Proliferating cell nuclear antigen (pcna)

IHC staining of tumors was performed as described previously [13 (link)]. Primary antibodies were HK2 (Proteintech #22029-1-AP, China), HK2 (Proteintech #66974-1-Ig, China), AIMP2 (Proteintech #10424-1-AP, China), Ki-67 (Cell Signaling Technology #9449, America), Cyto c (Proteintech #10993-1-AP, America), PCNA (Abclonal #a0264, China), LC3 II (Proteintech #18725-1-AP, China), CD8 (Abcam #ab217344), CD163 (Proteintech #16646-1-AP, China), CD206 (Cell Signaling Technology #24595, American). Staining intensity was independently evaluated by two senior pathologists. The immunohistochemical score was calculated based on the positive reaction area and the intensity of staining. Each group quantified at least five different views.

Lung tissues and PASMCs were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (containing 1% PMSF) on ice to extract total proteins. Nuclear and cytoplasmic proteins were extracted following the manufacturer's recommendations of the nuclear and cytoplasmic extraction kit (Beyotime). After protein quantification, the same amount of protein from each sample were separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and then the proteins on the gel were transferred to PVDF membranes (Millipore) by semi‐dry electrophoretic transfer system (Bio‐rad). The membranes were then blocked with 5% BSA and incubated with primary antibodies against calpain‐1 (1:1000, proteintech), HIF‐1α (1:1500, ABclonal), P65 (1:2000, proteintech), β‐actin (1:5000, proteintech), Lamin B (1:10000, proteintech), VEGF (1:1000, ABclonal), TGF‐β1 (1:800, Wanleibio), PCNA (1:1500, ABclonal), MMP2 (1:1000, ABclonal) and collagen I (1:1000, Wanleibio) overnight at 4°C. On the second day, membranes were incubated with HRP‐conjugated secondary antibodies (1:10000, ABclonal). The immune response bands were visualized with a chemiluminescence reagents (Biosharp).

After incubation with α-smooth muscle actin (α-SMA, 1 : 100; Bosterbio, Wuhan, China), proliferating cell nuclear antigen (PCNA, 1 : 100; Abclonal, Wuhan, China), and collagen I and collagen III (1 : 100; Bosterbio, Wuhan, China) antibodies overnight at 4°C, skin fibroblasts were incubated with Cy3- or Alexa Fluor 488-conjugated IgG (1 : 500; Beyotime, Shanghai, China) for 2 h in the dark. Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China). The fluorescence was observed and photographed with a laser confocal microscope (Leica, Wetzlar, Germany). The fluorescence intensity was evaluated with the ImageJ software and expressed as the fold of that in control group.

Total protein was extracted using RIPA lysis buffer from Yeasen (China). Cytoplasmic and nuclear protein extraction was carried out using the Nuclear and Cytoplasmic Protein Extraction Kit (Yeasen, China). ACTIN and LaminA/C were used as internal controls for cytoplasmic and nuclear proteins, respectively. Primary antibodies used in this study were, c-Myc(ab32072, Abcam, USA), PCNA (A0264, ABclonal, China), SRSF1 (sc-33652, Santa Cruz Biotechnology, China), β-catenin (51067-2-AP, Proteintech, USA), cyclin D1(ab134175, Abcam, USA), LaminA/C (A0249, ABclonal, China), and ACTIN (AC026, ABclonal, China).

In our study, 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS solution), 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N,N′,N′-tetramethylethylenediamine (TEMED), acrylamide/bisacrylamide, ammonium persulfate (APS), ascorbic acid (vitamin C), bovine serum albumin (BSA), ethanol (96%), glycine, methanol, Ponceau S, resazurin sodium salt (RES), sodium dodecyl sulfate (SDS), Trolox, trypsin-EDTA solution, Tris-HCl, Tris-Base, and Tween-20 were purchased from Merck KGaA (Darmstadt, Germany). The PVDF membrane with 0.45 µm-pore size and primary antibodies against SOD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary antibodies against GAPDH, NF-κB, IκBα, SRC, GLUT4, PCNA, PGC-1α, PPARγ, p-ERK1/2, and ERK1/2 were purchased from ABClonal (Woburn, MA, USA). The primary antibodies against NLRP3, anti-mouse- and anti-rabbit-HRP-conjugated antibodies were purchased from ThermoFisher (Waltham, MA, USA). Potassium persulfate (Warchem, Zakręt, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), DMEM (Dulbecco’s Modification of Eagle’s Medium, Biological Industries, Beit Haemek, Israel), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), and phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Ślaskie, Poland) were also used during the analyses.

OC cells lysis with PMSF containing lysis buffer (Beyotime, China) and then centrifugation at 10,000 g for 5 min at 4 °C was done to collect the supernatant. A BCA Protein Assay Kit (Beyotime) was employed for protein quantification. The protein separation was performed via SDS-PAGE (Beyotime, China), followed by PVDF membranes (Millipore, MA, USA) transfer and blocking for 1 h with non-fat milk. Primary antibodies were incubated with membranes at 4 °C overnight. After TBST washing, secondary antibodies (1:5000) were incubated with membranes for 45 min at 37 °C, then washed with TBST. ECL reagents (Beyotime, China) were used for blot visualization. Primary antibodies included OSR1, PCNA, and cyclin D1 from ABclonal, China; caspase-3, cleaved-caspase-3, Bcl-2, and Bax from CST, USA; p-p65, p65, p-IκBα, and IκBα from Wanleibio, China. Secondary antibodies used were goat anti-rabbit IgG and goat anti-mouse IgG from Beyotime.