Additional file 2: of Preclinical validation of 3-phosphoinositide-dependent protein kinase 1 inhibition in pancreatic cancer

Figure S1. Effect of pharmacological inhibition of PDK1 on CFPAC-1 cells. CFPAC-1 cells were treated with different concentrations of PDK1 inhibitors and their effects on cell viability (a, b) and anchorage independent growth (c, d) were assessed. Data are expressed as percentage of control cells treated with DMSO and are means ± SEM of n ≥ 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control. Figure S2. Effect of pharmacological PDK1 inhibition on PANC-1 cells. PANC-1 cells were treated with different concentrations of PDK1 inhibitors and their effects on cell viability (a, b) and anchorage independent growth (c, d) were assessed. Data are expressed as percentage of control cells treated with DMSO and are means ± SEM of n ≥ 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs control. Figure S3. Representative images of 3D colonies of AsPC-1 and PANC-1 cells treated with PDK1 inhibitors. Images of AsPC-1 colonies treated with different concentrations of GSK2344470 (a) as well as PANC-1 colonies treated with MP7 (b) and GSK2344470 (c) were acquired using 4X magnification lens. (d) Images of the 6-well plates of PANC-1 colonies treated with GSK2344470 (left) and MP7 (right), as visualized by the ChemiDoc system (BioRad). Figure S4. Effect of pharmacologicalinhibition of PDK1 on HPAF-II cells anchorage–independent growth. HPAF-II cells were treated with the indicated concentrations of the PDK1 inhibitors GSK2334470, 2-O-Bn-InsP5 (a) and MP7 (b) and their effects on anchorage-independent growth were determined. Data are expressed as percentage of control cells treated with DMSO. Data in (a) are means ± SEM of n = 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01. Data in (b) are means of n = 2 independent experiments performed in duplicate. Figure S5. Effect of p110δ/γ and p110γ inhibition on PDAC cells. (a,b) AsPC-1 cells were treated with the indicated concentrations of the p110δ/γ inhibitor IPI-742 or the selective p110δ inhibitor CAL-101 (a). Alternatively, PDAC cells AsPC-1 and HPAF-II, together with the two non-malignant epithelial pancreatic cell lines hTERT-HPNE and HPDE, were treated with increasing concentrations of the selective p110γ inhibitor IPI-549 (b). Cell viability was assessed after 72 h. Data are expressed as percentage of cells treated with vehicle alone and are means ± SEM of n ≥ 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001 vs control. (c) AsPC-1 and PANC-1 were plated on soft agar and treated with the indicated concentrations of IPI-549. Data are expressed as percentage of cells treated with vehicle alone and are means ± SEM of n ≥ 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs corresponding control. (d) Representative images of the effect of different concentrations of IPI-549 on AsPC-1 cells colony formation (4X magnification lens). Figure S6. PDK1 inhibitors enhance the effect of p110δ/γ inhibitors. (a-c) AsPC-1 (a,b) and CFPAC-1 (c) cells were treated with the indicated inhibitors alone or in combination. Cell viability was assessed after 72 h. Data are expressed as percentage of cells treated with vehicle alone and are means ± SEM of n ≥ 3 independent experiments performed in duplicate. Statistical analysis was performed using GraphPad Prism version 6.0 and one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. (d,e) AsPC-1 (d) and CFPAC-1 (e) cells were plated on soft agar and treated with the indicated inhibitors and their combination. Data are from n = 2 independent experiments performed in duplicate. (ZIP 2460 kb)