Figure 5.

Palb inhibits proliferation and BA induces apoptosis. MDA-MB-231, Panc1 and T47D cells were used to analyze the mechanistic effects of P (1 µM) or BA (25 µM). Culture medium or 0.1% D were used as controls. (A) The BrdU cell proliferation assay was performed after drug treatments for 72 h. (B) The RealTime-Glo Annexin V assay was utilized to measure apoptosis 48 h after addition of drug and presented as Annexin V (absorbance)/cell number (fluorescence). Cell number was determined using the CellTiter-Fluor Cell Viability Assay. (C) Target validation of BA. The product of the ACLY enzyme (acetyl-CoA) was shown to reverse toxicity induced by BA using the CellTox Green Cytotoxicity Assay. Error bars display standard deviation of the mean of n=8. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test. **P<0.01 compared with all other treatments. (D) Western blotting was performed on cells treated for 96 h as aforementioned using antibodies to p-Rb (780), total Rb, apoptosis markers cPARP and p-c-Jun and the proliferation markers Mcm7, cyclin D3 and PCNA. β-actin was used as a loading control. Results shown were repeated twice. CT, control; p, phosphorylated; cPARP, cleaved poly (ADP-ribose) polymerase; PCNA, proliferating cell nuclear antigen; BA, bempedoic acid; Palb, palbociclib; ACLY, ATP citrate lyase; BrdU, 5-bromo-2-deoxyuridine; Rb, retinoblastoma; Mcm7, minichromosome maintenance complex component 7.