Journal: PLoS ONE

Article Title: Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

doi: 10.1371/journal.pone.0268635

Figure Lengend Snippet: (A) Heatmap of ATARiS gene sensitivity scores of two KRAS dependent cell lines (NCI-H358 and MDA-MB-231) and a BRAF dependent cell line (A375). Negative values (shaded red) indicate sensitivity of the cell line proliferation to the knockdown of shown genes, whereas positive scores (shaded blue) indicate insensitivity. (B) IC 50 values for indicated compounds in Ras pathway mutant cell lines NCI-H358, MDA-MB231 and A375 grown as 3D spheroids under serum free conditions. Compounds were tested either as single agent at a concentration range of 0.2 μM– 10 μM (calmidazolium), 0.6 μM– 40 μM (DT-061), 0.2 μM– 40 μM (trifluoperazine) and 0.6 μM– 40 μM (ARS-1620) or in combination, applying the whole concentration range of calmidazolium combined with 1 μM or 2 μM of DT-061, as indicated. Data represent mean values ± SD, n = 2. Statistical comparisons were done using the average values across all three cell lines. (C) Bliss synergism scores for combinatorial effects of calmidazolium and DT-061 in KRAS- or BRAF-mutant cancer cell lines. Data represent mean values ± SD, n = 2. Positive scores indicate synergistic drug interactions whereas negative scores would denote antagonistic drug interactions. A score of zero would indicate no antagonistic or synergistic effect. ( D ) Flow cytometric analysis of apoptosis in MDA-MB-231 cells. MDA-MB-231 cells were treated with DT-061 (10 μM) or calmidazolium (5 μM) or their combination for 24 h and then labelled with Annexin V-FITC and 7AAD. Benzethonium chloride and DMSO treated cells were used as positive and negative controls, respectively, for setting the gates. Percentage of total apoptotic cells (early and late stage) from two biological repeats (mean ± SD) is presented. Statistical analysis was performed using Fisher’s exact test comparing the total number of viable and apoptotic cells from two biological repeats. ( E ) Effect of inhibitors on cellular apoptosis studied using Caspase-3/7Glo assay. MDA-MB-231 cells were treated with DT-061 (5 μM), calmidazolium (5 μM), combination of DT-061 and calmidazolium (5 μM each), and staurosporine (1 μM) for 24 h. The caspase-3/7 activity was measured as a luminescence read-out and normalized to the vehicle control, 0.05% (v/v) DMSO. Data represent mean values ± SD, n = 2. ( F ) Western blot analysis of PARP-cleavage activity in MDA-MB-231 cells treated with DMSO (0.2% (v/v)), DT-061 (20 μM), calmidazolium (5 μM), combinations of DT-061 (10 μM) and calmidazolium (5 μM), and staurosporine (1 μM) for 24 h. GAPDH detection was used as loading control.

Article Snippet: The normalized viability data for the siRNA knockdown of each gene of interest were downloaded from the project DRIVE and a double gradient heatmap plot was generated using Prism (GraphPad).

Techniques: Knockdown, Mutagenesis, Concentration Assay, Activity Assay, Control, Western Blot