Chemotaxis Assay of BMDMs

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.