Fig. 4.

Activating integrins increases pMLC and fibrillogenesis with hypoxia. (A) Immunoblots for activated and total β1 integrin in lysates from HK2 cells treated for 48 h with TGF-β, exposed to hypoxia or, as indicated, in the presence of 25 µM MnCl₂. (B) Quantification of the β1 integrin signals normalized to α-tubulin, calculated as the ratio of activated to total β1-integrin and expressed relative to normoxia controls in the absence of MnCl₂. Data are mean±s.e.m. of five independent cell preparations. (C) Immunoblots for pMLC-Thr18/Ser19 (pMLC) in lysates from HK2 cells treated for 48 h with TGF-β, exposed to hypoxia or, as indicated, in the presence of 25 µM MnCl₂. (D) Quantified pMLC signal normalized to α-tubulin and expressed relative to normoxia controls in the absence of MnCl₂. Data are mean±s.e.m. of three independent cell preparations. (E) Immunoblots for pFAK-Y397 and total FAK in lysates from HK2 cells treated as indicated and described in A and C. (F) Quantified pFAK signal normalized to α-tubulin and expressed relative to normoxia controls in the absence of MnCl₂. Data are mean±s.e.m. of three determinations for normoxia, TGF-β and hypoxia in the absence of MnCl₂ and averages of two determinations for normoxia and hypoxia with 25 µM MnCl₂. (G) HK2 cells with normoxia (controls) and upon hypoxia maintained in the absence or presence of 25 µM MnCl₂ for 48 h. Cells were labeled with antibodies for fibronectin (magenta) and stained for actin filaments with rhodamine (green). A magnified view on the boxed area is shown on the right. Scale bar: 20 µm.