Sp600125

Three primed hESC lines were subjected to naive conversion on MEFs in low oxygen conditions in either: (i) naive human stem cell medium (NHSM) composed of basal hESC media supplemented with AlbumaxI, N2 supplement (Invitrogen), PD0325901 (1 μM, Cayman) and CHIR99021 (3 μM, Axon Medchem; known together as 2i), leukaemia inhibitory factor (LIF, 1000U, Sigma), bFGF, (8 ng ml⁻¹), SP600125 (10 μM, Tocris), SB203580 (10 μM, Tocris) and TGFβ (1 ng ml⁻¹, Peprotech)10 (link); (ii) RT consisting of high glucose DMEM-F12 (Invitrogen) further supplemented with histone deacetylase inhibitors, sodium butyrate (0.1 mM) and SAHA (50 nM) for first three passages followed by 2i and bFGF (10 ng ml⁻¹)11 (link) or (iii) our novel medium naive conversion medium (NCM) containing hESC media with added 2i, LIF, forskolin (10 μM, Sigma), ascorbic acid (50 ng ml⁻¹, Sigma) and bFGF(12 ng ml⁻¹)8 (link). Upon the appearance of domed-shaped colonies, the culture was passaged using 0.05% trypsin and once the culture was well established, cells were split at a ratio of 1:4 to 1:8 every 3 days. Single-cell clonogenicity was determined from the number of single-cells plated (upon dissociating primed and naive hESCs using trypsin without ROCKi) and the subsequent number of colonies formed. Doubling time was calculated using the formula:

G1 was purchased from Tocris (Wiesbaden-Nordenstadt, Germany), dissolved (5 mM) in dimethyl sulfoxide (DMSO) (Roth, Karsruhe, Germany) and stored at −20 °C; Thapsigargin, SP600125, GSK2606414 were also purchased from Tocris. Indo-1 AM was from Thermo Fisher Scientific (Waltham, MA, USA). zVAD-fmk was bought from Santa Cruz (Santa Cruz, CA, USA). SB203580 and Kira6 were purchased from MERCK Millipore (Darmstadt, Germany). All substances were dissolved in DMSO. Antibodies were obtained from the following commercial sources: caspase 9 (Ca# 9502), cleaved PARP (Ca# 9541), IRE1α (Cat# 3294), PERK (Cat# 3192), eIF2α (Cat# 5234), phospho-eIF2α (Cat# 3398), BiP GRP78 (Ca# 3177), CHOP (Cat# 2895), p38 MAPK (Ca# 9212), phospho-p38 MAPK (Ca# 4511), phospho-SAPK/JNK (Ca# 4668), caspase 3 (Ca# 9662), BCL-2 (Ca# 2872), Cell Signaling (Danvers, MA, USA); ATF6 (Cat# 73500), BioAcademia (Osaka, Japan); puromycin (Ca# MABE343), cylophilin D (Ca# AP1035), MERCK Millipore (Darmstadt, Germany); phospho-IRE1α (Cat# NBP2-50067), Novus Biologicals (Littleton, CO, USA); cytochrome c (Ca# 556433), BD Biosciences (Franklin Lakes, NJ, USA); β-actin (Cat# A5441, Sigma-Aldrich (Steinheim, Germany)). Secondary, peroxidase-conjugated antibodies were purchased from Dianova (Hamburg, Germany). All other chemicals of analytical grade were obtained from Sigma-Aldrich or Roth.

Dulbecco's modified Eagle's medium (DMEM) and FBS were purchased from Gibco‐BRL (by Thermo Fisher Scientific Inc.; Waltham, MA, USA). MG‐132 was a product of Enzo Life Sciences (Exeter, UK). Cycloheximide (CHX) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were obtained from Sigma‐Aldrich (St. Louis, MO, USA). SP600125, U0126, and SB203580 were purchased from Tocris Bioscience (Bristol, UK). Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix was supplied by CORNING Inc. (Corning, NY, USA).

Peptidoglycan (PGN) from S. aureus, mouse anti-human immunoglobulin E antibody (ε-chain specific) (anti-IgE) were purchased from Sigma. Human Myeloma IgE was from Merck. Pam3CSK4 was purchased from Invivogen. SP600125, SB203580, PD98059, ciclosporin and Bay11-7821 were from Tocris. Wortmannin was from Cayman. FITC-conjugated anti-human FcεRI antibody and FITC-conjugated mouse IgG2b isotype control were purchased from eBioscience. When chemicals were dissolved in DMSO, the final concentration of DMSO did not alter the normal response of LAD2 cells.

SFII was obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, South Korea) [30 (link)]. Recombinant TNF-α and IFN-γ were purchased from R&D Systems (Minneapolis, MN, USA). Primary antibodies against p-STAT1 (sc-592, 1:5000), t-STAT1 (sc-7988, 1:2000), and CTSS (sc-6503, 1:1000) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and those against p-p65 (CST3031S, 1:6000), t-p65 (CST8242S, 1:2000), p-p38 (CST9211S, 1:2000), and t-p38 (CST9212S, 1:2000) were from Cell Signaling Technology Inc. (Danvers, MA, USA). Primary antibodies against β-actin (Thermo Fisher Scientific, Waltham, MA, USA, MA5-15739, 1:2000) and horseradish peroxidase-conjugated polyclonal secondary antibodies against mouse, rabbit, or goat IgG (GeneTex, Inc., Irvine, CA, USA, 1:10,000) were also purchased. The inhibitors SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and PD98059 (MEK1 inhibitor) were purchased from Tocris (Bristol, UK); InSolution™ JAK inhibitor I and BAY 11-7082 (NF-κB inhibitor) were purchased from Calbiochem (San Diego, CA, USA).

l-Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, Hanks’ salts, trypsin, DMEM, and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), HEPES, DMSO, d-glucose, MPP⁺, KCN, MPP⁺, l-NAME, tert-butyl hydroperoxide, and bovine serum albumin were from Sigma-Aldrich, St. Louis, MO, USA. 666-11, SB 202190, SP 600125, salirasib, FIPI, KN-93, ML-193, PSB C5, HA-1004, U-0126, KT-5720, and U-73 were from Tocris Bioscience, Bristol, UK. Total RNA Purification kit was from Jena Biosciences, Jena, Germany. MMLV reverse transcription kit and qPCR master mix qPCRmix-HS SYBR were from Evrogen, Moscow, Russia. cAMP determination kit and BrdU cell proliferation assay kit were from Abcam, Cambridge, MA, USA. DNase I was from Thermo Fisher Scientific, Waltham, MA, USA.
The peptide was synthesized by methods of classical peptide chemistry in solution using both protected and free L-amino acids, as described earlier [49 (link)]. Purity of the final compound was not less 98% (HPLC analysis, Supplementary S2).