Ravoxertinib

The antibody to Fgl2 was obtained from Novus Biologicals. Antibodies against Tyrobp, β-actin, LC3B, phospho-p44/42 MAPK (Erk1/2) and p44/42 MAPK (Erk1/2) were obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies were purchased from Abcam. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). The autophagic inhibitor CQ was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ravoxertinib was obtained from MedChemExpress (New Jersey, USA).

Migration assays of WT BMDMs toward SFM or CM from R7 cells with or without shRNA targeting of HGFL (1:1 dilution ratio of BMDM CM to SFM), or co-culture with R7 or R7 shHGFL cells (1:10 ratio of R7 cells to WT BMDM cells) using 4000 R7 cells and 40,000 BMDM cells were performed for up to 6 h using Boyden chambers [5 (link)]. For studies involving inhibitors, R7 cells were pretreated with inhibitors including 20 µM Ravoxertinib (ERK1/2 inhibitor; MedChemExpress, Monmouth Junction, NJ, USA, Cat#50-187-3397), 20 µM Afuresertib (Akt inhibitor; MedChemExpress Cat#50-187-3338), 20 µM STAT3 Inhibitor VII, S3I-201 (Calbiochem Cat#573103), or 10 µM Bay 11-7085 (EnzoLifeSciences, Farmingdale, NY, USA, Cat#573103) for 6 h prior to washing out and addition of fresh media, and addition of BMDM-containing Boyden chambers. The number of BMDMs that migrated toward SFM was used as a reference control. Following incubation, chambers containing migrated cells were removed, and the cells were fixed and stained using 0.5% w/v Crystal Violet (Sigma, Sofia, Bulgaria, Cat#C6158) in methanol. The upper part of the chamber was gently wiped clean using a cotton tip applicator to remove non-migrated cells prior to imaging and counting transmigrated cells.

The primary antibodies used for western blotting included: MMP9 (ab228402, Abcam, USA), β-actin (RM2001, Beijing Ray, Beijing, China).
The primary antibodies used for IHC included: S100A9 (ab242945, Abcam), Ly6G (ab238132, Abcam), CD11b (ab133357, Abcam), F4/80 (70,076 S, CST, Danvers, MA, USA), IL-1β (12,242 S, CST), MMP9 (ab228402, abcam).
The primary antibodies used for immunofluorescence (IF) included: CD11b (ab133357, Abcam), F4/80 (70,076 S, CST), IL-1β (12,242 S, CST), MMP9 (ab228402, abcam), SPC (ab211326, abcam).
The primary antibodies used for flow cytometry included: TruStain FcX anti-mouse CD16/CD32 antibody (Biolegend, 101,319), FITC anti-mouse/human CD11b (Biolgend, 101,205), PE/Cyanine7 anti-mouse Ly-6G (Biolegend, 127,617), Alexa Fluor 647 Rat anti-mouse S100A9 (BD Pharmingen, 565,833) and PE anti-mouse IL-1β (Thermo, 12-7114-80).
Other reagents included: Cultrex Basement Membrane Extract (R&D, Minneapolis, MN, USA); recombinant murine IL-1β (Peprotech, Rocky Hill, USA), murine SAA3 (General Biol, China); IKK 16, JNK-IN-7, Losmapimod and Ravoxertinib (MedChemExpress, Monmouth Junction, NJ, USA).