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. 2020 Nov 12;6(11):e05442. doi: 10.1016/j.heliyon.2020.e05442

Figure 3.

Figure 3

Determination of stem cell markers and gene expression in resistant and supraresistant cells compared to parental one. A) ALDH positive cells in cisplatin sensitive, resistant and supra resistant ovarian cancer cells. Cells were stained with ALDEFLUOR™ substrate represents a positive population, which is blocked by specific inhibitor DEAB. Data were expressed as mean Mean ± SD. ∗P < 0.01 between (i) A2780 andA2780-Cp or (ii) SKOV3 and SKOV3-Cp cells. B) Determination of side populations (SP) in cisplatin sensitive, resistant and supra resistant cells by dual-filter flow cytometry. Cells were stained with 2.5 μg/ml concentration of Hoechst dye 33342. The control cells were incubated in the presence of 50 μM verapamil in addition to Hoechst dye. Verapamil-sensitive, Hoechst-dye excluding cells were considered as side population. (i) A2780 and A2780-Cp cells, and (ii) SKOV3 and SKOV3-Cp cells. C) Sphere forming efficiency of SP and Non-SP (NSP) collected from A2780 and SKOV3 cell lines. Cells were allowed to grow in ultralow attachment, serum free culture condition in DMEM/F12 media supplemented with growth factors and counted after 2 weeks. The numbers of sphere with >50 μm in diameter obtained from 500-1000 cells were considered for the determination of efficiency. Means of 3 experiments ±SD are shown. ∗P < 0.01 between SKOV3 and SKOV3-Cp cells. D) mRNA expression of stemness related genes in cisplatin sensitive, resistant and supra resistant cells. Relative expression for POU5F1/OCT4, SOX2, NANOG, NES, BMI1, KLF4 and ALDH1A1 mRNA with respect to the housekeeping gene GAPDH presented as fold change. All samples were run in triplicate and relative gene expressions were determined by 2-ΔΔCt method with SYBR-green real-time PCR. (i) A2780 and A2780-Cp cells, and (ii) SKOV3 and SKOV3-Cp cells. Data were expressed as Mean ± SD. ∗P < 0.05 between A2780-A2780-Cp or SKOV3– SKOV3-Cp cells.