Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 2. Effects of RuCO on PERK, eIF2, ATF4, HO-1, and Nrf2 in HUVECs. A, Cells were exposed to 20 mol/L RuCO, 10 mol/L TG, or 10 mol/L TM for indicated periods of time. B, Cells were exposed to 20 mol/L RuCO or 20 mol/L arsenite (positive control) for 160 minutes. C, Cells transfected with siRNA against eIF2 were exposed to RuCO for 1 hour. D, Cells were exposed for 1 hour (PERK and eIF2 phosphorylations) or 6 hours (ATF4 expression) to RuCO in the presence or absence of 50 g/mL Hb. E, Cells trans- fected with ether PERK siRNA or eIF2 siRNA were exposed to RuCO for 1 hour (Nrf2 nuclear translocation) or 6 hours (HO-1 expres- sion). NF indicates nuclear fraction. Western blot analysis and densitometry analysis were performed as described in Materials and Methods. Blots shown are representative of 3 independent experiments. Values are meansSD from 3 independent experiments. *P0.05 with respect to each untreated group.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Positive Control, Transfection, Expressing, Translocation Assay, Western Blot

Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 6. Effects of RuCO and RuCO-induced HO-1 on TG-, TM-, and HCys-induced CHOP in HUVECs. A, Cells were preincubated for 6 hours with 20 mol/L RuCO and then were exposed for 6 hours to 10 mol/L TG, 10 mol/L TM, or 50 mol/L HCys. B, Cells trans- fected with either PERK siRNA or eIF2 were preincubated for 6 hours without or with RuCO and then were exposed to TG for 6 hours. C, Normal cells and the cells transfected with siRNA against HO-1 were preincubated for 6 hours with RuCO or 10 mol/L CoPP in the presence or absence of 50 g/mL Hb and then exposed to TG for 6 hours. D, Normal cells and the cells transfected with PERK siRNA, HO-1 siRNA, or eIF2 siRNA were preincubated for 6 hours with or without RuCO and then were exposed to TG for 18 hours. Western blot analysis for CHOP, HO-1, ATF6, and Xbp-1 and MTT assay for cell viability were performed as described in Materi- als and Methods. Blots shown are representative of 3 independent experiments. Each bar represents meanSD from 3 independent experiments. *P0.05 with respect to each untreated group. *P0.05.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, MTT Assay

Journal: Experimental & molecular medicine

Article Title: Sestrin2 protects against cholestatic liver injury by inhibiting endoplasmic reticulum stress and NLRP3 inflammasome-mediated pyroptosis.

doi: 10.1038/s12276-022-00737-9

Figure Lengend Snippet: Fig. 3 Bile acids upregulate Sestrin2 expression via an ATF4- and C/EBP-β-dependent mechanism. A, B HepG2 cells were treated with 200 µM CDCA or 750 µM CA for the indicated times. Cell lysates were immunoblotted with anti-p-eIF2α, anti-eIF2α, and anti-ATF4 antibodies. C Immunofluorescence staining of PDI (green) in HepG2 cells treated with 200 µM CDCA or 750 µM CA for 9 h (n = 3–4). Nuclei were stained with DAPI (blue). Scale bars, 10 μm. D, E Liver tissues were collected from mice 3 days after sham or BDL surgery (n = 4–5 mice per group) and analyzed by immunoblotting with the indicated antibodies. Band intensities were quantified and normalized to GAPDH or total protein intensities. F Immunohistochemical analysis of BiP in liver tissues from mice 3 days after sham or BDL surgery (n = 4–5 mice per group). The boxed areas are magnified in the bottom panels. Scale bars, 50 μm; 10 μm (insets). G, H HepG2 cells were infected with lentiviruses expressing shRNAs targeting luciferase (sh-Luc) or ATF4 (sh-ATF4) and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-ATF4 antibodies. I, J HepG2 cells were infected with lentiviral sh-Luc or sh-C/EBP-β and treated with 200 µM CDCA or 750 µM CA for 9 h. Cell lysates were immunoblotted with anti-Sestrin2 and anti-C/EBP-β antibodies. GAPDH or β-actin served as loading controls. Numbers below the immunoblot bands indicate fold changes normalized to the control band intensities. The data are representative of one (D–F) or at least three (A–C, G–J) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t test).

