Hepatocellular cell line, HepG2, and human prostate cancer cell line, PC3, were obtained from ATCC. HepG2 was maintained in MEM with supplements according to ATCC cell culture instructions. PC3 was maintained in DMEM with 10% FBS. Rat insulinoma cells, INS-1 832/13, were a gift from Dr. Christopher Newgard (Duke University Medical Center) and cultured in RPMI 1640 supplemented with 10% FBS. Ire1α−/− and Perk−/− mouse embryonic fibroblasts were gifts from Dr. David Ron (New York University School of Medicine) and maintained in DMEM 10% FBS. Mouse labyrinthine trophoblast SM10 cells were a gift from Dr. Joan Hunt (University of Kansas) and maintained in RPMI 1640 supplemented with 2 mM glutamine, 1% sodium pyruvate, 5×10−5 M 2-Mercaptoethanol and 10% FBS. Human embryonic kidney 293T cells and Mouse neuro2a cells were obtained from ATCC and maintained in DMEM with 10% FBS.
Mouse Atf4, mouse Xbp1-processed, mouse Perk, human Ire1α and human Ire1αKA (K599A) dominant-negative kinase mutant plasmids were provided by Dr. David Ron (New York University School of Medicine). Mouse ATF5 plasmid was a gift from Dr. Michael Green (University of Massachusetts Medical School). Cleaved processed ATF6(
373) plasmid was provided by Dr. R. Prywes (Columbia University).
Lentivirus expressing human IRE1α, human IRE1αKA and mouse Perk and GFP were generated by subcloning these fragments into lentiviral expression plasmid, pLenti-CMV/TO (Dr. Eric Campeau at the University of Massachusetts Medical School). Lentivirus was produced in HEK293T cells by transfection using Lipofectamine2000 (Invitrogen Carlsbad, CA). Lentiviral supernatant was collected 48 hours after transfection, and stored at −80°C. Details of this lentivirus system were described previously 
cells were infected with human IRE1α and mouse Perk lentivirus respectively and selected with puromycin 2 µg/ml to generate stable cell lines. SM10 cells were infected with human IRE1αKA and control GFP lentivirus and selected with Puromycin 1 µg/ml to generate stable cell lines.
siRNA and Plasmid Transfection
Small interfering RNA (siRNA) was transfected using the Nucleofector Device (Amaxa Biosystems, Gaithersburg, MD) into HepG2 cells according to manufacturer recommendations. Human HIF1-α smart pool siRNA was obtained from Dharmacon, (Lafayette, CO). SiRNAs directed against human PERK, human IRE1α, human ATF4 and human XBP1 were synthesized by IDT (Coralville, IA): for human PERK: CCAGAGAAGTGGCAAGAAA; for human IRE1: AGACAGAGGCCAAGAGCAA; for human ATF4: GCAAAGAGCTGGAAAAGAA; for human XBP1: GGTATTGACTCTTCAGATT. Cells were incubated in media for overnight after siRNA transfection, and then put under ER stress conditions. Mouse XBP1-processed plasmid was transfected using the Nucleofector device (Amaxa Biosystems, Gaitherburg, MD) into mouse embryonic fibroblasts according to manufacturer recommendations.
Cells were lysed in M-PER (Pierce, Rockford IL) for whole cell protein extraction, or NE-PER (Pierce, Rockford IL) for nuclear and cytoplasmic protein extraction, after adding protease inhibitor (Sigma, Saint Louis, MO) according to supplier protocol. Lysates were run on a 4%–20% linear gradient SDS-PAGE (BioRad, Hercules, CA) gel. Anti-Atf4 (anti-CREB-2 H-290), Anti-Xbp1, anti- CHOP/GADD153 and anti-Actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ire1α, anti-Perk antibodies was obtained from Cell Signaling (Danvers, MA). Anti-Hif1α, Anti-phospho Ire1 antibodies was obtained from Novus Biologicals (Littleton, CO).
