Gadd45g

Pancreatic tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned in 3-μm slices. Sections were stained with H&E and Masson’s trichrome to detect the interstitial volume expansion. Antigen retrieval was performed in sodium citrate buffer at 96°C. Sections were washed with PBS three times, and endogenous peroxidase activity was blocked by incubating sections in a solution of 3% H₂O₂ for 5 min. Sections were then blocked with 10% normal goat serum. The primary antibodies used were α-SMA (ab5694, Abcam), Desmin (A3736, Abclonal), GFAP (A19058, Abclonal), KRT19 (A19040, Abclonal), Gadd45g (A10286, Abclonal), and Tsc1 (BA2879, BOSTER). Incubation with the primary antibody was performed at 4°C overnight. Tissue sections were developed using the Ultra Vision Detection System (Thermo Fisher Scientific).

For western blotting analyses, pancreatic tissues or cell lines were lysed with RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitor (Thermo Fisher Scientific). Proteins were then subjected to SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked with 5% nonfat dry milk for 1 h, and incubated with the primary antibodies at 4°C overnight. The primary antibodies used were Tsc1 (BA2879, BOSTER), phospho-mTOR-S2448 (381557, ZENBIO), Fibronectin (ab2413, Abcam), α-SMA (ab5694, Abcam), Gadd45g (A10286, Abclonal), Stat3 (9139, CST), p-Stat3-Tyr705 (9145, CST), β-actin (AC026, Abclonal). Following washes with TBST buffer, membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody for 1 h at room temperature. Protein immunoreactivity was visualized using the SuperSignal West Pico Chemiluminescent kit (Thermo Fisher Scientific). Pictures were scanned by automatic chemiluminescence imaging using the analysis system Tanon5200, and were acquired using PVCAM software (Tanon, China). All experiments were performed in triplicate.