Article Snippet: Immunoblotting was performed using antibodies against human Sestrin2 (Proteintech Group, USA), mouse Sestrin2 (Dr. Jun Hee Lee, University of Michigan); cleaved caspase-3, phospho-eIF2α, PERK, phospho-p70 S6 kinase, p70 S6 kinase, phospho-S6 ribosomal protein, S6 ribosomal protein, phospho-AMPK, AMPK, TSC2, NLRP3 (Cell Signaling Technology, USA), ATF4, C/EBP-β, eIF2α, caspase-1, ASC (Santa Cruz Biotechnology, USA); GSDMD (Abcam), GAPDH (Aviva Systems Biology, USA), and β-actin (Developmental Studies Hybridoma Bank, University of Iowa).

Techniques: Expressing, Staining, Western Blot, Immunohistochemical staining, Infection, Luciferase, Control

Journal: BioMed Research International

Article Title: Role of PKR in the Inhibition of Proliferation and Translation by Polycystin-1

doi: 10.1155/2019/5320747

Figure Lengend Snippet: Effects of PKR/eIF2α overexpression on PC1-5TMC-inhibited proliferation and translation in HeLa cells . (a) Effects of eIF2 α overexpression on PC1-5TMC-inhibited proliferation. Transfected with GFP or PC1-5TMC, HeLa cells were cotransfected with eIF2 α . They were then plated in multiple wells of a 96-well plate and grown for 24 hr for cell proliferation assay; cells from the sample preparations were collected for immunoblotting. 5TMC, GFP-tagged PC1-5TMC; eIF2 α , WT eIF2 α ; Ctrl, GFP. Upper panel, averaged data (N=4, ∗ p< 0.05, ∗∗ p< 0.01). Lower panel, effectiveness of transfection of eIF2 α assessed by immunoblotting. (b) Effects of PKR overexpression on PC1-5TMC-inhibited protein synthesis. After transfected with GFP or PC1-5TMC, HeLa cells were cotransfected with WT PKR. They were then used for 35 S pulse labeling assays followed by SDS-PAGE and immunoblotting assays with the antibodies against P-eIF2 α , total eIF2 α , and P-PKR. β -actin served as loading control.

Article Snippet: Plasmid eIF2 α was from Santa Cruz (Santa Cruz, CA).

Techniques: Over Expression, Transfection, Proliferation Assay, Western Blot, Labeling, SDS Page, Control

Journal: PloS one

Article Title: Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

doi: 10.1371/journal.pone.0040994

Figure Lengend Snippet: Figure 3. Phosphorylation of eIF2a during glucose deprivation. Western blots of extracts from HepG2 cells incubated for 6 hr in media having either 0 mM or 5 mM glucose, and without (-) or with (+) 10 mM Sal003 as shown above lanes. Antibodies against phospho-eIF2a (eIF2a,P), total eIF2a and GAPDH as a loading control, were used as indicated to the right of each blot. doi:10.1371/journal.pone.0040994.g003 Figure 4. Glucose concentration affects secreted FVII antigen levels. HepG2 cells were cultured for 24 hr in media supplemented with 10% or 1% fetal bovine serum and either high (25 mM), standard (5 mM), or low/no (1 mM or 0 mM) glucose. The concentration of secreted FVII antigen, expressed as ng per ml, was determined by ELISA. For each experimental set, the average amount secreted by cells in 25 mM glucose was considered 100%, and amounts secreted at the lower glucose concentrations were expressed as percentages +/2 SD relative to that. The number of replicates assayed at each condition is shown below the bars. Reducing the concentration of glucose significantly increased the amount of FVII secreted for each experi- mental set (p,0.001). doi:10.1371/journal.pone.0040994.g004

Article Snippet: Thapsigargin (Calbiochem EMD Millipore, Billerica, MA) at 500 nM final and Sal003 (Santa Cruz Biotechnology, Santa Cruz, CA) at 10 mM final were added to cultures for 6 hr where indicated.

