Fig 2: Hormonal regulation of CBX2. a Establishment of the LTL313B/LTL313BR patient-derived xenograft model of CRPC; b Assessment of CBX2 mRNA levels in the LTL313B/LTL313BR xenograft model by qRT-PCR; c IHC staining of CBX2 in the LTL313B and LTL313BR xenografts ×20. Images are representative of multiple fields taken from two independent experiments; d Levels of CBX2 mRNA in androgen-dependent (AD, n = 10) and androgen-independent (AI, n = 5) PDXs from the LTL; e Relative CBX2 expression in PCa cell lines compared to benign control (BPH1) assessed by qRT-PCR; f CBX2 mRNA levels in LNCaP cells cultured in charcoaled-stripped media in the presence or absence of DHT supplementation (10 nM)

Fig 3: Correlation of CBX2 expression and autophagy, apoptosis, and EMT.Utilizing the TCGA (HGSOC, Nature, 2011) dataset, CBX2 expression was correlated to mRNA expression of all genes and 5838 genes (CBX2-associated genes) were identified to have a Spearman correlation of greater than r = 0.15. a The 5838 genes were cross-referenced with published gene sets for “Apoptosis”, “Autophagy”, and “Epithelial to Mesenchymal Transition (EMT).” Percentage indicates overlap with gene lists. PEO1, OVCAR4, and OVCAR8 cells were grown in adherent (Adh) or suspension (Sus) for 7 days, RNA was extracted, and used for RT-qPCR against MYLK (b), NOG (c), and TNFSF10 (d). Statistical test = two-sided t-test. shControl and shCBX2 #1 and 2 PEO1, OVCAR4, and OVCAR8 cells were grown in suspension, RNA was extracted, and used for RT-qPCR against MYLK (e), NOG (f), and TNFSF10 (g). Statistical test = ANOVA. Error bars = S.E.M.

Fig 4: CBX2 is overexpressed in high grade serous carcinoma and portends poor prognosis.a Overall survival analysis comparing expression of CBX2 (High = mRNA expression > 1.5 standard deviation; Low = SD < 1.5(=)). High (n = 31) vs. Low (n = 454) CBX2 expression (mean survival 34.0 months vs. 44.81 months, Log Rank p = 0.0011). b Same as (a), disease-free survival analysis, again comparing upregulation of CBX2, defined as above (High n = 25, Low n = 371). Increased expression of CBX2 was associated with statistically significant decreased disease-free survival (11.70 months vs. 17.64 months, Log Rank p = 0.0032). Number of patients differ from (a) due to available data within TCGA. c Examination of CBX2 mRNA expression in HGSOC patients alive (n = 51) and dead (n = 42) at 3 years after diagnosis. Data obtained from HGSOC Tothill Cohort47. Statistical test = two-sided t-test, F test p = 0.0057 d Correlation of CBX2 expression with FOXO3_PS318/321 in TCGA (“Provisional data” with RPPA data, n = 435) tumors (High = upper quartile and Low = bottom three quartiles). Statistical test = two-sided t-test, F test p < 0.0001. e Bioinformatic analysis evaluating relative intensity of CBX2 in high grade serous ovarian carcinoma (HGSOC) cases described as platinum sensitive (n = 3) or resistant (n = 3). Each tumor (color-coded) was examined in triplicate. Statistical test examined average CBX2 intensity for each tumor (GSE1926; one-sided t-test p = 0.0391, F test p = 0.29). f Relative intensity of CBX2 in benign human ovarian surface epithelium (HOSE, n = 10) compared to HGSOC, n = 53 (GSE18521; t-test p < 0.0001, F test p < 0.0001). g Relative intensity of CBX2 in fallopian tube epithelium (FTE, n = 24) compared to HGSOC, n = 13 (GSE10971; two-sided t-test p < 0.0001, F test p < 0.0001). h Protein lysates generated from the primary tissue of FTE, HGSOC, and mixed histotypes. Protein utilized for immunoblot against CBX2. (beta-actin = loading control) i Densitometric analysis of immunoblots. Intensities were normalized between immunoblot by indicated (*) sample (FTE vs. HGSOC, one-sided Rank Sum p = 0.0333)

Fig 5: Inhibition of CBX2 impairs HGSOC cell proliferation.a Model describing basic protocol for establishing adherent, suspension, and spheroid growth environments. For adherent and suspension, two verified high grade serous ovarian carcinoma cell lines were initially grown on tissue culture plastic, then distributed to normal tissue culture dishes (adherent) and polyHEMA coated culture dish (suspension) growth environments. Distributed at 1:3 or 1:5 ratio to account for forced suspension induced cell death. Photographs show PEO1 cells after 7 days in suspension (left) and HGSOC cells directly derived from patient ascites. For spheroid formation, cells were grown in 3D in Matrigel for 12 days. A representative image of a resulting spheroid is shown at upper right. b Immunoblots against CBX2 protein from OVCAR4, and PEO1 cells grown in adherent and suspension settings (described in (a)) over 7 days. c RT-qPCR for CBX2 in OVCAR4 cells transduced with small hairpin RNA (shRNA) specific for CBX2. Representative mRNA expression of CBX2 in shControl, shCBX2#1, and shCBX2#2. Statistical test = ANOVA. d Immunoblots against CBX2 protein derived from OVCAR4 shControl, shCBX2#1, and shCBX2#2 transduced cells (beta-actin = loading control). e Proliferation assay of OVCAR4 cells with CBX2 knockdown and shControl, grown in adherent setting (tissue culture plastic) over 96 h, evaluated using gLuc activity. Statistical test = ANOVA. f Same as (e), crystal violet staining and subsequent measurement of absorbance at 590 nm. Images of representative of stained cells from shControl, shCBX2#1, and shCBX2#2. Statistical test = ANOVA. g Proliferation assay of OVCAR4 cells with CBX2 knockdown and shControl, grown in suspension setting (poly-HEMA coated tissue culture plastic) over 96 h, evaluated using gLuc activity. Statistical test = ANOVA. h Same as (g), but cell viability was assessed via MTT after 96 h. Statistical test = ANOVA. i OVCAR4 cell lines (shControl, shCBX2 #1 and #2) grown in 3D using Matrigel over 12 days, leading to spheroid growth. Representative images of transduced OVCAR4 cells below. Scale bar = 100 µm. Spheroids measured across horizontal diameter. Diameter mean calculated from measurements of 50 spheroids per cell type. Statistical test = ANOVA. j OVCAR4 cells with CBX2 knockdown and shControl grown in adherent or suspension. Cells were subjected to AnnexinV/PI apoptosis assay. Statistical test = two-sided t-test, F test p = 0.9291. Error bars = S.E.M.