Genome-wide shRNA screen identifies genes required for arsenite-induced ER stress. A, CHOP promoter mCherry reporter construct. A human CHOP promoter fragment containing the CEBP-ATF composite site, AP-1 site, and ERSE element, which are involved in arsenite and ER stress induction of CHOP (64–66), was used to drive fluorescent mCherry expression. B, dose-dependent mCherry induction in a clonal HEK293 stable reporter cell line. Cells were treated with arsenite (5 μm (blue), 15 μm (red), or 30 μm (green)) or tunicamycin (5 μg/ml (purple)) for 16 h or left untreated (black) and then analyzed by FACS. Silver, control cells with no reporter. C, scheme of screen for genes required for reporter induction. Pools of shRNA lentivirus were stably transduced into the reporter cell line. Following arsenite treatment, weak activators were isolated by FACS, which were then regrown and subjected to further rounds of induction and FACS for enrichment of this “weak activator” cell population. shRNAs from this population were PCR-cloned and sequenced to identify target genes. D, primary screen results. Sequencing of 268 shRNA clones identified 168 annotated genes, which were selected for further analysis (for details, see “Results”). E, gene hits validated by shRNA and siRNA. Genes whose knockdown by shRNA and siRNA reduced arsenite-induced reporter activity or endogenous CHOP expression, respectively, were considered validated.

Reduced SNAT2 expression, inhibition of transporter activity, or glutamine deprivation suppresses arsenite-induced ER stress. A and B, SNAT2-targeting siRNA suppresses arsenite induction of endogenous CHOP and spliced XBP1 in HEK293T cells. Gene expression levels were analyzed by qPCR. Cells were untreated (UT) or treated with arsenite (As) (15 μm, 4 h). C, SNAT2-targeting siRNAs cannot suppress tunicamycin-induced CHOP. Cells were treated as in A but with tunicamycin (Tm) (5 ng/ml, 4 h). n = 2. D, SNAT2-targeting siRNA suppresses arsenite induction of CHOP protein. Cells were treated as in A. CHOP protein levels were analyzed by immunoblot and normalized to β-actin. Immunoblot analysis of SNAT2 protein levels confirmed SNAT2 knockdown. The arrowhead indicates a nonspecific band. *, p < 0.01 relative to arsenite-treated control. n = 3. E–G, inhibition of SNAT2 with saturating MeAIB (10 mm) or glutamine deprivation suppresses arsenite induction of CHOP and GRP78 in 3T3L1 adipocytes. Gene expression levels were analyzed by qPCR, E and F, CHOP protein levels were analyzed by immunoblot. G, cells were pretreated with MeAIB or glutamine-free medium for 3 h and then co-treated with arsenite (50 μm, 4 h). *, p < 0.01 relative to arsenite-treated control. n = 3. Error bars, S.E.

