Afuresertib

The PI3K inhibitors AZD8835, AMG319, HS173, and AS605240 were purchased from Selleck (Houston, TX, USA). The PI3K inhibitors GDC0941, LY294002, and BEZ235 and the Akt inhibitor afuresertib were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Each drug was dissolved with dimethylsulfoxide (DMSO) to a storage solution of 100 mM. We diluted 100 mM of each drug with DMSO to a concentration of 10 mM of drug. BmE cells were seeded in 96-well plates 24 h before treatment with drugs. We added 10 mM of each drug to the cell culture medium at a dilution ratio of 1:1000 (0.1% of total volume) to prepare 10 µM of the drug. BmE cells were incubated with 10 µM of the drugs for 72 h. BmE cells were also incubated with 0 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, and 100 µM of AZD8835 for 72 h. Each treatment with drugs and DMSO control comprised three replicates. Cell viability was measured using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI, USA) [22 (link)].

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.