Fig 1.
ACAT-1 expression and CE levels in a panel of ovarian cancer cell lines (OC-314, SKOV-3, A2780, IGROV-1) in comparison to primary ovarian epithelial cells, H-6036 (normal controls) (A) qRT-PCR analysis of ACAT-1 mRNA expression in a panel of ovarian cell lines. The threshold cycle (Ct) values were normalized to the housekeeping gene 18srRNA to quantify ACAT-1 mRNA (Comparative Ct method). Data represent mean fold change (FC) of ACAT-1 mRNA expression in cancer cell lines over normal control cells (H-6036), Mean FC ± SD of triplicate experiments, *P < 0.05 versus normal control cells, Student’s t-test. (B) ACAT-1 protein levels quantified by ELISA. Data represent mean FC ± SD of triplicate experiments. *P < 0.05 versus normal control cells, Student’s t-test. (C) Western blot analysis of the ACAT-1 and ACAT-2 proteins in a panel of ovarian cell lines. GAPDH is used as the loading control. (D) Immunocytochemical staining for ACAT-1 in various ovarian cancer cell lines using primary antibody against ACAT-1 with FITC labeled secondary antibody (green) and DAPI counterstain (blue). Representative images were taken with an inverted microscope (Olympus H4-100, CCD camera) and a 40X objective.
Fig 2.
Comparison of fold changes of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) levels between normal control cells and a panel of ovarian cancer cell lines.
Values represent mean FC ± SD of triplicate experiments, *P < 0.05 versus normal control cells, Student‘s t-test.
Fig 3.
Effect of ACAT-1 inhibition on viability of a panel of ovarian cancer cell lines.
(A) Cell viability assay of a panel of ovarian cancer cell lines treated with ACAT inhibitor avasimibe for 3 days. Vehicle treated cells (media with 1% DMSO) were considered as control against which treated cells were compared. Data expressed as mean ± SD of triplicate experiments. *P < 0.05, compared to control, Student’s t-test. (B) Confirmation of efficient stable ACAT-1 knock down assessed by western blot. β-actin was used as the loading control. (C) Assessment of cell viability upon stable knock-down of ACAT-1, in a panel of ovarian cancer cell lines. Data are expressed as mean ± SD of triplicate experiments. *P < 0.05, compared to the respective scrambled controls, Student’s t-test.
Fig 4.
Effect of shRNA mediated ACAT-1 inhibition on ovarian cancer cell proliferation, colony formation, migration and invasion properties (A) Cell proliferation assay of ovarian cancer cell lines stably transfected with control shRNA or ACAT-1 specific shRNA at 1, 2, 3, 4, 6, and 14 days post-seeding. Data are shown as mean ± SD of triplicate experiments. *P < 0.05, compared to the respective scrambled controls, Student’s t-test. (B) Left panel: Colony formation assay. Cells seeded at 200 cells/100 mm2 petri dish were incubated for 21 to 28 days. Surviving colonies were fixed, stained with crystal violet and observed under microscope. Right panel: Quantitation of number of colonies. Data were expressed as mean ± SD of triplicate experiments. *P < 0.05, as compared to respective scrambled controls, Student’s t-test (C) Cell migration abilities of control shRNA transfected cell lines vs. ACAT-1 shRNA transfected cell lines. (D) Cell invasion assay on control sh-RNA vs. ACAT-1 shRNA transfected cells. Data represent mean ± SD of triplicate experiments. *P < 0.05, as compared to respective scrambled controls, Student’s t-test.
Fig 5.
Effect of shRNA mediated ACAT-1 inhibition on cell cycle.
(A): Cell cycle analysis of ovarian cancer cell lines stably transfected with control shRNA or ACAT-1 shRNA. Arrows (Ap) represent sub-G1 (apoptotic) phase. (B): Graphical representation of cell cycle analysis. Data are presented as mean ± SD of triplicate experiments. *P < 0.05, compared to respective scrambled controls, Student’s t-test. (C) Effect of ACAT-1 inhibition on apoptosis. Nuclear condensation indicative of apoptosis was assessed using NucBlue Live Cell Stain Hoechst 33342. Representative images were taken with an inverted microscope (Olympus H4-100, CCD camera) and 20× objective.
Fig 6.
Effect of shRNA mediated ACAT-1 inhibition on apoptosis.
Quantification of apoptosis was done using Muse’s Annexin V & Dead Cell Assay Kit. The Muse Software Module performs calculations automatically based on Annexin-V and 7-AAD staining. Data represent mean ± SD of triplicate experiments. *P < 0.05, as compared to respective scrambled control, Student’s t-test.
Fig 7.
Effect of shRNA mediated ACAT-1 inhibition on mitochondrial transmembrane potential.
Detection of changes in mitochondrial trans-membrane potential upon ACAT-1 inhibition using Muse MitoPotential Assay Kit. Data represent mean ± SD of triplicate experiments. *P < 0.05, as compared to scrambled control, Student’s t-test.
Fig 8.
Effect of shRNA mediated ACAT-1 inhibition on apoptosis related proteins.
(A) Effect of ACAT-1 inhibition on casapses3/7 activities. Data were expressed as mean ± SD of triplicate experiments. *p < 0.05, compared to scrambled control, Student’s t-test. (B) Western blot analysis of the expression of proteins in the cell lysates. The blots were probed with the respective primary antibodies. β-actin was used as the loading control. All blots were quantified using Odyssey software. (C) Western blot band intensity was quantified by scanning densitometry. Data are means ± SD. *P<0.05, **P<0.01.
Fig 9.
Effect of shRNA mediated ACAT-1 inhibition on reactive oxygen species (ROS).
Detection of ROS was made by flow cytometry. Data were presented as relative-fluorescence intensity in a 2-dimensional FACS profile (standardized gating, 20,000 events). Enhanced ROS generation is observed as a shift the in peaks in ACAT-1 inhibited cell lines (blue) compared to scrambled controls (red). All experiments were performed in triplicate.
Fig 10.
Cisplatin dose response curves.
Cisplatin IC50 values were assessed and compared between ACAT-1 inhibited and scrambled controls. Cell viability was determined by PrestoBlue cell viability reagent. Dose response curves to calculate IC50 values were plotted using GraphPad Prism Software. Data were expressed as mean ± SD of triplicate experiments.