Triton x 100

Male C57BL/6 mice with average weights of 23–26 g were purchased from the Corporation of Lingchang Biological Technology (Shanghai, China). The animals were allowed to acclimate to their surroundings for 1 week. LPS was purchased from Sigma (USA). LFM-A13 was purchased from Topscience (Shanghai, China). Kits for protein extraction, BCA protein assay, SDS-PAGE gel preparation, Western blot analysis, hematoxylin and eosin (H&E staining), TUNEL apoptosis assay, mouse IL-4 enzyme-linked immunosorbent assay (ELISA), mouse IL-6 ELISA, mouse TNF-α ELISA, and myeloperoxidase (MPO) colorimetric activity assay were purchased from KeyGEN (Nanjing, China). Neutral gum, methanol, ethanol, and xylene were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Triton-X100 was purchased from KeyGEN (Nanjing, China). Rabbit anti-GAPDH (10B8) and goat antirabbit IgG-HRP were purchased from KeyGEN (Nanjing, China). Rabbit anti-p-BTK (ab52192) was purchased from Abcam (Shanghai, China). Rabbit anti-BTK (DF6472) was purchased from Affinity Biosciences (Cincinnati, USA).

The MSCs were seeded onto slides and cultured for 10 days. And then the MSCs were fixed with 4% paraformaldehyde for 30 min on ice, washed twice with PBS, and incubated with 3% H₂O₂-methanol solution at room temperature for 10 min. Micromass pellets were washed twice with PBS, fixed for 24 h in 10% formalin, embedded in paraffin, and cut into 5-μm thick sections. The latter were deparaffinized, rehydrated, and then washed with PBS. After a 5-min incubation with 0.5% Triton X-100 (KeyGEN), the cells/sections were blocked with 10% goat serum in PBS for 30 min and incubated overnight with anti-LC3B antibody (1:200; Novus Biological) at 4 °C. The slides were washed thrice with the blocking solution, incubated with fluorochrome-conjugated secondary antibody, and counterstained with diamidine phenylindole (DAPI; Molecular Probes, Waltham, MA, USA). The LC3 punctae were observed and counted under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany) [25 (link), 26 (link)].

When cell confluence reached approximately 80%, Cell-Light™ EdU fluorescence microscope detection kit (Keygen, Nanjing, China) was employed for BCPAP and TPC-1 cell proliferation detection in accordance with the manufacturer’s instructions. The cells were exposed to 50 μM EdU (5-ethynyl-2′-deoxyuridine; 100 μl/well, Syngene, Nanjing, China) for 2 h, fixed with PBS containing 4% paraformaldehyde (100 μl/well, Syngene, Nanjing, China) at room temperature for 15 min and incubated with 2 mg/ml glycine for 10 min. The cells were permeabilized with PBS comprising 0.5% Triton X-100 (100 μl/well) (Keygen, Nanjing, China) and stained with 100 μl of 1× Apollo dye liquor (Keygen, Nanjing, China) at room temperature for 30 min under conditions void of light. Incubation was continued following the addition of 100 μl of 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) staining solution at room temperature for 10–30 min in dark. After treating with DAPI, microscopic observation was performed under the guidance of a fluorescence microscope (Leica DM16000B, Germany). At least three fields were then selected from each well.

The EdU assay was conducted as described in our previous study. Briefly, cells were added to a 96-well plate. Ten hours later, EdU medium (RiboBio, Guangzhou, China, catalog No. C00003) was added to the plate and cultured for 2 h. After being fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.4%, KeyGen, Shanghai, China, catalog No. KGF001), cell nuclei were stained with Hoechst33342 (Beyotime, Shanghai, China, catalog No. C1002). Finally, a fluorescence microscope (Olympus, Tokyo, Japan) was used to observe the EdU-positive cells. EdU incorporation rate was expressed as the ratio of EdU-positive cells (red cells) to total Hoechst33342-positive cells (blue cells).

Cell proliferative ability was evaluated by EdU assay, which was performed with an EdU cell proliferation assay kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Cells were incubated with EdU solution for 2 h in an incubator containing 5% CO₂ at 37 °C and later fixed in paraformaldehyde (4%) for 15 min, then, cells were treated with Triton X-100 (0.4%, KeyGen, Shanghai, China) for 10 min and incubated with Apollo reagent for 30 min. Subsequently, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China) for 5 min. Finally, a fluorescence microscope was employed to capture the images.

The chemicals and reagents used were the standard substances kaempferol, quercetin, hesperetin, and hesperidin (National Institutes for Food and Drug Control), Cyclophosphamide (Baxter Oncology GmbH, Halle, Germany; No. 8K274A), Dehydroepiandrosterone (General Nutrition Corporation Pittsburgh, PA, USA; No. 61411H18), Rat FSH enzyme-linked immunosorbent assay (ELISA) kit (Cloud-Clone Corp. Wuhan, China; No. CEA830Ra), Rat AMH ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA228Ra), Rat GnRH ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA843Ra), Rat E₂ ELISA kit (Cloud-Clone Corp. Wuhan, China; No. CEA461Ge), TUNEL apoptosis assay kit (KeyGENBio TECH. Jiangsu, China; No. KGA 7072), 4′, 6-diamidino-2-phenylindole (DAPI) staining kit (KeyGENBio TECH. Jiangsu, China; No. KGA 215-10), Normal sheep serum (ZSGB-BIO. Beijing, China; No. ZLI-9022), Triton X-100 (KeyGENBio TECH. Jiangsu, China; No. KGF011), Caspase-1 rabbit polyclonal antibody (Proteintech Group, Inc. Chicago, USA; No. 22915-1-AP), GAPDH mouse monoclonal antibody (Proteintech Group, Inc. Chicago, USA; No. 60004-1-lg), Anti-GSDMD antibody (Abcam Cambridge, MA, USA; No. ab219800), Rat IL-18 ELISA kit (RayBiotech, Inc. Georgia, USA; No. P97636), and CoraLite594-conjugated donkey anti-rabbit IgG (H+L) (Proteintech Group, Inc. Chicago, USA; No. SA00013-8).

The SGC7901 cells were seeded onto 24-chamber culture slides and treated with AdMax-pDC315-DRAM-EGFP. Following fixation in methanol for 10 min, cells were blocked with a buffer containing 1% bovine serum albumin (BSA; Hangzhou Sijiqing Biological Engineering Material Co., Ltd.) and 0.1% Triton X-100 (Nanjing KeyGen Biotech., Co., Ltd., Nanjing, China) for 1 h. The cells were then incubated with the primary antibody against LC3 (diluted 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and PBS containing 1% BSA at 4°C overnight, and then incubated for 1 h with secondary ghost against rabbit cy3 fluorescence conjugated antibodies (1:500; Sigma) to visualize the binding sites of the primary antibody with laser confocal microscopy (Leica Microsystems Wetzlar GmbH).