Figure 2. TRIM28 knockdown does not affect breast cancer cell proliferation, cell viability and the percentage of CD44⁺/CD24^−/low breast cancer stem cell population in vitro.

A. 6 breast cancer cell lines (MDA-MB-231, HS-578T, BT-549, MDA-MB-468, T-47D and MCF-7) are characterized by different proportion of CD44-positive and CD24-positive cells as determined using FACS analysis. B. Identification of a CD44⁺/CD24^−/low subpopulation in breast cancer cell lines by flow cytometry. Relatively to MDA-MB-231 breast cancer cell line, MCF-7 and T-47D luminal breast cancer cell line and MDA-MB-468 basal-like breast cancer cell line are poor in the population of CD44⁺/CD24^−/low cancer stem cells. Error bars, SD; n = 3; ** p < 0.01; *** p < 0.001. C. The level of OCT3/4 pluripotency marker in selected breast cancer cell lines in vitro was determined using RT-qPCR analysis. Relatively to MDA-MB-231 breast cancer cell line, MCF-7 cells express OCT3/4 at the lowest level in vitro. Error bars, SD; n = 4; ** p < 0.01. D, E. MDA-MB-231 and MCF-7 cells stably infected with lentiviral vectors expressing TRIM28 shRNA (sh#1 or sh#2) or with empty vector (CTRL) were analyzed using RT-qPCR (A) and Western blot (B) for TRIM28 gene and protein levels. Error bars, SD; n = 3; *** p < 0.001. F, G. TRIM28 downregulation does not affect cell proliferation (F) and viability (G) in vitro as determined using an ³H-thymidine-incorporation assay and ATPlite™ Luminescence Assay, respectively. Error bars, SD; n = 4; p > 0.05. H. The comparison of CD44⁺/CD24^−/low cells frequency in breast cancer cell lines upon reduction of TRIM28 level in vitro. TRIM28 knockdown does not affect the percentage of breast cancer stem cell population in vitro. I. TRIM28 downregulation does not affect the expression of pluripotency markers OCT3/4, SOX2 and NANOG in vitro in MDA-MB-231 (upper panel) and MCF-7 (bottom panel) breast cancer cell lines as determined using RT-qPCR. Error bars, SD; n = 4; p > 0.05.