SNAT2 expression and activity is induced by arsenite in an ATF4-dependent manner. A and B, arsenite induces SNAT2 expression. 3T3L1 adipocytes were treated with arsenite (50 μm) or water for the times indicated (A) or with arsenite (16 h) at the concentrations indicated (B). SNAT2 Western blot (top) and qPCR analysis (bottom) (n = 2). The arrowhead denotes a nonspecific band. C, other forms of ER stress do not induce SNAT2 protein levels. 3T3L1 adipocytes were treated with tunicamycin (Tm) (5 μg/ml) or thapsigargin (Tg) (0.5 μm) or DMSO for the times indicated. CHOP induction demonstrates ER stress activation by these stressors. D, arsenite increases [¹⁴C]MeAIB uptake. 3T3L1 adipocytes were treated with various stimuli, and then uptake was measured. Cells were treated with arsenite (6 h) at the indicated concentrations. Tunicamycin (5 μg/ml, 6 h) and thapsigargin (0.5 μm, 6 h) do not increase uptake. Insulin (Ins) (100 ng/ml, 1.5 h) is a positive control. Preincubation with saturating cold MeAIB (Me) (10 mm, 3 h) inhibits uptake. n = 3. E, arsenite stimulates a sustained increase in [¹⁴C]MeAIB uptake. 3T3L1 adipocytes were treated with arsenite (50 μm) for up to 24 h, and then the uptake assay was performed. n = 3. F, N-acetyl cysteine (NAC) or 4-phenylbutyric acid (4PBA) inhibits arsenite induction of SNAT2. 3T3L1 adipocytes were pretreated with N-acetyl cysteine (10 mm), 4-phenylbutyric acid (10 mm), or water (con) for 30 min and then co-treated with arsenite (50 μm, 6 h). SNAT2 expression was analyzed by qPCR. *, p < 0.05 relative to arsenite-treated control. n = 2. UT, untreated. G, ATF4-targeting siRNA blunts SNAT2 induction by arsenite. SNAT2 expression was analyzed by qPCR (bottom) and immunoblot analysis (top). ATF4 Western blot demonstrates knockdown. HEK293T cells were untreated or treated with arsenite (15 μm, 16 h). *, p = 0.01 relative to arsenite-treated NTsi control. n = 3. H, SNAT2- and ATF4-targeting siRNAs inhibit arsenite-induced [¹⁴C]MeAIB uptake. HEK293T cells were untreated or treated with arsenite (15 μm, 4 h), and then the uptake assay was performed. n = 3. Error bars, S.E.

mTOR activation links SNAT2 activity and arsenite-induced ER stress. A and B, arsenite induces S6K1 phosphorylation. 3T3L1 adipocytes were serum-starved (16 h) and then treated with arsenite (50 μm) for the times indicated. A, arsenite induction of S6K1 phosphorylation is dose-responsive and rapamycin-sensitive. B, starved 3T3L1 adipocytes were treated with arsenite at the concentrations indicated for 6 h. Rapamycin (Rap) (20 nm, 30 min) pretreatment can inhibit S6K1 phosphorylation induced by arsenite or insulin (Ins) (100 nm, 90 min). Phospho-S6K1, total S6K1, phospho-AKT, and total AKT were analyzed by immunoblot. UT, untreated. C, inhibition of System A or L transport, glutamine deprivation, and rapamycin suppresses arsenite-induced CHOP. 3T3L1 adipocytes were pretreated with MeAIB (10 mm, 3 h), glutamine-free medium (3 h), BCH (10 mm, 3 h), or rapamycin (20 nm, 30 min) and then co-treated with arsenite (50 μm, 4 h). CHOP induction was analyzed by immunoblot. D, rapamycin or BCH (System L transport inhibitor) suppresses arsenite-induced ER stress. HEK293T cells were pretreated with rapamycin (20 nm, 30 min) or BCH (10 mm, 3 h) and then co-treated with arsenite (15 μm, 3 h). CHOP expression was analyzed by qPCR (top) and immunoblot. *, p < 0.01 relative to arsenite-treated control. n = 3. E, siRNA knockdown of SNAT2 reduces arsenite-induced S6K1 phosphorylation. HEK293T cells transfected with SNAT2-targeting siRNA were serum-starved (16 h) and then treated with arsenite (15 μm, 3 h). Phospho-S6K1 values were normalized to untreated, non-targeting siRNA control. *, p < 0.05 relative to arsenite-treated control. n = 3. F, MeAIB, BCH, or glutamine deprivation suppresses arsenite-induced S6K1 phosphorylation. Serum-starved (16 h) HEK293T cells were pretreated with MeAIB, BCH, or glutamine-free medium, as in E, and then co-treated with arsenite (15 μm) for 3 h. Phospho-S6K1 levels were normalized to untreated control. *, p < 0.05 relative to arsenite-treated control. n = 2. G, proposed model of SNAT2 activation of mTOR and ER stress during arsenite exposure. Error bars, S.E.