Ly2228820

HuH‐7 cells were treated with 50 mm d‐allose (Rare Sugar Research Center, Kagawa University, Kagawa, Japan) for 48 h, and cell lysate was immunoprecipitated against anti‐TXNIP. COS‐7 cells were transfected with FLAG–TXNIP expression plasmid, and cell lysate was immunoprecipitated with anti‐FLAG agarose as described above. The immunoprecipitates were separated by SDS/PAGE. To visualize phosphoprotein, the gel was stained using the Pro‐Q Diamond phosphoprotein gel stain (Thermo Fisher Scientific (Invitrogen)) following the manufacturer's protocol. The p38 MAPK activity was inhibited by pre‐incubation in 500 nm LY2228820 (Selleck Chemicals, Houston, TX, USA) for 2 h before preparing the cell lysate. The samples for liquid chromatography–mass spectrometry (LC‐MS/MS) analyses were prepared by in‐gel‐digestion of the proteins by trypsin, chymotrypsin, and aspartic protease. The phosphorylation state was analyzed by LC‐MS/MS using Micromass Q‐TOF (Waters, Milford, MA, USA), followed by a Mascot search (Matrix Science, Boston, MA, USA).

U2OS, A549, Saos-2, and HEK293 cells (purchased from ATCC) were cultured either in Dulbecco’s modified Eagle medium (Sigma, D5796) or Roswell Park Memorial Institute Medium (RPMI)-1640 supplemented with 10% fetal bovine serum (Thermo Scientific, E6541L), 2 mml-Glutamine (LabClinics, M11–004) and 100 μg/ml penicillin–streptomycin (LabClinics, P11–010). For the inhibition of p38α, we used 1 μm PH797804 (Selleckchem, S2726) or 500 nm BIRB0796 (Axon MedChem, 1358), and 200 nm LY2228820 (Selleckchem, S1494). MK2 was inhibited using 10 μm MK2 Inhibitor III (Calbiochem, 475864–5MG) or 10 μm PF 3644022 (Sigma, PZ0188). The following additional treatments were used: 0.5 μm MitoQ (kindly provided by M. Murphy, Cambridge, UK), 250 nm Doxorubicin hydrochloride (Sigma, D1515–10mg), 50 μm Z-VAD (OMe)-FMK (SM Biochemicals LLC SMFMK001), 200 or 50 nm bafilomycin A1 (Santa Cruz, sc-201550), 20 μm chloroquine (Sigma, C6628), 10 μm Spautin-1 (Axon MedChem, 2512), 15 μm palbociclib (Selleckchem, S1116), 500 nm camptothecin (Sigma, C9911). The compounds were dissolved in dimethyl sulfoxide (DMSO) or water and the total concentration of DMSO in the culture medium never exceeded 1%.

GSK2118436 (S2807), INCB018424 (S1378), LY2228820 (S1494), LY294002 (S1105), PLX-4032 (S1267), and SB202190 (S1077) were purchased from Selleckchem. GF-120918 (HY-50879) and KO-143 (HY-10010) were purchased from MedChemExpress. All compounds were used following 24 h serum withdrawal in low serum conditions (1% FBS) at the indicated concentrations and time.
Other drugs used were CHX (100 μg/ml) (Sigma–Aldrich), mithramycin A (200 nM) (Merck Millipore), λ−protein phosphatase (100U) (New England BioLabs), and MG-132 (100 nM) (Sigma–Aldrich).

One hundred LT‐HSCs per well (96‐well format) were lentivirally transduced (multiplicity of infection (MOI) 100) and cultured in serum‐free expansion medium (SFEM) (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) supplemented with 100 ng/ml SCF and Thrombopoietin (TPO) (both PeproTech, Rocky Hill, NJ, http://www.peprotech.com). Cells were analyzed by fluorescence activated cell sorting (FACS) (antibodies against CD48, CD117, CD16/32, CD11b). For Tetracycline (TET)‐inducible GADD45A expression studies, 300 LT‐HSCs were seeded per well in SFEM supplemented with stem cell factor (SCF), TPO, and Doxycycline at the indicated concentrations. The cells were lentivirally transduced and analyzed after 8 days of culture as described above.
For analyzing the LT‐HSC fate upon DNA‐damage inducing conditions, 1,000 LT‐HSCs per well (96‐well format) were seeded in SFEM supplemented with SCF and TPO. After 24 hours, the cells were stimulated with γ‐IR (2 Gy) and were analyzed for their marker expression after 5 days of culture as described above.
For inhibitor studies 20 ng/ml Interleukin 3 (IL3) (PeproTech) was additionally supplemented. Inhibitors were used as follows: VX‐702 (1 μM), LY2228820 (0.1 μM, all from Selleck, http://www.selleckchem.com). FACS antibodies are listed in Supporting Information Table S1.