Fig. 6: Treatment with MB augments Tubulin polymerization, induces G2/M arrest, negatively enriches translation pathways, inhibits nascent protein synthesis, and induces synergistic loss of viability with PLK1 inhibitor, Volasertib, in GMR-AML1 cells.

A GMR-AML1 cells were treated with the indicated concentration of MB for 16 h. Cells were fixed, permeabilized, and stained with β-Tubulin antibody. DNA were stained with DAPI. Cells were imaged by confocal microscopy. Original magnification is 60×. B GMR-AML1 cells were treated with the indicated concentration of MB for 24 h. Cells were fixed with 70% molecular grade ethanol, stained with propidium iodide, and cell cycle analysis was determined by flow cytometry. C GMR-AML1 cells were treated with the indicated concentrations of MB for 72 h. Then the percentage apoptotic cells were determined by Annexin V and TO-PRO-3 iodide staining and flow cytometry. D–F GMR-AML1 cells were treated with 1000 nM of MB for 16 h and bulk RNA-Seq analysis was performed. Gene set enrichment analysis (GSEA) in MB treated cells compared with HALLMARK and REACTOME pathway datasets. G GMR-AML1 cells were treated with 3000 nM MB for 16 h in biologic triplicates. Reverse phase protein array (RPPA) analysis was conducted. Log2 fold-changes in selected significantly altered proteins are shown. H GMR-AML1 cells were treated with the indicated concentrations of MB, Volasertib, or Alisertib for 16 h. Nascent polypeptide elongation was detected by OPP puromycin incorporation assay and flow cytometry. Column represents mean of six independent experiments; bar represents SEM. I GMR-AML1 cells were treated with the indicated concentrations of MB and/or Volasertib for 48 h. Then the percentage annexin V-positive apoptotic cells were determined by flow cytometry. Delta synergy scores (by ZIP method) were determined using SynergyFinder v3.0.