Evaluation of TGF-β signalling activation in HG-treated tubular primary cell cultures.

(A) Representative western blot analysis of Phospho-Smad2 and Smad2/3 protein level in nuclear and cytoplasmic fractions obtained from primary tubular cells cultured in control (ctrl) and HG medium for 24h, 96h and 7 days. The Phospho-Smad2 level and the prevalent cytoplasmic localization of Smad2/3 proteins does not change between control and HG-treated cells at the different time points analysed. Nuclear and cytoplasmic protein fractions of control and HG-treated primary cultures have been obtained as described in Di Stefano et al. [25]. The absence of Tubulin protein band in nuclear fractions and of Histone-H3 protein band in cytoplasmic fractions ensured the quality of sub-fractions obtained. Total homogenate proteins from a Renal Cell Carcinoma cell line, treated with TGF-β1 and with TGF-β1+SB431542 as described [39], are used as positive (ctrl+) and negative (ctrl-) control of TGF-β1 signalling activation, respectively. To detect the specific protein bands, antibodies against Phospho-Ser 465/467 Smad2 (1:1000, Cell Signaling Technology), Total Smad2/3 (1:1000, Cell Signaling Technology), α-Tubulin (1:1000, Cell Signaling Technology) and Histone-H3 (1:4000, clone 96C10, Cell Signaling Technology) have been used. (B) PAI-1 transcript expression evaluated by Real-time PCR in primary tubular cell cultures at the indicated time points in control (ctrl) and high glucose (HG) conditions. The expression of PAI-1, a well known transcriptional target of TGF-β/Smad signalling [39], did not significantly increase in HG-treated primary tubular cells. The relative transcript amounts, calculated as 2−ΔΔCt, are represented as fold change with respect to the corresponding control sample considered equal to 1. (n = 3). Real-time quantitative PCR assays were carried out, as indicated in the Material and Method section, with the following TaqMan Gene Expression Assays: PAI-1 (Hs01126606_m1) and GAPDH (Hs99998805_m1) (Applied Biosystems) used as housekeeping gene. (TIF)