Journal: Nutrients

Article Title: Identification of Amino Acids That Regulate Angiogenesis and Alter Pathogenesis of a Mouse Model of Choroidal Neovascularization

doi: 10.3390/nu17183006

Figure Lengend Snippet: ATF4-dependent endothelial VEGFA expression and angiogenesis under Met, Lys, and Thr restriction. ( A – F ) Western blot ( A ) and quantification ( B – F ) of eIF2α (p-S51, total), ATF4, GCN2 (p-T899, total), PERK (p-T982, total), PKR (p-T446, total), HRI, AKT (p-S473, total), HIF1α and mTORC1 pathway in HRMVECs cell line cultured in normal control (NC), methionine-deficient (-Met), tryptophan-deficient (-Trp), lysine-deficient (-Lys), valine-deficient (-Val), isoleucine-deficient (-Ile), leucine-deficient (-Leu), isoleucine-deficient (-Ile), phenylalanine-deficient (-Phe) or threonine-deficient (-Thr) media for 6 h (n = 3 replicates). ( G ) Relative ATF4 mRNA expression in HRMVECs cell line incubated in the indicated media for 6 h (n = 4 replicates). ( H ) Tube formation assay: representative capillary-like structures in ATF4 KD, CTH KD, and vector control in HRMVECs cell line incubated in normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys) or threonine-deficient (-Thr) media for 6 h (n = 4 replicates). Scale bar, 100 μm. ( I – L ) Quantifying the number of master junctions, meshes, tubes, and the length of tubes in ATF4 KD, and vector control incubated in the indicated media for 6 h (n = 4 replicates). ( M ) VEGFA secreted protein concentration in the media of HRMVECs cell line in ATF4 KD, and vector control incubated in the indicated media for 6 h (n = 4 replicates). Data are represented as means ± SEM. Asterisks indicate the significance of the difference by one-way ANOVA with Dunnett’s multiple comparisons test or two-way ANOVA with Bonferroni’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Proteins were transferred to PVDF membrane (Sigma-Aldrich, St. Louis, MO, USA) and blotted for ATF4 (11815, Cell Signaling Technology, Danvers, MA, USA), HIF1a (36169, Cell Signaling Technology), p-eIF2a (9712, Cell Signaling Technology), total eIF2a (9722, Cell Signaling Technology), HRI (A3415, ABclonal, Woburn, MA, USA), total PKR (A19545, ABclona), p-PKR (AP1134, ABclonal), total PERK (A188196, ABclonal), p-PERK (AP1501, ABclonal), total GCN2 (3302, Cell Signaling Technology), p-GCN2 (AP1356, ABclonal), p-AKT (9271, Cell Signaling Technology), total AKT (9272, Cell Signaling Technology), p-mTORC1 (2971, Cell Signaling Technology), total mTORC1 (2972, Cell Signaling Technology), p-P70 S6k (9204, Cell Signaling Technology), total P70 S6k (9202, Cell Signaling Technology), p-4E-BP1 (9451, Cell Signaling Technology), total 4E-BP1 (9452, Cell Signaling Technology), and Actin (4970, Cell Signaling Technology) and secondarily with HRP-conjugated anti-rabbit antibody (Cell Signaling Technology).

Techniques: Expressing, Western Blot, Cell Culture, Control, Incubation, Tube Formation Assay, Plasmid Preparation, Protein Concentration

Journal: Nutrients

Article Title: Identification of Amino Acids That Regulate Angiogenesis and Alter Pathogenesis of a Mouse Model of Choroidal Neovascularization

