Gsk650394

NDRG1 or GSK3B genes were inhibited by transient transfection with siRNA (siNDRG1 and siGSK3β) from Santa Cruz (50 ng/ml) and lipofectamine RNAiMAX (Invitrogen) following the manufacturer's instructions for 48h after stimulation with TGFβ1 (10 ng/ml) (PeproTech) for 8h or 14 days. TGFβ1 was replenished every 72h. Scrambled siRNA (SCR) was used as negative control 20 (link). Galunisertib (LY2157299, TGFβR1 inhibitor) (5 μM), CHIR99021 (GSK3α/β inhibitor) (10 μM), LY294002 (PI3K inhibitor) (10 μM), MK2206 (Akt inhibitor) (1 μM), GSK650394 (SGK1/2 inhibitor) (10 μM), and Rapamycin (mTOR inhibitor) (10 μM) were from MedChemExpress. Cells were pre-stimulated with TGFβ1 for 8h and treated with each inhibitor for 24, 48, and 72h.

The mouse recombinant LIGHT protein (rLIGHT, cat#HY-P73837), PI3Kγ specific inhibitor (Eganelisib, IPI549, cat# HY-100716), and SGK1 inhibitor (GSK650394, cat#HY-15192) were purchased from MedChemExpress (MCE, New Jersey, USA). The primary antibodies anti-phospho-Akt (Ser473) (cat#4060) and anti-Akt (cat#4691)used for western blotting were purchased from Cell Signalling Technology (Danvers, Essex County, MA, USA); anti-PIK3CG (cat#A0266), anti-SGK1 (cat#A3936), and anti-phospho-Smad2/Smad3 (cat#AP1343) were purchased from ABclonal (Wuhan, China); anti-Smad2 (cat#ET1604-2), anti-Smad3 (cat#ET1607-4) and anti-PI3K p85α (cat# ET1608-70)were purchased from HUABIO (Hangzhou, China); and anti-F4/80 (cat#28463-1-AP), anti-CD3 (cat#17617-1-AP), anti-CD163 (cat#16646-1-AP), anti-ARG1 (cat#66129-1-Ig), anti-CD206(cat#60143-1-Ig), anti-iNOS (cat#18985-1-AP), anti-MCP1 (cat#66272-1-Ig), anti-TGFβ1 (cat#21898-1-AP), anti-collagen I (cat#14695-1-AP), anti-collagen III (cat#68320-1-Ig), anti-α-SMA (cat#14395-1-AP), anti-β actin (cat#20536-1-AP), and anti-GAPDH(cat#10494-1-AP) were purchased from Proteintech (Wuhan, China).

GFP-AZI2 -4OHT cells were seeded in 24-well plates and left overnight before treatment with respective inhibitors. Upon treatment, images of cells were acquired every 2 h with an Incucyte live-cell imaging system (Essen Bioscience) to monitor GFP-AZI2 puncta formation. Inhibitor concentrations used in this screen were; SB203580, 10 µM (MedChemExpress, HY-10256); Tozasertib, 5 µM (MedChemExpress, HY-10161); Chloroquine, 100 µM (Sigma Aldrich, C6628); GDC0994, 5 µM (Cayman Chemical, 21107); Prexasertib, 5 µM (MedChemExpress, HY-18174); LTURM34, 10 µM (MedChemExpress, HY-101667); GSK650394, 5 uM (MedChemExpress, HY-15192); Amlexanox, 100 µM (MedChemExpress, HY-B0713); U0126, 10 µM (Cayman Chemical, 70970); IRAK 1–4 Inhibitor I, 10 µM (MedChemExpress, HY-13329); SP600125, 25 µM (MedChemExpress, HY-12041); SBI0206965, 10 µM (Cayman Chemical, 18477); BMS345541, 10 µM (MedChemExpress, HY-10519); Triciribine, 10 µM (MedChemExpress, HY-15457); Silmitasertib, 10 µM (MedChemExpress, HY-50855); PP242, 5 µM (MedChemExpress, HY-10474); Lys05, 20 µM (provided by Dr. Ravi Amaravadi); Palbociclib, 5 µM (MedChemExpress, HY-50767); and D4476, 50 µM (MedChemExpress, HY-10324). The puncta/image at 24 h were quantified and plotted.