Sb202190

NPCs were serum-starved for 12 hours and then divided into four groups for subsequent experiments—control group: cells were cultured in defined medium for 24 hours; glucose group: cells were cultured in defined medium with various concentrations of glucose (5, 15, or 25 mM) for 24 hours; glucose + N-acetylcysteine (NAC) group: cells were pretreated with antioxidant NAC (10 mM, Sigma) for 12 hours before addition of 25 mM glucose; glucose + SB 202190 group: cells were pretreated with a specific p38 MAPK inhibitor SB 202190 (10 μM, Sigma) for 12 hours before addition of 25 mM glucose.

Human CRC organoid was cultured as described previously^{60 (link),61 (link)}. In brief, fresh CRC tumor tissues were washed with cold PBS containing penicillin-streptomycin, and cut into 3–5 mm fragments. Pieces were digested with EDTA (5 mM) on ice for 60 min with mixing. After being digested into clumps of cells, the sample was mixed with Matrigel and seeded into a 24-well plate. After Matrigel polymerization (10 min at 37 °C), 500 μL/well advanced DMEM/F12 medium containing 10 mM HEPES, 100 U/mL penicillin/streptomycin, 2 mM GlutaMAX, 1× B27, 1× N2 (Life Technologies), 10 nM gastrin I (Biogems), 500 ng/mL R-spondin1 (Peprotech), 10 μM SB202190 (Sigma), 10 μM Y-27632 (Abmole), 50 ng/mL recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/mL recombinant Noggin (Peprotech), 10 mM nicotinamide (Sigma), 1 mM N-acetylcysteine (Sigma) was added to each well containing organoids. On the second day, the organoids were transduced with shCTL or shCHD6 lentivirus using polybrene (10 μg/mL) (Millipore, TR-1003-G).

Human EGF and bFGF were from Thermo Fisher Scientific (NY, USA). Human TGFβ1 and TNF-α were from R&D systems, Inc. (MN, USA). Human TGFβ2 and TGFβ3 were from Prospec-Tany TechnoGene Ltd. (East Brunswick, NJ, USA). Thrombin, PD98059, SB202190, SP600125 were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Human recombinant VEGF was purchased from Prospect Biotech (Rehovot, Israel). Estrogen and progesterone were purchased from Cayman Chemical Co. (Ann Arbor, Michigan, USA). Recombinant human CTGF was obtained from Thermo Fisher Scientific eBioscience (Waltham, MA, USA). The antibodies (Abs) raised against vimentin (sc-6260), HSP47 (sc-8352) and phospho-Smad2/3 (sc-11769) were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The Ab for total Smad2/3 (#3102) was purchased from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). The Ab for α-SMA (GTX100034) was purchased from GeneTex (Hsinchu City, Taiwan). The Abs for collagen (ab34710), CTGF (ab6992) and α-tubulin (ab7291) were purchased from Abcam (Cambridge, MA). TGFβ isoforms were dissolved in 4 mM HCl/0.1% BSA (vehicle).

MB109 is a recombinantly expressed human BMP-9 cytokine. It contains a methionine residue in front of the mature domain of human BMP-9 (Ser320-Arg429, 110 residues). MB109 was prepared as described previously [13 ]. Recombinant BMP-2 was purchased from joint Protein Central (jointproteincentral.com, Incheon, Korea). The antibodies against p21, P-SMAD1/5/8 and P-p38 were purchased from Cell Signaling Technology (Massachusetts, USA), ID3 from Santa Cruz Biotechnology (California, USA), β-actin from Sigma Aldrich (Missouri, USA), CD90 from Novus Biologicals (CO, USA), AFP from Abcam (Cambridge, UK), PE-conjugated CD90 from BD Biosciences (New Jersey, USA) and FITC-conjugated CD44 from Miltenyi Biotec (Bergisch Gladbach, Germany). Chemical inhibitors, LDN193189 was purchased from Sellekchem (Texas, USA) and SB202190 was purchased from Sigma Aldrich (Missouri, USA). The cell culture media, fetal bovine serum (FBS), Penicillin-Streptomycin Solution (P/S) and Trypsin-EDTA were purchased from Hyclone (Utah, USA).

Sal (purity >99%) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). The enhanced chemiluminescence (ECL) detection kit was from Millipore (Burlington, MA, USA). Cadmium acetate (Cd), Lucifer yellow (LY), 18-β-glycyrrhetinic acid (GA), U0126, SP600125, SB202190 and the anti-ERK, anti-P-ERK, anti-JNK, anti-P-JNK, anti-p38, anti-P-p38 and anti-β-actin antibodies were purchased from Sigma–Aldrich (Shanghai, China). PrimeScript RT Reagent kit and SYBR Premix Ex Taq were obtained from TaKaRa Biotechnology (Dalian, China). Oligonucleotide primers were synthesized by Invitrogen (Shanghai, China). All other reagents were of analytical grade.

SB202190, GSK2606414, thapsigargin (Tg), FK506, chloroquine (CQ), and cyclosporin A (CsA) were purchased from Sigma-Aldrich (St Louis, MO, USA). Torin 1 was from (Tocris-Biotechne, Minneapolis, MN, USA).