Figure 6.
ILK inhibition and knockdown upregulates necroptotic signal in cultured renal tubular epithelial cells. (A, a) LLC-PK1 relative cell viability was reduced with the treatment of cpd22 in a dose-dependent manner as measured by MTT assay. Cpd22 (0.25–4 μM) was incubated with cells for 24 hours. *P<0.05, ***P<0.001 versus control (con). (b) ILK siRNA knockdown (ILK KD) or treatment with TNF-α or cpd22 (2 μM) caused cell loss as measured by DNA quantification assay in mCCDC11. ***P<0.001 versus control. (B) Increased plasma membrane accumulation of phospho-MLKL (p-MLKL, red) was clearly detected in mCCDC11 cells transfected with ILK siRNA. Similar membrane accumulation of phospho-MLKL was also present in cells treated with cpd22 or TNF-α as detected by immunofluorescence staining. Scale bar, 10 μm. (C) Immunofluorescence staining revealed that cpd22 treatment caused translocation of MLKL signals (red) from cytosol to the plasma membrane in LLC-PK1, compared with the control. Nuclei were stained blue with DAPI. Scale bar, 20 μm. (D) Representative immunoblotting showed the reduction of phosphorylated Akt (Ser 473, p-Akt) and upregulation of MLKL and RIPK3 in LLC-PK1 with cpd22 treatment for 24 hours (left panel). Bar graph represents the ratio of p-Akt over total Akt, and the ratio of MLKL and RIPK3 over β-actin expression as quantified by densitometry (right panel). *P<0.05, **P<0.01 versus control. (E) Nec-1 treatment of LLC-PK1 cells reversed the reduction of cell viability induced by cpd22. LLC-PK1 cells were treated with cpd22 (2 μM) in the presence or absence of Nec-1 (50 μM) for 24 hours. Relative cell viability was assessed by measuring the cellular DNA content. *P<0.05 versus control; ###P<0.001 versus 2 μM cpd22 treatment group. Data presented as mean±SEM. Experiments were repeated at least three times. Statistics were performed using one-way ANOVA.