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. 2022 Feb 27;14(5):1230. doi: 10.3390/cancers14051230

Figure 1.

Figure 1

The down-modulation of the heme synthesis-export system, obtained by FLVCR1a silencing, sensitizes colorectal cancer cells to metformin. (A)Western blot analysis of FLVCR1a and ALAS1 expression in SKCO1 cells. Vinculin and beta-actin expression are shown as a loading control, respectively. Cells in which the expression of FLVCR1a was down-regulated using a specific shRNA are compared to cells expressing a scramble shRNA. A representative blot is shown. For statistical analyses, a Student t-test was used. Densitometry shows mean ± SEM, n = 3 (FLVCR1a blot) and 2 (ALAS1 blot) biological replicates, ** p < 0.01, **** p < 0.0001. (B,C) Cell viability of SKCO1 cells untreated (NT) or treated with 7 uM 5-FU, 5 uM Oxaliplatin and 50 nM SN38 (B) or with 2.5 mM metformin (C), measured after two days (5-FU, Oxaliplatin and SN38) or two and three days (metformin) of drug exposure without medium refresh. Cells in which FLVCR1a expression was down-modulated using a specific shRNA are compared to cells expressing a scramble shRNA. Cell viability is measured as a fold increase over an untreated calibrator sample and expressed relative to day 0. For statistical analyses a two-way analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons. Values represent mean ± SEM, n = 4 biological replicates, *** p < 0.001, **** p < 0.0001. (D) Cell viability of CACO2 cells was assessed by crystal violet staining on the indicated days. Cells in which the expression of FLVCR1a was down-regulated using a specific shRNA are compared to cells expressing a scramble shRNA; 5 mM metformin administration occurred at day 1 of cell proliferation and medium was maintained without refresh till the end of the experiment. Staining quantification is reported in the graph as the percentage of crystal violet stained area versus image total area. For statistical analyses, a two-way analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons. Values represent mean ± SEM, n = 3 biological replicates, **** = p < 0.0001.