Figure 3 | Cell Death & Disease

Figure 3

From: Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

Figure 3

Potential BRAFi resistance candidates. Silencing of FLI1 or EPHA2 re-sensitizes the resistant A375VR4 and SKMEL24 sublines to vemurafenib. (a) Protein expression levels of MET, FLI1, pEPHA2, EPHA2 and EFNA1 analyzed by immunoblotting in presence of 1 μM of vemurafenib or DMSO as control treatment for 24 h. β-actin was used as control. (b) Basal mRNA expression of FLI1 and EPHA2 in A375 and A375VR4 cell lines. (Error bars represent S.D. of the mean of three independent experiments). (c) Representative immunoblots showing downregulation of FLI1 and EPHA2 in A375VR4 and SKMEL24 after 48 h transfection with 100 nM siFLI1 or siEPHA2. (d) Silencing of FLI1 re-sensitized the A375VR4 cells to 5 μM of vemurafenib. Bar graph showing cytoxic effect as % of DMSO control measured by MTS colorimetric assay in A375 and A375VR4 cells transfected with siFLI1 or sicontrol in presence of vemurafenib (or DMSO). (Error bars represent S.D. of the mean of three independent experiments). (e) Silencing of FLI1 or EPHA2 in A375VR4 cells increased the number of apoptotic cells after 10 μM vemurafenib treatment. Bar graph showing percentage of apoptotic cells measured by annexin V-PI staining assay in A375VR4 cells transfected with siFLI1, siEPHA2 or sicontrol in presence of vemurafenib (or DMSO). (Error bars represent S.D. of the mean of three independent experiments). (f) Silencing of FLI1 or EPHA2 re-sensitized the SKMEL24 cells to vemurafenib. Bar graph showing percentage of apoptotic cells measured by annexin V staining assay in SKMEL24 transfected with siFLI1, siEPHA2 or sicontrol in presence of 10 μM vemurafenib (or DMSO). (Error bars represent S.D. of the mean of three independent experiments). (g) Silencing of FLI1 and EPHA2 with siRNA affects cell proliferation. Bar graph showing percentage of viable cells measured by MTS colorimetric assay in A375, A375VR4 and SKMEL24 cells transfected with siEPHA2, siFLI1 or sicontrol without any treatment. (Error bars represent S.D. of the mean of at least three independent experiments). Paired t-test was used to determine differences between groups and P<0.05 was considered significant. P<0.05 *; P<0.01 **; P<0.001 ***

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