Hs 173

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)].

Human MG-63 and U2OS osteosarcoma cell lines were purchased from Cells Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco-modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), at 37 °C in a humidified atmosphere with 5% CO₂. MG-63 and U2OS cells were plated onto 6-well cell culture clusters (Costar) and grown to 80% confluence, and then serum-starved for 24 h. These cells were subsequently treated with HS-173 (PI3Kα inhibitor), MK-2206 (Akt inhibitor), A-674563 (Akt1 inhibitor), or CCT128930 (Akt2 inhibitor) (Selleck, Houston, TX) before RhoA activation assays and wound healing assays.

TC^ITGB1+ or TC^ITGB1− were digested and cultured in 96-well plates with the density of 5 × 10³ cells/well, 3–6 wells per group, followed by treatment with TGFβ1 at 0.5, 5.0, or 50 ng/ml, respectively, for 24 h to characterize TGFβ1-induced TCs proliferation. In order to evaluate roles of PI3K catalytic isoform proteins, 5 μM HS173 (PI3K p110α inhibitor, SelleckChem Co., Houston, USA), 5 μM GDC0941(PI3Kα/δ inhibitor, SelleckChem), 0.25 μM Enzastaurin (PKCβ inhibitor, SelleckChem) or 0.5 μM SB216763 (a potent and selective GSK-3 inhibitor for both GSK-3α and GSK-3β, SelleckChem)on TC^ITGB1+ or TC^ITGB1− with or without TGFβ1 for 48 h. Ten microliters of CCK-8 reagents (Dojindo Molecular Technologies, Inc., Maryland, USA) was added to every well and incubated for 0.5 h at 37° C, with 5% CO₂. We determined the absorbance at 450 nm using SpectraMax M5 Microplate Reader (Molecular Devices Instruments Inc., Sunnyvale, California, USA).