Quantitative Polymerase Chain Reaction
Total RNA was isolated from the cells by using the RNeasy Mini Kit (Qiagen, Valencia, CA). 1 µg of total RNA from cells was reverse transcribed with Oligo-dT primer (Promega, Madison, WI). For the thermal cycle reaction, the iQ5 system (BioRad, Hercules, CA) was used at 95°C for 10 min, then 40 cycles at 95°C for 10 sec, and at 55°C for 30 sec. The relative amount for each transcript was calculated by a standard curve of cycle thresholds for serial dilutions of cDNA sample and normalized to the amount of the house-keeping beta-actin levels. The polymerase chain reaction (PCR) was performed in triplicate for each sample; all experiments were repeated three times. Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used for the quantitative PCR. PCR primer sequences are listed in Table S1
mRNA Stability Assay
Cellular mRNA transcription was attenuated by treating cells with 5 µg/ml Actinomycin D (Sigma A-4262) for 1 hr. followed by treatment with thapsigargin (0.2 µM) for different times. Total RNA was collected and quantitative PCR method described above was employed to measure levels of VEGFA gene transcripts. Time point zero for each condition was standardized to 1 and the subsequent rate of degradation of mRNA was measured.
Mouse Vegfa 1 Kb (−1039 to 0) promoter and 3.3 Kb Intron (460 bp Exon 1+2891 bp Intron 1) was cloned from Mouse BAC clone RP23 (Invitrogen, Carlsbad, CA) into Xho1/HindIII site of pGL3 luciferase vector (Promega, Madison, WI). 293T cells were transfected with both reporters as well as mock pGL3 vector along with Atf4, processed-Xbp1, processed-Atf6 (Δ373) and Atf5 by Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA). Twenty-four hrs post-transfection, lysates were prepared using a Luciferase Assay System kit (Promega, Madison, WI) according to manufacturer's protocol. The light produced from the samples was read by a standard plate reading luminometer. Each sample was read in triplicate and normalized against the signal produced from mock wells. β-galactosidase activity was measured by β-Gal Reporter Gene Assay, chemiluminescent (Roche Diagnostics, Mannheim, Germany). The assay was performed independently three times.
Chromatin Immunoprecipitation (ChIP)
Mouse neuro2a cells were transfected with pFlag-CMV-2 and processed-Xbp1 expression plasmids by Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA). After 36 hrs, cells were treated with or without thapsigargin for further 6 hrs and fixed in 1% formaldehyde. Untransfected Neuro2a cells were also treated with or without thapsigargin for 6 hrs and fixed in 1% formaldehyde ChIPs were performed using SimpleChIP™ Enzymatic Chromatin IP Kit (Agarose Beads) (Cell Signaling, Danvers, MA) as per manufacturer's recommendation. Antibodies against Xbp1 and ATF4 from Santa Cruz Biotechnology (Santa Cruz, CA) as well as a negative control, rabbit IgG, were used for ChIP assay. Purified DNA from cross-linked cells was dissolved in 25 µl TE; 2 µl was used for PCR. Inputs consisted of 10% chromatin before immunoprecipitation. Quantitative PCRs were performed as described in Quantitative polymerase chain reaction section using following primer sets: for mouse Vegfa promoter, ATTTCCTGGGAAAGGGAATTG and TCCACGGCCTCAAAATTATC; for mouse Vegfa intron 1, GCCACAGTGTGACCTTCAGA and CGTGGAGAAAGGGAACAGAA.
WT, Ire1α−/−, Perk−/−, Ire1 rescued and Perk rescued mouse embryonic fibroblasts were treated with media without glucose as indicated. Both supernatant medium as well as whole cell lysates were collected. Mouse VEGF secreted protein from supernatant and lysates were measured via ELISA analysis using Bio-Plex 200 System (BioRad, Hercules, CA) at the UMass Mouse Phenotyping Center at University of Massahusetts Medical School, Worcester, MA.
Ire1α+/− mice on 129 background are maintained at the animal medicine core at UMass Medical School (Worcester, MA).