Figure 5
Inhibition of HSPB1 expression by siRNA sensitizes to apoptosis in human podocytes. (a) HSPB1 siRNA downregulation of HSPB1. Representative western blot of the specific siRNA. (b) HSPB1 knockdown with HSPB1 siRNA increases high glucose-induced apoptosis and promotes Ang II-induced apoptosis. Cell death was assessed by flow cytometry of DNA content (hypodiploid cells) after culture in the presence of high glucose or Ang II (10−7 M) for 24 h. Mean±s.d. of three independent experiments. *P<0.05 vs control; **P<0.001 vs high glucose; #P<0.03 vs Ang II alone. (c) Representative western blot corresponding to the conditions of the experiment shown in b. (d) HSPB1 silencing increased apoptosis in human podocytes as assessed by morphology. Representative image of apoptotic cells with typical nuclei (shrunk, bright; DAPI staining) among cells transfected with HSPB1 siRNA (arrows). Anti-HSPB1 staining in green confirmed upregulation of HSPB1 expression by high glucose or Ang II and effective downregulation by siRNA. (e) Quantification of apoptotic cells expressed as percentage of cells with apoptotic nuclei as assessed by DAPI staining. Mean±s.d. for three independent experiments. *P<0.009 vs high glucose and Ang II alone; **P<0.0001 vs control; #P<0.04 vs control.
