cholesterol cell-based detection assay kit  (Cayman Chemical)

Journal: Cancer Research

Article Title: Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer

doi: 10.1158/0008-5472.CAN-24-0970

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 screen identifies synergy with combined AKT inhibition and cholesterol homeostasis gene knockout in TNBC cells. A, Schematic of CRISPR/Cas9 screen. SUM159 cells were transduced with a Cas9-expressing lentivirus containing 94,495 sgRNAs with 3 to 4 sgRNAs per gene. Infected cells were allowed to grow for approximately 1 week before seeding the treatment arms. Cells were treated with DMSO, BYL719 (PI3Kα-selective inhibitor, 0.4 µmol/L), or GDC-0068 (catalytic AKT inhibitor, 3 µmol/L) for 72 hours ( N = 3 technical replicates for each treatment arm). B, Rank plots showing the log 2 -fold change of each gene plotted against the dropout gene rank for the BYL719 and GDC-0068 treatments arms compared with the DMSO arm. Expected changes in PI3K/AKT signaling genes are highlighted, including TSC2 , PTEN , and FOXM1 . Plots were generated using MAGeCK with a read count cutoff of 50 ( N = 3 technical replicates). C, Plot of the top pathways selectively perturbed in the GDC-0068 arm of the CRISPR/Cas9 screen. Analysis was performed via gene set enrichment analysis. D, Rank plot showing the log 2 -fold change of each gene plotted against the dropout gene rank for the GDC-0068 treatment arm of the CRISPR/Cas9 screen compared with the DMSO arm. The transcription factors SREBF1 and SREBF2 are highlighted. The plot was generated using MAGeCK with a read count cutoff of 50.

Article Snippet: Cells were incubated for 24 hours and then treated with 10 μL of DMSO or 1 μmol/L U18666A (Cayman Chemicals, Cholesterol Cell-Based Detection Assay Kit, 10009779) and 10 μL of DMSO or 2 μmol/L pitavastatin (Selleckchem, S1759) for 24 hours.

Techniques: Genome Wide, CRISPR, Inhibition, Gene Knockout, Transduction, Expressing, Infection, Generated

Journal: Cancer Research

Article Title: Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer

doi: 10.1158/0008-5472.CAN-24-0970

Figure Lengend Snippet: Disruption of cholesterol homeostasis synergizes with AKT inhibition in TNBC cells. A, Cholesterol is synthesized in multiple steps from acetyl-CoA. HMGCR catalyzes the first rate-limiting step of cholesterol biosynthesis. This pathway also generates the prenylation substrates FPP and GGPP. SREBP-1/2 sense low endoplasmic reticulum cholesterol levels and translocate from the endoplasmic reticulum to the Golgi where they are cleaved and activated. N-terminal active SREBP-1/2 enter the nucleus to regulate the transcription of target genes. Drugs targeting this pathway are highlighted, including HMGCR inhibitors (statins) and inhibitors of protein farnesylation (FTI-277) and geranylgeranylation (GGTI-298). B and C, TNBC cell lines (SUM159, MDA-MB-468, and BT20) were treated with increasing doses of GDC-0068 (SUM159, 0–10 µmol/L; MDA-MB-468, 0–20 µmol/L; and BT20, 0–5 µmol/L; B ) or AZD5363 (SUM159, 0–5 µmol/L; MDA-MB-468, 0–40 µmol/L; BT20, 0–5 µmol/L; C ) and pitavastatin (0–2,000 nmol/L) for 72 hours, and cell density was measured by SRB assay. Data are represented as mean ± SD ( N = 3 technical replicates). D, TNBC cell lines (SUM159, MDA-MB-468, and BT20) were treated with DMSO, AZD5363 (SUM159, 2.5 µmol/L; MDA-MB-468, 10 µmol/L; BT20, 1.25 µmol/L), pitavastatin (SUM159, 4 µmol/L; MDA-MB-468, 2 µmol/L; BT20, 0.5 µmol/L), or a combination of AZD5363 and pitavastatin for 72 hours, and cell density was measured daily by SRB assay. Data are represented as mean ± SD ( N = 3 technical replicates). Statistical analysis was performed using two-way ANOVA with Dunnett multiple comparison test. *, significant differences compared with the AZD5363 and pitavastatin combination treatment on day 4. **, P = 0.0021; ***, P = 0.0002; ****, P < 0.0001.

Article Snippet: Cells were incubated for 24 hours and then treated with 10 μL of DMSO or 1 μmol/L U18666A (Cayman Chemicals, Cholesterol Cell-Based Detection Assay Kit, 10009779) and 10 μL of DMSO or 2 μmol/L pitavastatin (Selleckchem, S1759) for 24 hours.

Techniques: Disruption, Inhibition, Synthesized, Sulforhodamine B Assay, Comparison

Journal: Cancer Research

Article Title: Targeting Cholesterol Biosynthesis with Statins Synergizes with AKT Inhibitors in Triple-Negative Breast Cancer

doi: 10.1158/0008-5472.CAN-24-0970

Figure Lengend Snippet: 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). IC 50 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.

Article Snippet: Cells were incubated for 24 hours and then treated with 10 μL of DMSO or 1 μmol/L U18666A (Cayman Chemicals, Cholesterol Cell-Based Detection Assay Kit, 10009779) and 10 μL of DMSO or 2 μmol/L pitavastatin (Selleckchem, S1759) for 24 hours.

Techniques: Inhibition, Clone Assay, Sulforhodamine B Assay, Comparison, Transfection, Expressing, Staining, Two Tailed Test