Ly6g fitc

Murine IL‐6 cytokine was purchased from R&D Systems. Murine GM‐CSF cytokine GM‐CSF was purchased from Abcam. Anti‐Ly6G (1A8) was purchased from eBioscience. STAT3 inhibitor was purchased from BioVision. Ethanol was from Sigma–Aldrich. Anti‐phosphorylated STAT3 (p‐STAT3) was purchased from CST. APC‐CD11b, FITC‐Ly6G, and PE‐Cy7‐Ly6C antibodies were purchased from BioLegend. APC‐CD3, FITC‐CD4, and APC‐Cy7‐CD8 antibodies were purchased from BD Biosciences. Berberine hydrochloride was purchased from Sigma–Aldrich. Lieber‐DeCarli liquid diet was purchased from Bio‐Serv.

Tumors and blood of tumor-carrying mice were collected. Peripheral blood lymphocytes and tumor-infiltrating lymphocytes were isolated, and red blood cells were lysed using Red Blood Cell Lysis Buffer (Beyotime Biotechnology) for 15 mins according to the manufacturer’s instruction manual. Cells were washed and counted using an automated cell counter (Countstar). 1 × 10⁶ cells were blocked with anti-mouse CD16/32 (BioLegend). To identify the percentage of macrophages and myeloid-derived suppressor cells (MDSCs), cells were stained with antibodies: Ax647-CD11b, FITC-Ly-6G, PE-Gr-1,PerCP/Cy7-F4/80 and PerCP/Cy5.5-Ly-6C, purchased from BioLegend company. Flow cytometry acquisition was performed on CytoFLEX (BD Biosciences), and data were analyzed using CytExpert software (2.4.0.28).

Rabbit monoclonal antibodies to murine IL-1β (clone D3H1Z), neutrophil elastase (clone E8U3X), PTP-PEST (clone D4W7W), and rabbit polyclonal antibody to SHIP1 (D1163) were from Cell Signaling Technology, rabbit polyclonal antibody to GAPDH (#G9545) from Sigma-Aldrich. The monoclonal antibodies to phosphotyrosine (clone 4G10) and PSTPIP2 (clones PSTPIP2-01 and PSTPIP2-03 (14 (link))) were produced in-house with the use of respective hybridomas. Flow cytometry antibodies Ly6G-FITC (catalog # 127606, also used for Western blot), Ly6C-PE-Cy7 (# 128018), CD11b-PE (# 101208) were from Biolegend and CD62L-APC (# 177-0621-81) was from eBioscience (ThermoFisher).

Cells in BALF were stained with fixable viability dye and anti-murine CD45-PE Fluor61 (eBioscience, San Diego, CA), SiglecF-Alexa647, CD11b-PE Cy7 (BD Bioscience, Franklin Lakes, NJ), CD11c-PerCP Cy5.5, Ly-6C AlexaFluor700 (BD Pharmingen, San Jose, CA), and Ly-6G-FITC (Biolegend, San Diego, CA). Cells were fixed and red blood cells were removed using fix/lyse solution according to manufacturer’s instruction (eBioscience, San Diego, CA). Quantification of total cell content was performed by addition of precision count beads (Biolegend, San Diego, CA) before measurement. Expression levels of activation markers were quantified via the median fluorescent intensity (MFI). Data were collected on a Canto II flow cytometer (BD Bioscience, Franklin Lakes, NJ) and analyzed using FlowJo software v10.7.1 (Treestar, Palo Alto, CA, USA). The full gating strategy can be found in Fig. S1.

The following antibodies were used: LIVE/DEAD Fixable Near-IR dead cell stain (Life Technologies), CD45:PB (Biolegend, clone 104), CD11B:PE-Cy7 (Biolegend, clone M1/70), Ly6G:FITC (Biolegend, clone 1A8), Ly6C:BV510 (Biolegend, clone HK1.4), and CD45.2:Alexa700 (Biolegend, clone 104). Cells were resuspended in 100 µL of antibody mix and incubated for 30 min at 4 °C in the dark and then washed with FACS buffer and spun down at 450× g for 8 min at 4 °C. Cells were fixed in 5% paraformaldehyde for 15 min and then washed twice with FACS buffer and centrifuged at 450× g for 8 min at 4 °C. The cells were finally resuspended in 100 µL of FACS buffer. All the data were collected using a Gallios flow cytometer (Beckman Coulter) and analyzed using FlowJo software version 10 (FlowJo LLC). The gating of the flow cytometric data was performed according to the guidelines for the use of flow cytometry and cell sorting in immunological studies [48 (link)], including pre-gating on viable and single cells for analysis. Absolute cell numbers of each cell population were calculated based on a defined number of counting beads (BD Bioscience, Calibrite beads) which were added to the samples before the flow cytometric acquisition.