Figure S6 from PAK5-Mediated Suppression of β-Hydroxybutyrate Production Promotes Breast Cancer Metastasis and Can Be Overcome with Ketogenic Diet

Supplementary Figure 6. HMGCS2 and its non-phosphorylatable mutants antagonize PAK5-driven breast cancer cell proliferation, migration and invasion in vitro (a) GST pull-down analysis of the direct binding of HMGCS2 and SIRT3. Black stars indicate GST and GST-fusion proteins. (b) IP and IB analysis of the interaction of endogenous HMGCS2 and SIRT3 in BT474 cells. (c) GST pull-down analysis of the direct binding of PAK5 and SIRT3. Black stars indicate GST and GST-fusion proteins. (d) IP and IB analysis of the interaction of endogenous PAK5 and SIRT3 in MDA-MB-231 cells. (e) IP and IB analysis of endogenous HMGCS2 acetylation in BT474 cells transfected with the indicated plasmids. (f) IP and IB analysis of the interaction of SIRT3 and GFP-HMGCS2 WT, GFP-HMGCS2 S138A or GFP-HMGCS2 S311A. (g) CCK8 proliferation analysis of HS578T cells transfected with the indicated plasmids, the data were presented as a histogram of the mean and SEM of three independent experiments, *P < 0.05, **P < 0.01,****P < 0.0001. (h) Colony formation analysis of HS578T cells transfected with the indicated plasmids, representative pictures of the colonies were shown. The data were presented as a histogram of the mean and SEM of three independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001. (i) Transwell analysis of HS578T cells transfected with the indicated plasmids. Results were representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (j) Wound-healing analysis of HS578T cells transfected with the indicated plasmids. Results were representative of three independent experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001.