Fig. 6

MED12 regulated the balance of F-actin polymerization and depolymerization via LIMK2/cofilin pathway in NSCLC cells. a F-actin was detected using FITC-phalloidin. Representative micrographs were shown. Nucleus was counterstained by DAPI. Arrowheads indicated F-actin location abnormally. Scale bar: 10 μm. b Live-cell images displayed the arrangement of F-actin cytoskeleton. Red rectangle indicated disordered F-actin cytoskeleton of MED12 KO1 cells stably expressed pLifeAct-TagGFP2. Scale bar: 25 μm. c Live-cell images displayed the cytokinesis process of MED12 WT and MED12 KO1 of PC9 cells stably expressed pLifeAct-TagGFP2. MED12 KO1 cell was unable to pass abscission of intercellular bridge and eventually rejoined to one cell again. Arrowheads indicated F-actin abnormally located at the contractile ring and ellipses indicated the intracellular bridge. Scale bar: 25 μm. d Western blotting assay of G-actin and F-actin expression and quantification of the F-actin/G-actin ratio in WT, MED12 KO and MED12 reconstitution single clones of PC9 cells. *P < 0.05. e HE staining showed multinucleation in control and JPK-treated MED12 WT of PC9 cells. Arrowhead indicated multinucleated cell. Scale bar: 100 μm. *P < 0.05. f Western blotting analysis of G-actin and F-actin expression and quantification of the F-actin/G-actin ratio in control and JPK-treated MED12 WT of PC9 cells. *P < 0.05. g HE staining showed multinucleation in control and LatA-treated MED12 KO1 cells. Arrowhead indicated multinucleated cell. Scale bar: 100 μm. h Western blotting analysis of G-actin and F-actin expression and quantification of the F-actin/G-actin ratio in control and LatA-treated MED12 KO1 of PC9 cells. *P < 0.05. i Schematic of the function of LIMK2/cofilin in actin assembly. j Western blotting assay of LIMK2/cofilin pathway in WT, MED12 KO and MED12 reconstitution single clones of PC9 cells. k Map of the 1200 bp test promoter region relative to LIMK2 gene. l Dual-luciferase reporter analysis verified the targeting relationship between MED12 and LIMK2 in HEK-293FT and MED12 KO1 of PC9 cells. A series of deleted constructs containing the individual sequence D1- D4 were designed. Values are expressed as means ± SE of three replicates. *P < 0.05. m The schematic describes a proposed model for the role of MED12 in the regulation of actin dynamics and intracellular bridge abscission during cytokinesis, and that MED12 deletion resulted in aborted cytokinesis