P stat3

MAC sorted, CD11b⁺ wound Mφ cells were subjected to cell lysis buffer and protease inhibitor cocktail. Protein suspensions were then standardized for protein concentrations using a Bradford protein assay (Bio-Rad). Equal amounts of protein were mixed with loading buffer and subjected to 4–18% Tris-glycine gel electrophoresis under reducing conditions. Proteins were then wet-transferred at 100 V for 1 hour in Tris-glycine transfer buffer (Invitrogen) to nitrocellulose membranes and probed with primary antibodies (all Cell Signaling; p-JAK1 (D7N4Z), JAK1 (6G4), p-JAK3 (D44E3), JAK3 (D7B12), p-STAT3 (D3A7), STAT3 (D3Z2G), STING (D2P2F), IL-1β (3Α6), β−actin (8H10D10), p-NFκB p65 (Ser536; 93H1), NFκB p65 (D14E12), p-TBK1 (D52C2), TBK1 (E8I3G), p-IRF3 (D6O1M), IRF3 (D83B9) and GAPDH (D16H11)) diluted to 1:500 v/v in 5% bovine serum albumin in Tris buffered saline with Tween buffer overnight at 4 °C with agitation. Nitrocellulose membranes were then washed and incubated with anti-rabbit IgG or anti-mouse IgG HRP-conjugated secondary antibody (Cell Signaling, Inc.) for 1 h at RT with shaking and visualized with timed chemiluminescence (Thermo Fisher Scientific). Densitometry was calculated using ImageJ (NIH), and statistical significance was obtained using unpaired Student’s t test.

We collected wound tissues for total protein extraction and analyzed STAT3 and p-STAT3 levels after 3 days of cutaneous-wound healing in vivo. Traumatized cutaneous tissues from mice were scraped and resuspended in 0.5 ml RIPA; tissue lysates were placed on ice for 1 h. After centrifugation at 10,000g for 20 min, we determined total protein concentration using a Bicinchoninic Acid (BCA) Protein Determination Kit (Yeasen). We electrophoresed 20-ct samples via 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Yeasen) and transferred them onto the PVDF membrane. Samples were blocked with blocking buffer at RT for 2 h and then probed with STAT3 (dilution, 1:500; Invitrogen), p-STAT3 (dilution, 1:1000; Invitrogen), and β-actin (dilution, 1:10000; AB clonal Technology, Woburn, MA, United States) at 4°C overnight. Secondary aBs used for detection included horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin G (IgG; dilution, 1:10000; ABclonal Technology). We detected target protein expression using an Enhanced Chemiluminescence (ECL) Detection Kit (Boster).

The following reagents and materials were used: TAC and monobenzone (Cat. No. 1642802 and 1445506, respectively; Sigma-Aldrich, China), specific antibodies against chemokine (C-X-C motif) ligand CXCL9, CXCL10, CXCR3 and HSP70 (Cat. No. ab202961, ab271208, ab181013 and ab181606, respectively; Abcam, Cambridge, MA, USA), anti-CD8 monoclonal antibody (Cat. No. 55397; Cell Signaling Technology, Danvers, MA, USA), Trizol and cDNA synthesis kits (Cat. No. 15596018 and 4374967, respectively; Invitrogen, Carlsbad, CA, USA), primers for reverse transcription–quantitative polymerase chain reaction ([RT-qPCR]; Invitrogen, Shanghai, China) and specific antibodies against p-STAT3, STAT3 and GAPDH (Cat. No. 9145 T, 91395 and 5174S, respectively; Cell Signaling Technology).

Total protein was obtained from cells or tissues using a lysis buffer (Cell Signaling Technology, California, USA), and protein levels were quantified using the BCA kit (Shanghai Ze Ye Biotechnology Co., Ltd, Shanghai, China). Subsequently, approximately 30 μg of protein was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride membrane (Millipore, Massachusetts, USA), followed by incubation with 5% bovine serum albumin and incubation with the primary antibodies against JAK1 (1:800, Invitrogen, Massachusetts, USA), p-JAK1 (1:800, Invitrogen, Massachusetts, USA), JAK2 (1:800, Invitrogen, Massachusetts, USA), p-JAK1 (1:800, Invitrogen, Massachusetts, USA), STAT3 (1:800, Invitrogen, Massachusetts, USA), p-STAT3 (1:800, Invitrogen, Massachusetts, USA), STAT5 (1:800, Invitrogen, Massachusetts, USA), p-STAT5 (1:800, Invitrogen, Massachusetts, USA), and GAPDH (1:800, Invitrogen, Massachusetts, USA). Subsequently, the membranes were incubated with a secondary antibody (1:2000; Invitrogen, Massachusetts, USA). Finally, enhanced chemiluminescence (Invitrogen, Massachusetts, USA) was used for the visualization of bands, followed by quantification using the ImageJ software (National Institutes of Health, USA) [19 (link)].

Pretreatment serum SCCA levels were evaluated by the ARUP National Reference Laboratory (Salt Lake City, Utah, USA) using ELISA, and the TMA was generated from untreated human tumor specimens, as described previously (14 (link)). TMA sections were sent to HistoWiz for IHC staining for CD11b (1:100, Abcam, ab224800), and IHC for p-STAT3 (1:200, MilliporeSigma, SAB4300033) was performed by the Washington University AMP Core Laboratories. Mouse tumor sections were stained with p-STAT3 (1:150, Invitrogen, Thermo Fisher Scientific, PA5-121259) and CD11b (1:500, Invitrogen, Thermo Fisher Scientific, PA5-79532), using the Pierce peroxidase IHC detection kit (Thermo Fisher Scientific). QuPath, version 0.3.2, software was used for automated analysis of surface and cytoplasmic staining to determine the percentage of cells positive for CD11b. IHC for p-STAT3 was evaluated by a board-certified pathologist with gynecologic expertise, and staining scores were calculated for a minimum of 2 tissue cores for each patient (the percentage of positively stained tumor cells × staining intensity ranged from 0 to 3). Values from at least 2 cores from each sample were considered valid, and an average score was taken.

The supernatant was collected from the NALF for quantitating cytokine release. Cytokine quantitation kits were used to measure cytokine levels including those of IL-10, IL-17, IL-6, TNF-α (R&D Systems, Inc., Minneapolis, USA); Nrf-2, HO-1, MDA, Foxp-3 (BD Biosciences, San Diego, CA, USA); STAT3, p-STAT3 (Invitrogen, Carlsbad, California); and RORγ (Fine Biotech, Wuhan, Hubei, China). All assays were performed in duplicate, standard solutions and NALF samples were transferred to 96-well plate which was prior coated with a target cytokine capture antibody, incubated for 2 h at room temperature, then washed for 4 times. After incubated 2 h with an appropriate biotin-conjugated antibody, the wells were aspirated and washed again 4 times, and then a horseradish peroxidase (HRP) was added to each well. Removing the excess HRP conjugate by washing, a substrate solution was added to convert the solution to a detectable form. Add stop solution to each well, read the optical density of each well at 450 nm immediately^{47 (link),48 (link)}.