P eif2α

Antibodies against STIM1, STIM2, ORAI1, and TRPC1 were obtained from Alomone (Jerusalem, Israel). Bcl-2, Bax, GRP78, caspase 12, β-actin, flag tag, and myc tag antibodies were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to α-actinin, CHOP, ATF4, ATF6, p-eiF2α, eiF2α, p-PERK, PERK, p-JNK, and JNK were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Adenovirus carrying human STIM1 (Ad-STIM1), negative control Lacz adenovirus (Ad-Lacz), STIM1 siRNA, and non-specific siRNA oligonucleotides (negative siRNA) were synthesized from RiboBio Co., Ltd (Guangzhou, China). Mammalian expression plasmids for myc-tagged GRP78 and flag-tagged STIM1 were constructed by BioVector NTCC Inc. (Beijing, China). Dulbecco’s minimal essential medium (DMEM), OptiMEM I medium, fetal bovine serum (FBS), penicillin, streptomycin, and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified.

Compound K (purity > 98%) used in the present study was isolated from P. ginseng, and its structural identities were determined spectroscopically (¹H and ¹³NMR, IR, MS) as previously described [21] (link). RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), penicillin, streptomycin, and Fura-2/AM were obtained from Life Technologies Inc (Chicago, IL, USA). 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tertazolium bromide (MTT), dimethyl sulfoxide (DMSO), RNase A, phenylmethylsulfonylfluoride (PMSF), propidium iodide (PI), 4-phenylbutyrate (4-PBA), 2-aminoethoxydiphenyl borate (2-APB), dantrolene, BAPTA-AM, ethylene glycol tetraacetic acid (EGTA), and CGP37157 were purchased from Sigma Chemical Co. The following antibodies for caspase-3, caspase-7, caspase-9, poly (ADP-ribose) polymerase (PARP), XBP-1, c-FLIP_L, Bid, Bcl-2, Bcl-xL, p-eIF2α, GADD 153, m-calpain, and β-actin were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). The antibodies for X-linked inhibitor of apoptosis protein (XIAP), caspase-8, and caspase-12 were purchased from BD Biosciences, Pharmingen (San Jose, CA, USA). The antibodies for IRE1α and GRP78 were purchased from Cell Signaling Technology (Denvers, MA, USA). z-VAD-fmk, z-ATAD-fmk were purchased from Calbiochem (San Diego, CA, USA).

BSC40 and U266 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). BTZ-sensitive (Dox40) and -resistant (Dox40^BTZ) human MM cells35 (link) were a kind gift from Dr Bei Liu at the Medical University of South Carolina. BSC40 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% fetal bovine serum plus 1× penicillin-streptomycin-L-glutamine (Mediatech, Inc., Manassas, VA, USA). U266, Dox40, and Dox40^BTZ cells were cultured at concentrations below 5×10⁵ cells/mL in Roswell Park Memorial Institute (RPMI)-1644 with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) plus 20% fetal bovine serum plus 1× penicillin-streptomycin-l-glutamine. Cell viability was performed by CellTiter^® 96 Non-Radioactive Cell Proliferation Assay (MTT) (Promega Corp, Fitchburg, WI, USA) and read on a FLUOstar Optima Plate reader (BMG LABTECH GmbH, Ortenberg, Germany). The following antibodies were used: from Cell Signaling Technology, Inc. (Danvers, MA, USA), Mcl1 (5453) activating transcription factor (ATF)4 (11815), CHOP (CCAAT/-enhancer-binding protein homologous protein) (2895), Actin (8457), and p-eIF2α (3398); and from Santa Cruz Biotechnology (Dallas, TX, USA), protein kinase RNA-like ER kinase (PERK) (13073), eIF2α (11386), and ATF6 (22799).

RPMI 1640 was obtained from Thermo-Fisher Biochemical Products Co. Ltd (Beijing, China). Fetal bovine serum (FBS), thapsigargin (TG), protein kinase A (PKA) inhibitor H89 and KT5720, 4-phenylbutyric acid (PBA) and PGE1 were purchased from Sigma (St. Louis, MO, United States). Antibodies against GRP78, PERK, eukaryotic translation initiation factor-2α (eIF-2α), phospho-PERK (p-PERK) and phospho-eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), spliced X box-binding protein 1 (sXBP1), growth arrest and DNA damage-inducible gene 34 (GADD34) and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was purchased from Dojindo Laboratories (Kumamoto, Japan). Small interfering (si)RNA scramble control and validated human GRP78-siRNA were purchased from Santa Cruz Biotechnology. All other chemicals and reagents were obtained from Sigma, unless stated otherwise.

Cells were washed with phosphate-buffered saline (PBS) and split using RIPA assay (Pierce, Rockford, IL, USA). The protein was quantified by a BCA assay (Pierce). Denatured proteins were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose (Amersham, Bensheim, Germany). nitrocellulose was blocked with 5%-BSA in TBST and incubated with Bax, BiP, CHOP, p-eIF2α, LC3, Beclin-1, Atg 7, DAPK3, p-Akt, p-mTOR and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. nitrocellulose was washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated appropriate secondary antibodies and reacted with ECL detection reagents (Amersham Bioscience).