Fig 4. VPS34 inhibition decreases intracellular levels of cholesteryl-esters, blocks cholesterol uptake and enhances lysosomal degradation of LDLR.

(A) Lipidomic analysis of RKO cells upon VPS34 inhibition. RKO CTRL cells were treated with 1 μM PIK-III for 24 hours and lipids were extracted and subjected to liquid chromatography mass spectrometry analysis. The table shows PIK-III-induced log2 fold change for aggregated and averaged lipid classes in RKO cells. The adjusted p-values were calculated between PIK-III and vehicle treatment group. Data represents the mean of three biological replicates and the entire dataset is reported in S4 Table. (B) RKO.Cas9 cells were treated with vehicle or 1 μM PIK-III for 24 hours followed by incubation with BODIPY FL LDL probe for 2 hours at 37°C. Nuclei were labeled with Hoechst and live cell imaging was performed. The white bar represents 10 μm in length. (C) BODIPY FL LDL probe cellular uptake in (B) was quantified as percent average intensity of total cells. Data in the form of technical replicates were averaged and presented as the mean ± SD from 6 wells. (D) RKO CTRL cells were treated with the indicated concentrations of PIK-III for 1, 6, 12, or 24 hours. (M) stands for mature protein and (P) stands for precursor protein. (E, F) RKO CTRL cells were treated with the indicated concentrations of PIK-III with or without 50 nM bafilomycin A1 (E) or 30 μM chloroquine (F) for 12 and 24 hours. Lysates were immunoblotted with the specified antibodies. (M) and (P) indicate the mature and precursor protein, respectively. (G) RKO CTRL cells were treated with indicated concentrations of PIK-III along with vehicle, 2 μg/ml and 10 μg/ml soluble cholesterol (Chol) for 5 days and cell viability was assessed using the CellTiter-Glo assay. Data presented is the mean from three independent experiments ± SD. NS, not significant (two-way ANOVA).