in 24-well chambers was performed by ImageJ. 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.