Supplemental Materials
Molecular Biology of the Cell
Wei et al.
SUPPLEMENTARY DATA
FIG S1. Effects of GCN2 expression silencing on ouabain, oleandrin and foliandrin‐induced cell apoptosis. A549 cells transfected with control or GCN2 siRNA were treated in the absence or presence of these chemicals at 200 nM for 24 h. Cell apoptosis were expressed as the mean±SD; n=3, ns means no significance, *P<0.05, ***P<0.001. as compared with control cells. The siRNA‐mediated depletion of GCN2 was examined by immunoblot analysis using the indicated antibodies on the right panel. 1
FIG S2. Effect of the knockdown of GCN2 expression on GCN2 ubiquitination. The A549 cells were co‐transfected with HA‐Ub plus GCN2 or control siRNA. After 30 h, cells were further treated with MG132 (50 μM) for 5 h before being harvested. Lysates were immunoprecipitated using anti‐HA IgG, followed by immunoblot analysis using anti‐GCN2 and anti‐tubulin. Similar results with three independent experiments are shown. 2
FIG S3. Effect of ouabain on the ubiquitination of the phosphorylated GCN2. A549 cells after transfection with HA‐tagged GCN2 (WT) or GCN2 (T899A) were treated in the absence or presence of 100 nM ouabain for 6 h. Lysates were immunoprecipitated using anti‐HA IgG, and the immunpellets were detected by immunoblot analysis with the indicated antibodies. 3
FIG S4. Effects of GCN2(WT) and GCN2(T899D) on cell apoptosis. A549 cells were transfected with YFP tagged GCN2 (WT), GCN2 (T899D) or empty vector. After 36 h, further treated in the absence or presence of 200 nM ouabain for an additional 24 h. Cells were collected and stained with Annexin V‐RFP, then analyzed the YFP and RFP both positive cells by flow cytometry analysis. n=3, ns means no significance, ***p<0.001, as compared with control cells. 4
TABLE S1. Effect of ouabain on the mRNA expressions of CHOP, amino acid synthetases and transporters by microarray assay * Genes Ouabain (400 nM)/Control C/EBP homologous protein 5.146874 Genes Ouabain (100 nM)/Control Amino acid Synthetases asparagine synthetase 4.347826 cysteinyl‐tRNA synthetase 2.415459 threonyl‐tRNA synthetase 2.114165 tyrosyl‐tRNA synthetase 2.008032 Amino acid transporters solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 "solute carrier family 7 (cationic amino acid transporter, y+ system), member 5 " "solute carrier family 1 (glial high affinity glutamate transporter), member 3 " "solute carrier family 1 (neutral amino acid transporter), member 5 " "solute carrier family 7 (cationic amino acid transporter, y+ system), member 6 " 5
2.04499 2.074689 2.544529 2.762431 2.906977 "solute carrier family 6 (neutral amino acid transporter), member 15 " "solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 " "solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 " "solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 " 2.816901 3.076923 3.194888 3.333333 * A549 cells were treated with 100 nM or 400 nM ouabain for 12 h. After treatments, RNA was extracted, and the hybridization was performed at 42 for 14h with NimbleGen Hybridization System. The arrays were scanned using MS200 scanner (NimbleGen) with 2 µm resolution, and NimbleScan software was used to extract fluorescent intensity raw data from the scanned images. The ratio of gene expressions in 100 nM ouabain‐treated cells relative to control cells is shown. 6

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