doi: 10.3390/nu17183006

Figure Lengend Snippet: HRI-dependent endothelial VEGFA expression and angiogenesis under electron transport chain inhibition. ( A ) Rectome pathways enrichment analysis of Robust DEGs, and the red box highlights the signal pathways exhibiting consistent alterations. ( B ) Heatmap of DEGs for GSE23447 , GSE211583 , and GSE233164 datasets. ( C ) The Column Chart Illustrating DEGs in the GSE94019 Dataset. ( D – G ) OCR in HRMVECs cell line pretreated for 16 h with normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys), or threonine-deficient (-Thr) media (n = 5 replicates). OCR ( D ), basal respiration ( E ), maximal respiration ( F ), ATP production ( G ). ( H – J ) Western blot ( H ) and quantification ( I , J ) of eIF2α (p-S51, total), ATF4, AKT (p-S473), HRI, PKR, PERK, and GCN2 in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line treated with Oligomycin (1 μmol/L), Rotenone (0.5 μmol/L), and FCCP (2 μmol/L) for 6 h (n = 3 replicates). ( K ) Relative ATF4 mRNA expression in HRMVECs cell line treated with Oligomycin (1 μmol/L), Rotenone (0.5 μmol/L), and FCCP (2 μmol/L) for 6 h (n = 4 replicates). ( L ) Tube formation assay: representative capillary-like structures in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line treated with Oligomycin (1 μmol/L), Rotenone (0.5 μmol/L), and FCCP (2 μmol/L) for 6 h (n = 4 replicates). Scale bar, 100 μm. ( M – P ) Quantifying the number of master junctions, meshes, tubes, and the length of tubes in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line treated with Oligomycin (1 μmol/L), Rotenone (0.5 μmol/L), and FCCP (2 μmol/L) for 6 h (n = 4 replicates). Data are represented as means ± SEM. Asterisks indicate the significance of the difference by unpaired two-tailed Student’s t -test, one-way ANOVA with Dunnett’s multiple comparisons test, or two-way ANOVA with Bonferroni’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Proteins were transferred to PVDF membrane (Sigma-Aldrich, St. Louis, MO, USA) and blotted for ATF4 (11815, Cell Signaling Technology, Danvers, MA, USA), HIF1a (36169, Cell Signaling Technology), p-eIF2a (9712, Cell Signaling Technology), total eIF2a (9722, Cell Signaling Technology), HRI (A3415, ABclonal, Woburn, MA, USA), total PKR (A19545, ABclona), p-PKR (AP1134, ABclonal), total PERK (A188196, ABclonal), p-PERK (AP1501, ABclonal), total GCN2 (3302, Cell Signaling Technology), p-GCN2 (AP1356, ABclonal), p-AKT (9271, Cell Signaling Technology), total AKT (9272, Cell Signaling Technology), p-mTORC1 (2971, Cell Signaling Technology), total mTORC1 (2972, Cell Signaling Technology), p-P70 S6k (9204, Cell Signaling Technology), total P70 S6k (9202, Cell Signaling Technology), p-4E-BP1 (9451, Cell Signaling Technology), total 4E-BP1 (9452, Cell Signaling Technology), and Actin (4970, Cell Signaling Technology) and secondarily with HRP-conjugated anti-rabbit antibody (Cell Signaling Technology).

Techniques: Expressing, Inhibition, Control, Western Blot, Plasmid Preparation, Tube Formation Assay, Two Tailed Test

Journal: Nutrients

Article Title: Identification of Amino Acids That Regulate Angiogenesis and Alter Pathogenesis of a Mouse Model of Choroidal Neovascularization

doi: 10.3390/nu17183006

Figure Lengend Snippet: HRI-independent endothelial VEGFA expression and angiogenesis under Met, Lys, and Thr restriction. ( A ) Western blot and quantification ( B – F ) of eIF2α (p-S51, total), ATF4, AKT (p-S473), GCN2 (p-T899, total), HRI, PKR, and PERK in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line incubated in normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys), or threonine-deficient (-Thr) media for 6 h (n = 3 replicates). ( G ) Relative ATF4 mRNA expression in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line incubated in normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys), or threonine-deficient (-Thr) media for 6 h (n = 4 replicates). ( H ) Tube formation assay: representative capillary-like structures in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line incubated in normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys), or threonine-deficient (-Thr) media for 6 h (n = 4 replicates). Scale bar, 100 μm. ( I – L ) Quantifying the number of master junctions, meshes, tubes, and the length of tubes in HRI KD, PKR KD, PERK KD, GCN2 KD, and vector control HRMVECs cell line incubated in normal control (NC), methionine-deficient (-Met), lysine-deficient (-Lys), or threonine-deficient (-Thr) media for 6 h (n = 4 replicates). Data are represented as means ± SEM. Asterisks indicate the significance of the difference by two-way ANOVA with Bonferroni’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Proteins were transferred to PVDF membrane (Sigma-Aldrich, St. Louis, MO, USA) and blotted for ATF4 (11815, Cell Signaling Technology, Danvers, MA, USA), HIF1a (36169, Cell Signaling Technology), p-eIF2a (9712, Cell Signaling Technology), total eIF2a (9722, Cell Signaling Technology), HRI (A3415, ABclonal, Woburn, MA, USA), total PKR (A19545, ABclona), p-PKR (AP1134, ABclonal), total PERK (A188196, ABclonal), p-PERK (AP1501, ABclonal), total GCN2 (3302, Cell Signaling Technology), p-GCN2 (AP1356, ABclonal), p-AKT (9271, Cell Signaling Technology), total AKT (9272, Cell Signaling Technology), p-mTORC1 (2971, Cell Signaling Technology), total mTORC1 (2972, Cell Signaling Technology), p-P70 S6k (9204, Cell Signaling Technology), total P70 S6k (9202, Cell Signaling Technology), p-4E-BP1 (9451, Cell Signaling Technology), total 4E-BP1 (9452, Cell Signaling Technology), and Actin (4970, Cell Signaling Technology) and secondarily with HRP-conjugated anti-rabbit antibody (Cell Signaling Technology).

