Figure 4. The SRI31277 peptide overcomes resistance to vemurafenib plus sorafenib therapy in PTC-derived cells harboring the heterozygous BRAF^V600E mutation.

A) Western blot analysis of proteins expression levels in pericytes at 5 hrs treatment with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib and combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide that blocks TSP-1/TGFβ1 activation (cells were grown in 0.2% FBS DMEM growth medium during treatment). These results were validated by two measurements. B) Measurements of secreted TSP-1 and TGFβ1 total protein levels in pericytes treated for 48 hrs with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib (cells were grown in 0.2% FBS DMEM growth medium during treatment) in the presence or absence of the SRI31277 peptide (10 μM). The secretome (0.2% FBS DMEM growth medium enriched by cell-derived secreted protein factors) was collected and protein levels (ng/mL or pg/mL) were determined by ELISA (enzyme-linked immunosorbent assay). Secreted protein levels were normalized to cell growth medium (DMEM supplemented with 0.2% FBS) which was measured to determine subtracted background. These data represent the average ± standard deviation (error bars) of 2 independent replicate measurements (*p<0.05, **p<0.01). C) Western blot analysis of proteins expression levels in BRAF^WT/V600E-KTC1 cells at 5 hrs direct treatment with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide; or 5 hrs treatments with pericyte-derived secretome treated with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib as shown in A in the presence or absence of the SRI31277 peptide. Cells were grown in 0.2% FBS DMEM growth medium during treatment. These results were validated by two independent replicate measurements. D) Measurements of secreted TSP-1 and TGFβ1 total protein levels in both BRAF^V600E-KTC1 cells and co-culture with BRAF^WT/V600E-KTC1^mCherry cells and pericytes treated for 48 hrs with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide (10 μM); cells were grown in 0.2% FBS DMEM growth medium during treatment. The secretome (0.2% FBS DMEM cell growth medium enriched by cell-derived secreted protein factors) was collected and protein levels (ng/mL or pg/mL) were determined by ELISA (enzyme-linked immunosorbent assay). Secreted protein levels were normalized to cell growth medium (DMEM supplemented with 0.2% FBS) that was measured to determine subtracted background. These data represent the average ± standard deviation (error bars) of 2 independent replicate measurements (*p<0.05, **p<0.01, ***p<0.001). E) Fluorescence imaging for fixed BRAF^WT/V600E-KTC1^mCherry cells (highlighted by mCherry and Hoechst staining, with red and white signal, respectively) treated for 48 hrs with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide (10 μM); cells were grown in 0.2% FBS DMEM growth medium during treatment. Quantification of only BRAF^WT/V600E-KTC1^mCherry cells number is reported in the histogram. Fluorescence imaging for fixed co-culture with BRAF^WT/V600E-KTC1^mCherry cells (highlighted by mCherry and Hoechst staining, with red and white signal, respectively) and pericytes (highlighted by Hoechst staining, white signal) treated for 48 hrs with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide (10 μM); cells were grown in 0.2% FBS DMEM growth medium during treatment. Quantification of only BRAF^WT/V600E-KTC1^mCherry cells number is reported in the histogram. These data represent the average ± standard deviation (error bars) of three independent replicate measurements (*p<0.05, **p<0.01). F) Fluorescence imaging for fixed co-culture with BRAF^WT/V600E-KTC1^mCherry cells (highlighted by mCherry and Hoechst staining, with red and white signal, respectively) and pericytes (highlighted by Hoechst staining, white signal) treated for 48 hrs with DMSO (vehicle), 10 μM vemurafenib, 2.5 μM sorafenib, or combined therapy with 10 μM vemurafenib plus 2.5 μM sorafenib in the presence or absence of the SRI31277 peptide (10 μM); cells were grown in 0.2% FBS DMEM growth medium during treatment. Cell coculture was stimulated every 8 hrs for 48 hrs with recombinant human active TGFβ1 (7.75 ng/500μL in 24 well dishes) and compared to cell coculture without recombinant human active TGFβ1 as a control. Quantification of only BRAF^WT/V600E-KTC1^mCherry cells numbers are reported in the histogram. These data represent the average ± standard deviation (error bars) of three replicate measurements (**p<0.01).