MakA inhibits tumour cell proliferation in a PIP5K1α-dependent manner.A MakA and PIP5K1α inhibitor, ISA-2011B synergistically inhibited HCT8 cell proliferation as quantified by colony forming assay. Data are representative of two independent experiments; bar graphs show mean ± s.d. Significance from replicates was determined using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (post-test) against vehicle (Veh). *p ≤ 0.05 and **p ≤ 0.01. B Western blot analyses of cyclin D1 and pAkt levels in HCT8 cells upon 48 h treatment with MakA (500 nM), ISA-2011B (25 μM) or a combination of MakA (500 nM) and ISA-2011B (25 μM). β-actin was used as a loading control. C Histograms showing the normalized quantification of cyclin D1 and pAkt relative to total actin. Data are representative of at least two independent experiments; bar graphs show mean ± s.d. Significance from replicates was determined using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (post-test) against vehicle (Veh). *p ≤ 0.05 and **p ≤ 0.01. D Confocal microscopy analysis of vehicle (Veh), MakA (500 nM), ISA-2011B (25 μM), or a combination of MakA (500 nM) and ISA-2011B (25 μM) treated HCT8 cells (48 h treatment). Cells were stained with cell proliferation marker, Ki67 (green). Nuclei were counterstained with DAPI (blue). Inhibition of HCT8 cell proliferation was quantified by an increase in the number of Ki67-negative stained cells from four individual microscopy fields of view (65-165 cells per field of view). *p ≤ 0.05 and **p ≤ 0.01. Scale bar = 20 μm.

Effect of MakA in HCT8 cells with PIP5K1α overexpression and schematic summaries of the MakA effects on PIP5K1α-mediated signalling.A HCT8 cells were transfected with EGFP-vector control (upper panel) or PIP5K1α-EGFP (lower panel) plasmids as described in “Materials and methods”. For co-localization experiments of MakA and PIP5K1α, the transfected HCT8 cells were subsequently treated with Alexa568MakA (250 nM, red) for 48 h. Nuclei were counterstained with DAPI (blue). Graphs on the right show fluorescence intensity profiles of the corresponding images along the dotted white lines. Pearson correlation co-efficient was used for calculation of Alexa568MakA (red) co-localization with EGFP or PIP5K1α-EGFP (green) along the cell membrane. Scale bars = 10 μm. B Western blot analysis of PIP5K1α levels in EGFP-vector control or PIP5K1α-EGFP HCT8 total cell lysates. C Confocal microscopy analysis of vehicle (Veh) or MakA (250 nM) treated (48 h treatment) HCT8 cells transfected with EGFP-vector control or PIP5K1α-EGFP plasmids. Cells were stained with cell proliferation marker, Ki67 (green). Nuclei were counterstained with DAPI (blue). Effects on HCT8 cell proliferation was assessed by quantifying the number of Ki67-negative cells from eleven individual microscopy fields of view (65–200 cells per field of view). Significance was determined using an unpaired t test, ns non-significant. D Schematic representations showing (i) the downstream signalling of PIP5K1α during tumour cell proliferation and survival, (ii) the effect of MakA and ISA-2011B inhibition.

MakA induces G2/M cell cycle arrest in HCT8 cells and alters the expression of cell cycle regulatory proteins.A Western blot analysis shows levels of E2F1, CDK1, CDK2, cyclin D1, and cyclin A in HCT8 cells treated with increasing concentrations of MakA (24 h). B, C Histograms indicate the normalized quantification of E2F1, CDK1, CDK2, cyclin D1, and cyclin A1 relative to total actin. Data is representative of at least two independent experiments and is expressed compared to cells treated with vehicle (20 mM Tris-HCl); bar graphs show mean ± s.d. Significance was determined using a one-way analysis of variance (ANOVA) followed by Dunnett’s post-test in comparison to the vehicle control *p ≤ 0.05 and **p ≤ 0.01. D Cell cycle distribution flow cytometric analysis of HCT8 cells treated with MakA protein (500 nM, 24 h). E Histogram showing the distribution of HCT8 cells during each phase of the cell cycle after treatment with vehicle or MakA. Data are representative of three independent experiments. *p ≤ 0.05 and **p ≤ 0.01.