Techniques: Expressing, Western Blot, Plasmid Preparation, Control, Incubation, Tube Formation Assay

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA suppresses B56α expression and promotes BCL2 phosphorylation. A, Western blot analysis was performed using antibodies against PKR, B56α, p-eIF2 α, eIF2 α, BCL2, and actin on total lysate (1 × 106 cell equivalents) from REH cells transfected with control shRNA or PKR shRNA. B, REH cells transfected with control shRNA or REH cells transfected with PKR shRNA were labeled with [32P]orthophosphoric acid (32P). BCL2 was immunoprecipitated using polyclonal rabbit antisera (Santa Cruz Biotechnology), electrophoresed using 10% SDS-PAGE, and transferred to a nitrocellulose filter, and phosphorylated bands were detected by autoradiography. The identity of the BCL2 32P-labeled band was established by using a mouse monoclonal antibody against BCL2 (Dako) on the same filter. C, Western blot analysis was performed using antibodies against B55, PP2A/A, and GAPDH on total lysate (1 × 106 cell equivalents) from REH cells transfected with control shRNA or PKR shRNA.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Phospho-proteomics, Western Blot, Transfection, Control, Labeling, Immunoprecipitation, SDS Page, Autoradiography

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA suppresses mitochondrial PP2A activity and promotes chemoresistance. A, mitochondrial (Mito) and nuclear (Nuc) PP2A activity was determined using a molybdate dye assay for REH cells transduced with control shRNA or with shRNA against PKR. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from PP2A activity from REH cells transduced with control shRNA (p < 0.05, standard t test). B, REH parental cells were treated with 10 μm PKR inhibitor for 3 h, and mitochondrial PP2A activity was determined using the molybdate dye assay. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from PP2A activity in untreated REH cells (p < 0.05, standard t test). C, Western blot analysis was performed to determine PKR activity in untreated REH cells and cells treated with 10 μm Calbiochem PKR inhibitor for 3 or 20 h using antibody against p-PKR. PKR antibody was used to measure total kinase.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Activity Assay, Transduction, Control, Western Blot

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: Suppression of PKR expression by shRNA promotes chemoresistance. Apoptosis of shRNA-transduced REH cells treated with 1 μm etoposide for 24 h was examined using FACScan analysis of sub-G0 content of propidium iodide-stained cells. Error bars represent the mean ± S.D. from three separate experiments. *, statistically significant differences from cell viability in untreated REH cells (p < 0.05, standard t test).

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, shRNA, Staining

Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: eIF2α and AKT promote B56α protein expression. A, Western blot analysis was performed to determine the effect of salubrinal on B56α expression. HA antibody was used to detect HA-GFP protein in REH/GFP cells and exogenous HA-B56α protein in REH/WT B56α cells on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 75 μm salubrinal for 24 h. Antibodies against eIF2α, p-eIF2α, and tubulin were used as controls. B, Western blot analysis was performed to determine the effect of LY294002 on B56α expression in REH cells transfected with control shRNA and REH cells transfected with PKR shRNA. Rabbit polyclonal antibody against B56α protein was used on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 1 μm LY294002 for 24 h. Antibodies against p-AKT, AKT, and tubulin were used as controls.

Article Snippet: A shRNA plasmid targeting PKR (designated TI379192) is commercially available from OriGene.

Techniques: Expressing, Western Blot, Transfection, Control, shRNA