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. 2021 Feb 9;10:584024. doi: 10.3389/fonc.2020.584024

Figure 5.

Figure 5

Upregulation of VCX2 expression by epigenetic treatment in breast cancer and melanoma cells. (A) VCX family gene expression was investigated by RNA sequencing in MDA-MB-231 breast cancer cells after treatment with either vehicle (black bars) or guadecitabine (white bars) for 96 h. All VCX genes were significantly upregulated by treatment with guadecitabine. The experiment was performed with three biological replicates. (B) The effect of combining DNA methyltransferase inhibitors and histone deacetylase inhibitors were investigated using guadecitabine and valproic acid. MDA-MB-231 cells were treated with increasing concentrations of guadecitabine (0.3 to 10 μM) with or without 1 mM valproic acid for 96 h and VCX2 protein expression was subsequently evaluated using immunocytochemical staining. The experiment was performed with two biological replicates. A representative replicate is shown. (C) Quantification of cells from (B). (D) MCF7 breast cancer cells were treated with 0.5 mM of guadecitabine with or without 1 mM valproic acid (VPA) for 96 h and VCX2 protein expression was subsequently evaluated using immunocytochemical staining. The experiment was performed with two biological replicates. A representative replicate is shown. Approximate frequency of positive cells is shown in bottom left corner. (E) PDX tumors from a metastatic TNBC patient were established in NOG mice and when tumors reached a size of 2–3 mm, the mice were randomized into treatment groups based on tumor size. The mice were treated with either 24.4 mg/kg guadecitabine or vehicle every fifth day for a total of four times and VCX2 expression was subsequently evaluated by immunohistochemistry. The experiment was performed with three biological replicates. A representative replicate is shown. Approximate frequency of positive cells is shown in bottom left corner. Scale bars = 50 µM. (F) A375 and MZ2-MEL melanoma cells were treated with 0.5 mM of guadecitabine (gua) with or without 1 mM valproic acid (VPA) for 96 h and VCX2 protein expression was subsequently evaluated using immunocytochemical staining. Representative pictures are shown. Approximate frequency of positive cells is shown in bottom left corner. Scale bars = 50 µM.