Fig 2: Methylation status of actinin alpha 4 (ACTN4) in HPC cells and ACTN4 gene demethylation induced by EGCG. M: Methylation-specific PCR. U: Unmethylation-specific PCR. (A): Methylation status of ACTN4 in HPC cells. Line 1: Methylation status of ACTN4 in HPC without HG treatment. Line 2: Methylation status of ACTN4 in the diabetic nephropathy (DN) cell model. Line 3: Unmethylated positive control. Line 4: Methylated positive control. Line 5: Negative Control. (B): ACTN4 demethylation induced by EGCG.

Fig 3: Signaling pathway molecules associated with WAVE2 and ACTN4. A, Human phosphoprotein arrays showing the differential phosphorylation of proteins between WAVE2 rescue construct-transfected S2-013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA, and WAVE2 rescue construct-transfected S2-013 cells that had been transfected with WAVE2 siRNA alone. Data are representative of three independent experiments. B, Densitometric analysis of the results of A. The level of phosphorylated p27 in WAVE2 rescue construct-transfected S2-013 cells that had been transfected with WAVE2 siRNA alone was compared to that in WAVE2 rescue construct-transfected S2-013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA. Data are derived from three independent experiments. Columns, mean; bars, SD. *P < 0.01 compared with WAVE2 rescue construct-transfected S2-013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA (Student's t test).

Fig 4: Roles of WAVE2 in the translocation of ACTN4 to actin filaments in cell protrusions. A, Immunoprecipitation of WAVE2 from S2-013 cells cultured on fibronectin. Proteins within immunoprecipitates were examined on Western blots probed with antibodies against WAVE2, ACTN4, and actin. Rabbit IgG isotype control antibody was used as the control. B, Confocal immunofluorescence microscopic images. Oligonucleotides (siRNAs targeting WAVE2 (siWAVE2) or scrambled siRNAs (Scr) as the negative control) were transiently transfected into S2-013 cells. Transfected cells were incubated on fibronectin and were subsequently stained with anti-WAVE2 antibody (green), anti-ACTN4 antibody (red), and phalloidin (violet). Arrows, ACTN4 bound to peripheral actin structures in cell protrusions. Blue, DAPI staining. Scale bars, 10 µm. C, Confocal immunofluorescence microscopic images. A myc-tagged WAVE2 rescue construct was transfected into S2-013 cells that had been transfected with WAVE2 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti-myc antibody (green), anti-ACTN4 antibody (red), and phalloidin (violet). Arrows, exogenous WAVE2 localized in cell protrusions. Blue, DAPI staining. Scale bar, 10 µm. D, Oligonucleotides (siRNAs targeting ACTN4 (siACTN4) or Scr) were transiently transfected into S2-013 cells. Western blotting was performed using anti-ACTN4 antibody. E, Confocal immunofluorescence microscopic images. A myc-tagged WAVE2 rescue construct was transfected into S2-013 cells that had been transfected with both WAVE2 siRNA and ACTN4 siRNA; 48 h later, cells were incubated on fibronectin. Cells were stained with anti-myc antibody (green), anti-ACTN4 antibody (red), and phalloidin (violet). Blue, DAPI staining. Scale bars, 10 µm

Fig 5: Effects of WAVE2 and ACTN4 on p27 activity. A, Confocal immunofluorescence microscopic images. S2-013 and HPNE cells were cultured on fibronectin and then labeled with anti-WAVE2 (green) and anti-phosphorylated p27 (red) antibodies. Actin filaments were labeled with phalloidin (violet). Arrows, phosphorylated p27 in the nucleus. Blue, DAPI staining. Scale bar, 10 µm. B, S2-013 and HPNE cells were incubated on fibronectin and fractionated into cytosolic (c) and nuclear (n) fractions. Western blotting of the fractions was performed using anti-phosphorylated p27 and anti-p27 antibodies. C, Scr-transfected S2-013 cells and siWAVE2-transfected S2-013 cells were incubated on fibronectin. Western blotting was performed using anti-WAVE2, anti-phosphorylated p27, and anti-p27 antibodies. D, A myc-tagged WAVE2 rescue construct was transfected into S2-013 cells that had been transfected with scrambled control siRNA or WAVE2 siRNA with or without ACTN4 siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies