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. 2024 Jul 18;84(19):3250–3266. doi: 10.1158/0008-5472.CAN-24-0970

Figure 6.

Figure 6.

NPC1 inhibition causes lysosomal cholesterol accumulation and rescues pitavastatin sensitivity. A, ER-negative (MDA-MB-468 and T47D fulvestrant-resistant clones 1 and 2) and ER-positive (T47D and parental T47D) breast cancer cell lines were treated with a range of concentrations of OSW-1 (0–10 nmol/L) for 72 hours, and cell density was measured by SRB assay. Data are represented as mean ± SD (N = 3 technical replicates). IC50 values for each cell line are reported. B, TN (MDA-MB-468) and ER-positive (T47D) breast cancer cells were seeded into media supplemented with 10% lipid-depleted serum and treated for 1 hour with vehicle or high dose of pitavastatin (10 µmol/L). Media were removed and replaced with media supplemented with 10% lipid-depleted serum and vehicle or low dose pitavastatin (2 µmol/L) for 72 hours, and cell density was measured by SRB assay. Data are represented as mean ± SD (N = 3 technical replicates). Statistical analysis was performed using two-way ANOVA with Šidák multiple comparison test. C, TNBC cells (SUM159, MDA-MB-468, and BT20) were transfected with siControl (siCtrl) or siNPC1 and then treated with DMSO or 1 µmol/L U18666A and DMSO or 2 µmol/L pitavastatin for 72 hours, and cell density was measured by SRB assay. Data are represented as mean ± SD (N = 3 technical replicates). Statistical analysis was performed using two-way ANOVA with Šidák multiple comparison test. D, TN (MDA-MB-468) and ER-positive (T47D) breast cancer cells expressing red fluorescent protein in the endoplasmic reticulum (ER-RFP; red) were treated with DMSO or 1 µmol/L U18666A for 24 hours. Cells were fixed with 4% formaldehyde and stained with Filipin III (blue) and a LAMP1 antibody (green). Representative images are shown. Scale bars, 50 µm. E, Quantification of Filipin III and LAMP1 colocalization normalized to total LAMP1 from 12 nonoverlapping fields. Statistical analysis was performed using an unpaired, two-tailed parametric t test. *, P = 0.0332; **, P = 0.0021; ***, P = 0.0002; ****, P < 0.0001; ns, not significant.