Dlg5 depletion promotes JNK and p38 activation.

<p><b>A:</b> LLc-PK1 cells were stimulated with 4 ng/ml TGF-β and incubated for the indicated periods. Cells were then lysed and immunoblotted using the indicated antibodies. <b>B, C, D, E:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (KD) or control siRNA and incubated for two days. Cell lysates were then immunoblotted using the indicated antibodies, and the results were quantitated. The values represent the mean ± S.E. from at least three independent experiments. Dlg5 depletion promoted JNK and p38 activation but not Smad2/3 activation.</p

Dlg5 depletion disrupts epithelial cell morphology and induces the expression of mesenchymal marker proteins.

<p><b>A:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (siDlg5#1: KD) or control siRNA and then incubated with 4 ng/ml of TGF-β for three days. The cells were lysed and immunoblotted using the indicated antibodies. Expression of β-tubulin was examined as a loading control. The upper band observed in immunoblotting with anti-Dlg5 is the larger variant of Dlg5. <b>B:</b> Cells were treated as in A and incubated for 60 hours. The cells were then photographed using phase contrast microscopy to examine cell morphology. <b>C:</b> Cells were transfected with Dlg5 siRNA or control siRNA and then immunostained using anti-Dlg5 or anti-E-cadherin antibodies. The asterisks indicate the cells that were transfected with Dlg5 siRNA. The scale bar indicates 10 µm. <b>D:</b> LLc-PK1 cells were transfected with Dlg5 siRNA or control siRNA and incubated for 24 hours. A GFP expression plasmid was then transfected into the cells with FLAG-Dlg5 or control plasmids. After incubation for two days, the cells were immunostained using an anti-SMA antibody. Fifty GFP-expressing cells were randomly selected, and the fluorescent intensity of SMA staining was examined. The graph shows the ratio of cells with higher SMA expression than background. <b>E:</b> LLc-PK1 cells were transfected with FLAG-Dlg5 or control plasmid and incubated for six hours. The cells were further transfected with Dlg5 siRNA or control siRNA. After two days of incubation, the cells were lysed and immunoblotted using the indicated antibodies. Arrows indicate endogenously expressed Dlg5, and an arrow head indicates FLAG-Dlg5. Dlg5 depletion induced the expression of mesenchymal marker proteins, and the re-expression of Dlg5 suppressed it.</p

Inhibition of the TGF-β receptor suppresses the expression of mesenchymal marker proteins in Dlg5-depleted cells.

<p><b>A:</b> LLc-PK1 cells were transfected with Dlg5 siRNA (KD) or control siRNA and stimulated with or without 4 ng/ml TGF-β. Two days after the addition of 2 µM ALK5 inhibitor II (TGF-β receptor inhibitor), cells were lysed and immunoblotted using the indicated antibodies. <b>B:</b> LLc-PK1 cells were transfected with a Smad7 expression plasmid or control plasmid and incubated for six hours. The cells were then treated with or without TGF-β (left panel) or transfected with Dlg5 siRNA or control siRNA (right panel). Two days after further incubation, cells were lysed and the expression of the indicated proteins was investigated by immunoblotting. Expression of β-tubulin was detected as a loading control. The results are representative of at least three independent experiments. Both pharmacological and physiological inhibition of TGF-β receptor suppressed the expression of SMA and fibronectin induced in Dlg5-depleted cells.</p

Dominant-negative mutants of JNK and p38 suppress the expression of mesenchymal marker proteins in Dlg5-depleted cells.

<p><b>A:</b> Six hours after transfection of a dominant-negative JNK mutant or a control plasmid into LLc-PK1 cells, the cells were further transfected with control or Dlg5 siRNA. After incubation for two days, expression of the indicated proteins was investigated by immunoblotting. <b>B:</b> Dominant-negative p38 mutant or a control plasmid was transfected into LLc-PK1 cells and treated and analyzed as in A. Expression of β-tubulin was detected as a loading control. The results are representative of at least three independent experiments. The expression levels of dominant-negative mutants were affected by Dlg5 knockdown by an unknown mechanism. Dominant-negative mutants of JNK and p38 suppressed the expression of fibronectin and SMA.</p

TGF-β-mediated EMT decreases Dlg5 expression.

<p><b>A:</b> LLc-PK1 cells were incubated with 4 ng/ml of TGF-β for three days. Images were taken using phase contrast microscopy. TGF-β treatment induced morphological changes of LLc-PK1 cells. <b>B:</b> Three days after incubation with TGF-β, cells were lysed and immunoblotted using the indicated antibodies, which include an antibody for the epithelial marker E-cadherin, antibodies for the mesenchymal markers SMA and fibronectin, and antibodies for Dlg5 and β-catenin. As a loading control, vinculin expression was detected. <b>C:</b> LLc-PK1 cells were incubated with 4 ng/ml of TGF-β for three days. The cells were immunostained with anti-Dlg5 or anti-E-cadherin antibody. The scale bar indicates 10 µm. TGF-β treatment induced EMT and decreased Dlg5 expression. The results are representative of at least three independent experiments.</p