After three washes with phosphate-buffered saline (PBS), tissue samples were prepared into a suspension in radioimmunoprecipitation assay buffer (Biyuntian, Shanghai, China) using a tissue homogenizer, followed by centrifugation at 10,000×g for 20 min at 4 ˚C, and then the resulted supernatants were kept at -80 ˚C for further analysis. The concentration of protein was measured using the bicinchoninic acid protein assay (Biyuntian, Shanghai, China). All samples were adjusted to 40 μg total protein, then denatured in loading buffer [10% glycerol, 62 mM Tris, 0.003% bromophenol blue, 2% sodium dodecyl sulfate (SDS), pH 7.4], separated on an SDS–polyacrylamide gel (8%) at 100 V for 2 h, and subsequently transferred to polyvinylidene difluoride-plus membranes (60 min at 80 V; MSI, Westborough, MA, USA) using a semi-dry protein transfer system (UVP Inc., Upland, CA, USA). Bovine serum albumin (5%) or 5% non-fat dry milk in Tris-buffered saline (TBS; pH 7.4) with 0.2% Tween-20 (TBST) was used to block the membranes at room temperature for 1 h, which was subjected to three washes in TBST (10 min each), and subsequent overnight incubation at 4 ˚C with one of the following primary antibodies: anti-STAT3, anti-phospho-STAT3 (p-STAT3) (both 1:1000 dilution; Cell Signalling Technology, Danvers, MA, USA), anti-Bcl2, anti-Bax (both 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-type I collagen, and anti-α-SMA (both 1:1,000 dilution; Sigma, St. Louis, MO, USA). Next, the membranes were again rinsed in TBST for three times, followed by incubation with appropriate HRP-conjugated secondary antibody at room temperature for 2 h, followed by another three washes (10 min each) in TBST. Final visualization of bands was performed with the aid of an enhanced chemiluminescence kit (Amersham, NJ, USA). GAPDH (1:1000 dilution; Santa Cruz, CA, USA) was included as the loading control. The concentrations of protein were determined with an image analysis program (Bio-Rad, MD, USA).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
The quantity of cell nuclei with fragmented DNA, for instance apoptotic cells, was determined using the TUNEL assay kit (Roche Diagnostics, Mannheim, Germany). After deparaffinization and hydration, tissue sections were washed in PBS for three times (10 min each). Treatment with 0.3% hydrogen peroxide in methanol for 30 min was used to quench endogenous peroxidase, and 10 μg/ml proteinase-K was used to treat the sections for 15 min, which were then rinsed with PBS, followed by 1 h incubation with digoxigenin-dNTP and deoxynucleotidyl transferase. After termination of the reaction, the sections were rinsed with PBS again, then added with anti-digoxigenin antibody at ambient temperature and allowed to react for 30 min. After another 3 rinses with PBS, the sections were developed with 3,3′-diaminobenzidine, followed by counterstaining with hematoxylinn (10%). Apoptosis-positive nuclei were manually counted at ×400 magnification from ten random fields. The numbers of apoptotic cells in the interstitium as well as tubules were pooled. In every group, the apoptosis-positive nuclei distributed approximately evenly in the interstitial and tubular cells.
All results were presented as the means ± standard deviation (SD). Statistics was performed using the SPSS version 10.5 (Chicago, IL, USA). Sufficiency of animal group size was verified by Cohen’s d method . The means of data from all groups were divided by their standard deviation to calculate the standardized effect size, the largest of which was then referenced to Cohen’s d power table to determine minimum group size , compared to which our group size of 10 is sufficient. Comparisons of mean values were conducted using a one-way analysis of variance with Dunnett’s multiple comparison test. P values less than 0.05 were considered statistically significant.