Nuclear and cytoplasmic extraction reagents

Human gastric adenocarcinoma SGC‐7901 cells (the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were grown in DMEM containing penicillin and streptomycin (each 100 mg/L) and supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. ATPR was synthesized by the School of Pharmacy, Anhui Medical University, with a purity of 99%. ATPR was prepared as a stock solution of 10⁻² mol/L in dehydrated alcohol and kept at −20°C. Cells were treated with ATPR (10⁻⁵ mol/L final concentration, the ATPR group) for 48 hours and then collected. The vehicle group (0.1% alcohol diluted with DMEM) was set up, and the cells were treated under the same conditions. Cell nuclear and cytoplasmic proteins were extracted using nuclear and cytoplasmic extraction reagents (KeyGEN BioTECH, Nanjing, China).

Kidney tissue or NRK-52E cells were ground and homogenized with a protein lysis solution (KeyGen BioTech, China). Nuclear and cytoplasmic proteins were extracted by using nuclear and cytoplasmic extraction reagents according to the manufacture’s instructions (Keygen Biotech. Co., LTD, Nanjing, China). The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% non-fat milk for two hours at room temperature, followed by overnight incubation with the rabbit polyclonal anti-TLR4 antibody (1:2,000, Santa Cruz Biotechnology, USA), the rabbit polyclonal anti-nuclear factor-kappa B (NF-kB) p65 antibody (1:500, Santa Cruz, USA), the rabbit polyclonal anti-Histone H3A polyclonal antibody (1:500, Santa Cruz, USA) and the rabbit polyclonal anti-β-actin (1:500, Santa Cruz, USA). The membrane was then washed and incubated with a secondary antibody (goat anti-rabbit IgG, HRP-linked antibody 1:4,000, Cell Signaling Biotechnology, USA) for two hours. Blotted protein bands were visualized by enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham, USA). Protein density measurement was correlated to protein expressions, and normalized with respect to Histone H2A or β-actin band density.

Total protein was extracted with lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail. Cytoplasmic/nuclear proteins were extracted with nuclear and cytoplasmic extraction reagents (KeygenBiotech, Nanjing, China). Proteins were quantified with Coomassie Brilliant Blue, and BSA served as the standard. Proteins were separated by SDS-PAGE on a 10% gel and transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with primary antibodies for SEMA3A, NRP1, active-caspase-3, Plexin A1, A2, A3, A4, D1 (Abcam, USA), P65, p-P65, IκB, p-IκB, CDK2/4/6, cyclin E1, cyclin D1/D3, P21, P27, caspase-7 (CST, USA), E-cadherin, N-cadherin, Vimentin, β-catenin, β-actin, H1, cleaved-caspase-5, and SNAI2 (Bioworld, China). All antibodies were used at a 1:1000 dilution. The membranes were incubated with anti-goat IgG HRP-conjugated secondary antibodies (ZhongshanGoldenbridge Bio) for 1 h at room temperature. Immunoreactive bands were detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using an ImageQuantLAS 4000 mini imaging system (General Electric). Analyses of the bands were performed using ImageJ software.