Data Supplement from Epigenetic Targeting of Ovarian Cancer Stem Cells
Authors: Wang Yinu , Cardenas Horacio , Fang Fang , Condello Salvatore , Taverna Pietro , Segar Matthew , Liu Yunlong , Nephew Kenneth P. , Matei Daniela 
Published: March 2023

<p>Supplemental Figure S1. Low dose SGI-110 induces sustained cell cycle arrest on OC cells. A. Time-course cell cycle analysis of platinum sensitive A2780 ovarian cancer cells and platinum resistant A2780_CR5 and SKOV3 OC cells after 72 hour daily treatment with low dose SGI-110 (100nM). Mean values {plus minus} SD of three independent experiments are shown (*: P<0.05, **: P<0.01, ***: P<0.001). Supplemental Figure S2. Low dose SGI-110 treatment has no impact on ALDH(+) OC cells growth rate. 1x 104 ALDH(+) cells were directly sorted from DMSO or 3-day 3-day SGI-110 (100nM) pretreated A2780_CR5 ovarian cancer cells and plated into 15cm plates. The average number of cells was counted and calculated on the Day 4, 5, 6 and 7 after ALDH(+) cells were cultured. Mean values {plus minus} SD of three independent experiments are shown (*: P<0.05, **: P<0.01, ***: P<0.001). Supplemental Figure S3. Low dose SGI-110 inhibits sphere formation by ovarian CSCs. Representative images of tumor spheres (10X) formed from 500 ALDH(+) cells (derived A2780-CR5) that were treated with CDDP (1.67μM) or SGI-110 (100nM) alone or in combination. The number of sphere was counted on 14 days. Scale bar is 200μM. Supplemental Figure S4. Low dose SGI-110 inhibits ovarian CSCs self-renewal. Representative images (5X, 10X, 20X) of tumor spheres formed from 500 untreated and SGI-110 (100nM) treated ALDH(+)/ALDH(-) cells isolated from A2780_CR5. The number of spheres was counted on day 14. Scale bar is 200μM. Supplemental Figure S5. Low dose SGI-110 blunts sphere formation over passages. Average number of spheres formed in the serial passaged assay. ALDH (+)/(-) (10,000 cells) were treated with SGI-110 (100μM) or control (DMSO) for 3 days and allowed 4 days of recovery. Then cells were trypsinized and passaged every 7 days. The average number of spheres formed by 500 ALDH(+)/(-) cells in passage 2 and 3. Mean values {plus minus} SD of three independent experiments performed in triplicate are shown (*: P<0.05, **: P<0.01, ***: P<0.001). Supplemental Figure S6. Low dose SGI-110 induces differentiation of ovarian CSCs. Representative images of ALDH(+) cell differentiation assay. ALDH(+) derived from A2780_CR5 were treated with SGI-110 (100nM) or control (DMSO) and cultured in RPMI or DMEM for 7, 14, 21 , 28 and 42 days. The average percentage of ALDH(+) cells are shown. (B) The quantification of ALDH (%) in the cell differentiation assay. Mean values {plus minus} SD of three independent experiments in triplicate are reported. Supplemental Figure S7. ALDH isoforms are overexpressed in platinum-resistant A2780 OC cells. RNA-Seq analysis demonstrated overexpression of ALDH1A1, ALDH1B1, ALDH3A2, ALDH3B1, ALDH5A1, ALDH6A1, ALDH7A1 and ALDH18A in A2780_CR5 compared with parental (platinum-sensitive) A2780. Supplemental Figure S8. SGI-110 decreases expression of ALDH1A1 and DNMT isoforms in OC. (A) Average expression of DNMT3A and DNMT3B (B) were measured by qRT-PCR in A2780_CR5-derived ALDH(+) cells on the Day 4, 7 and 14 after the initial 3 days-SGI-110 (100nM) treatment compared with untreated ALDH(+) cells. (C) Protein level of ALDH1A1 and DNMT1 in (C) platinum-sensitive A2780 cells, (D) platinum-resistant A2780 cells and (E) SKOV3 cells were measured by western blot analysis. Three independent experiments were performed and mean values {plus minus} SD are calculated (*: P<0.05, **: P<0.01, ***: P<0.001).</p>