Txnip

The Western-blot assays described in reference with PMID 31839825¹⁴. HPV16 E6 (1:200, Bioss Biotechnology Co., Ltd, Beijing, China), HPV16 E7 (1:200, Bioss Biotechnology), PTEN (total protein, 1:500, Wanleibio Co., Ltd, Shenyang, China), p-PTEN-S380 (serine 380 phosphorylated protein, 1:1000, Zenbio Co., Ltd., Chengdu, China), TXNIP (1:500; Proteintech Co., Ltd, Wuhan, China), HIF-1α (1:1000; Wanleibio), GLUT1 (1:500; Wanleibio), and GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA). Membranes were further incubated with peroxidase-coupled anti-mouse or anti-rabbit IgG (1:5000; Proteintech) at 37°C for 2 h. Bound proteins were visualized using the ECL Western blot kit (advansta, USA), and their densities were measured using a BioImaging systems (UVP Inc., Upland, CA, USA). Protein bands were visualized using electrochemiluminescence substrate (Pierce) and detected by using BioImaging Systems (DNR, Jerusalem, Israel). GAPDH protein levels were used as the control group to calculate relative protein levels.

DHT (purity ≥ 98%) was purchased from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, China). MTS, N-Acetyl-L-cysteine (NAC), Ca²⁺ probe (Fluo-3/AM), and ROS probe (DCFH₂-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). z-vad-fmk and vx765 were purchased from Selleck Chemicals (Houston, TX, USA). COX-2, TOM20, and GAPDH were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Porimin was purchased from Bioss (Beijing, China). TXNIP was purchased from Proteintech (Chicago, IL, USA). UCP2 was purchased from Affinit (Cincinnati, OH, USA). β-tubulin was purchased from Abmart (Shanghai, China). LDH assay kit and 10% sodium azide were purchased from Beyotime Biotechnology (Shanghai, China). Mitochondrial membrane potential assay kit (JC-1) was acquired from Thermo Fisher Scientific (Waltham, MA, USA). BCA protein assay kit, PVDF membranes, and qRT-PCR kit were bought from Thermo Fisher (Waltham, MA, USA). Annexin V-EGFP apoptosis detection kit was bought from Shanghai Epizyme Biomedical Technology Co., Ltd. (Shanghai, China).

Four percent paraformaldehyde-fixed tissues were carried out with paraffin and sliced into 4 μm thick sections. Then 0.3% TritonX-100 penetrated tissues for 30 min and immunostained for Ki67, TXNIP (Proteintech, Wuhan, China) primary antibodies at 4°C overnight and universal secondary antibodies at 37°C for 60 min. Next, tissues were visualized by using the 3‐amino‐9‐ethylcarbazole was applied for 10 min. The tissues were washed with PBS for three times and tissues were counterstained with hematoxylin, then dehydrated and coverslipped according to the protocol.

Freshly harvested gastrocnemius SkM was fixed in 4% paraformaldehyde and embedded in paraffin, and sectioned into 5-μm-thick sections onto poly-l-lysine-coated glass slides. The sections were incubated overnight with primary antibodies against Txnip (Proteintech Group, IL, USA, 18243-1-AP, 1:100), Keap1 (sc-514914, 1:100), and Nrf2 (16396-1-AP, 1:200). Immunostaining was visualized with 3,3-diaminobenzidine substrate.

The immunohistochemical method of Pandurangan et al. was adopted with some modifications.
²⁰ Paraffin‐embedded tissue sections of 5 µm thickness were rehydrated in xylene and then in graded ethanol solutions. Then, the slides were incubated with HistoVT (10×, pH 7.0) (Nacalai Tesque) antigen retrieval solution for 20 min at 90°C and then cooled to room temperature. The slides were then blocked with 5% bovine serum albumin (BSA) in TBS‐Tween 20 (TBST) for 2 h. The sections were then incubated with primary antibody overnight at 4°C for immunostaining. The primary antibody: KLF4 (1:100; Proteintech, 11880‐1‐AP, China), TXNIP (1:100; Proteintech, 18243‐1‐AP, China), NLRP3 (1:100; Proteintech, 27458‐1‐AP, China), Caspase‐1 (1:100; Proteintech, 22915‐1‐AP, China), GSDMD (1:100; Proteintech, 20770‐1‐AP, China). After washing the slides thrice with TBST, the sections were incubated with the appropriate secondary antibodies in TBST with 5% BSA for 2 h at room temperature. The sections were then washed with TBST and incubated for 5 min with a peroxidase stain from a peroxidase stain DAB kit following the instructions provided by the manufacturer (Nacalai Tesque). Counterstaining was performed using hematoxylin (Cell Path), and the slides were photographed under a light microscope (Nikon ECLIPSE 80i; Nikon Corporation).

Protein samples were extracted from skin wound tissues or cultured cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane. Membranes were probed with primary antibodies against TXNIP (Proteintech, Chicago, Illinois, United States), NLRP3, caspase-1, IL-1β, phosphorylated Akt ser⁴⁷³ (p-Akt), Akt, phosphorylated GSK3β ser⁹ (p-GSK3β), GSK3β (Cell Signaling Technology, Beverly, Massachusetts, United States) and β-actin (Abbkine, Redlands, California, United States). Membranes were then washed and labeled with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Abbkine). β-actin and all secondary antibodies were used at a dilution of 1:10,000. All other antibodies were used at a dilution of 1:1,000.