Fig. 6: Inhibition of CBX2 decreases stemness.
From: CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer
a CBX2-associated genes were cross-referenced with a gene set for stemness. Percentage indicates overlap of stemness gene set with CBX2-associated genes. b OVCAR4 cells grown in adherent and suspended settings for 7 days. Aldefluor assay and flow cytometry were utilized to determine the percentage of cells that were positive for aldehyde dehydrogenase (ALDH), a marker of stemness. Diethylaminobenzaldehyde (DEAB), a potent ALDH inhibitor, prevented the increase in ALDH activity and served as negative control (left). c As above, OVCAR4 cells grown in adherent and suspended settings for 7 days. Bar graph compares the percentage of cells ALDH positive control (+DEAB) to cells without DEAB (experimental) in adherent and suspended settings. Statistical test = two-sided t-test. F Test p = 0.1136. d OVCAR4 cells with shControl, shCBX2#1 and #2 cultured in suspension over 7 days. Aldefluor assay and flow cytometry again utilized to determine the percentage of cells ALDH positive. Statistical test = two-sided t-test. F-test p = 0.83 e Utilizing the TCGA (HGSOC, Nature, 2011, n = 489) dataset, a scatter plot CBX2 expression was correlated to ALDH3A1 expression. Spearman correlation r = 0.2123 and p-value < 0.0001. f RT-qPCR of ALDH3A1 in OVCAR4 cells cultured in adherent and suspension conditions with CBX2 knockdown (shCBX2 #1). Statistical test = ANOVA. g Scatter plot of ALDH3A1 expression (x-axis, Z-score) compared to disease-free survival (y-axis, months). Pearson’s correlation r = −0.1258 and p-value = 0.0122. h Utilizing the TCGA (HGSOC, Nature, 2011, n = 489) dataset, a scatter plot CBX2 expression was correlated to SOX4 expression. Spearman correlation r = 0.154 and p-value = 0.0006. i PEO1, OVCAR4, and OVCAR8 cells grown in adherent (Adh) or suspension (Sus) settings for 7 days. RNA was extracted, and used for RT-qPCR against SOX4. Statistical test = two-sided t-test. Experiment performed in technical triplicates and biological duplicate. j shControl and shCBX2 #1 and #2 PEO1, OVCAR4, and OVCAR8 were cultured in suspension conditions, RNA was extracted and used for RT-qPCR against SOX4. Experiment performed in technical triplicates and biological duplicate. Statistical test = ANOVA. Error bars = S.E.M.