Techniques: Phospho-proteomics, Western Blot, Incubation, Control, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular Cell

Article Title: System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection

doi: 10.1016/j.molcel.2019.01.017

Figure Lengend Snippet: Application of RIC to HEK293 Cells Infected with SINV (A) Schematic representation of cRIC. (B) Schematic representation of SINV and chimeric SINV-mCherry genomes. (C) Analysis of the proteins synthesized in uninfected and SINV-infected HEK293 cells by [ 35 S]-Met/Cys incorporation for 1 h followed by autoradiography. (D) Analysis of total and phosphorylated eIF2α by western blotting. (E) Silver staining analysis of the “inputs” (i.e., total proteome, left) and eluates (i.e., RBPome, right) of a representative RIC experiment in SINV-infected cells. (F) qRT-PCR analysis of the eluates of a representative RIC experiment using specific primers against SINV RNAs, actb and gapdh (for normalization) mRNAs. Error bars represent SE. hpi, hours post-infection; MW, molecular weight. See also Figure S1 .

Article Snippet: EIF2α , Santa Cruz Biotechnology , Cat# sc-11386; RRID: AB_640075.

Techniques: Infection, Synthesized, Autoradiography, Western Blot, Silver Staining, Quantitative RT-PCR, Molecular Weight

Journal: Molecular Cell

Article Title: System-wide Profiling of RNA-Binding Proteins Uncovers Key Regulators of Virus Infection

doi: 10.1016/j.molcel.2019.01.017

Figure Lengend Snippet:

Article Snippet: EIF2α , Santa Cruz Biotechnology , Cat# sc-11386; RRID: AB_640075.

Techniques: Virus, Recombinant, Proliferation Assay, CRISPR, Cloning, Plasmid Preparation, Sequencing, Software

Journal: The Journal of Biological Chemistry

Article Title: Autoantibodies against the cell surface–associated chaperone GRP78 stimulate tumor growth via tissue factor

doi: 10.1074/jbc.M117.799908

Figure Lengend Snippet: Treatment with anti-GRP78 AutoAbs increases the expression of TF and UPR markers in DU145 human prostate cancer cells. A, flow cytometry analysis of csGRP78 amounts in DU145 cells. B, pathological doses of anti-GRP78 AutoAbs (60 μg/ml) increase protein expression of both TF and markers of UPR activation (PDI, IRE1, and phospho-eIF2α), compared with non-treated (0 μg/ml) cells or cells treated with a normal dose of anti-GRP78 AutoAbs (5 μg/ml). β-Actin was used as a loading control. C, protein bands of the immunoblot in B were quantified with ImageJ, and values were normalized to β-actin. Error bars are ± S.D. p-eIF2α, phosphorylation of the initiation factor eIF2 α. D, quantitative real-time PCR analysis of GRP78 and spliced XBP1 mRNA expression in DU145 cells treated with a pathological dose of anti-GRP78 AutoAbs (60 μg/ml). Results are expressed as -fold induction over non-treated (NT) cells (*, p < 0.05; n = 3). E, pathological doses of anti-GRP78 AutoAbs (60 μg/ml) do not increase GRP78 expression in PC-3 cells; thapsigargin (Tg; 300 nm) or tunicamycin (Tm; 2.5 μg/ml) was used as a control UPR inducer. β-Actin was used as a loading control.

Article Snippet: Membranes were blocked overnight in Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% skim milk and subsequently incubated with the following primary antibodies: anti-GRP78/Bip (catalog no. 610979, BD Transduction), anti-phospho-eIF2α (catalog no. 9721S, BD Transduction), anti-VEGF pathway sampler kit (catalog no. 8696, Cell Signaling, Danvers, MA), anti-TF (catalog no. 4502, American Diagnostica, Stamford, CT), anti-CHOP (catalog no. B3, Santa Cruz Biotechnology, Inc., Dallas, TX) followed by the appropriate HRP-conjugated secondary antibodies (Dako, Carpentaria, CA) diluted in TBST containing 1% skim milk.

Techniques: Expressing, Flow Cytometry, Activation Assay, Western Blot, Real-time Polymerase Chain Reaction