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: 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: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: Knocking down and inhibitor HBX41108 of USP7 activate ERS and NF- κ B signaling under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. (c) Quantitative measurement of (b). (d) Relative BiP and CHOP mRNA expression of wild ATDC5 cells under TNF- α -induced inflammation after 48 h chondrogenic induction in HBX41108. (e) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of wild ATDC5 cells under TNF- α -induced inflammation after 48 h chondrogenic induction in HBX41108. (f) Quantitative measurement of (e). (g) Immunofluorescent staining of p65 in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction. Scale bars = 20 μ m. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: ERS signaling inhibitor 4-PBA reverses chondrocyte proliferation, apoptosis, and inflammation caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (c) Quantitative measurement of (b). (d) Alcian blue and toluidine blue staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. Scale bars = 100 μ m. (e) Growth curves in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA, measured by CCK8-assay. (f) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (g) Col2a1, Sox9, Cleaved Caspase-3, Bax, and Bcl-2 protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (h) Quantitative measurement of (g). (i) Relative Caspase-3 activity in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (j) TUNEL staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. White arrows indicate TUNEL-positive cells. Scale bars = 50 μ m. (k) Quantitative measurement of (j). (l) Relative IL-6 , COX , NOS2 , and MMP13 mRNA expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. (m) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without 4-PBA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining, CCK-8 Assay, Activity Assay, TUNEL Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: si-CHOP reverses chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (c) Quantitative measurement of (b). (d) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (e) Col2a1, Sox9, Cleaved Caspase-3, Bax, Bcl-2, and PCNA protein expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (f) Quantitative measurement of (e). (g) Relative IL-6 , COX , NOS2 , and MMP1 3 mRNA expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. (h) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without si-CHOP. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: NF- κ B signaling inhibitor QNZ reverses chondrocyte proliferation, apoptosis, and inflammatory response caused by USP7 knockdown under TNF- α -induced inflammation. (a) Relative BiP and CHOP mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (b) p-eIF2 α , eIF2 α , ATF4, CHOP, p-p65, and p65 protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (c) Quantitative measurement of B. (d) Alcian blue and toluidine blue staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. Scale bars = 100 μ m. (e) Relative Col2a1 and Sox9 mRNA expression in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (f) Col2a1, Sox9, Cleaved Caspase-3, Bax, Bcl-2, and PCNA protein expression of in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (g) Quantitative measurement of F. (h) Relative Caspase-3 activity in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. (i) TUNEL staining in USP7 knockdown and its control groups under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. White arrows indicated TUNEL-positive cells. Scale bars = 50 μ m. (j) Quantitative measurement of (i). (k) Relative IL-6 , COX , NOS2 , and MMP1 3 mRNA expression in USP7 knockdown and its control groups under TNF- α- induced inflammation after 48 h chondrogenic induction, with and without QNZ. (l) IL-6 expression in USP7 knockdown and its control group supernatant under TNF- α -induced inflammation after 48 h chondrogenic induction, with and without QNZ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques: Expressing, Staining, Activity Assay, TUNEL Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: USP7 Attenuates Endoplasmic Reticulum Stress and NF- κ B Signaling to Modulate Chondrocyte Proliferation, Apoptosis, and Inflammatory Response under Inflammation

doi: 10.1155/2022/1835900

Figure Lengend Snippet: Schematic diagram of the effects and underlying mechanisms of USP7 on chondrocyte proliferation, apoptosis, and inflammatory response under TNF- α -induced inflammation. Under TNF- α -induced inflammation, USP7 promotes chondrocyte proliferation and suppresses chondrocyte apoptosis and inflammatory response, through inhibiting the BiP-eIF2 α -ATF4-CHOP signaling of ERS and NF- κ B signaling.

Article Snippet: The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2 α (1 : 1000, ABclonal, Wuhan, China), eIF2 α phosphorylation (p-eIF2 α , 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China).

Techniques:

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) Schematic representation of the parameters and reactions included in the mathematical model. The stress-sensing module represents the activation of stress kinases. PKR is activated by binding to dsRNA (viral stress, p-PKR*), HRI by arsenite treatment (oxidative stress, HRI*), and PERK by thapsigargin (ER stress, PERK*). Active stress kinases signal to the decision module, where upon crossing a p-eIF2α level threshold will trigger SG formation (SG-On). Elevated p-eIF2α levels activate the recovery module consisting of the GADD34 negative feedback loop, i.e., ppp15R1a promoter activation (P OFF to P ON ) with time delay (clock symbol), GADD34 transcription (mGADD34), and protein synthesis (GADD34). In turn, GADD34 dephosphorylates eIF2α and thereby resumes translation. Gray arrow, basal eIF2α dephosphorylation by CReP, the constitutive regulatory subunit of PP1. Ø, degradation. ( B and C ) Absolute quantification of eIF2α and PKR mean molecule numbers in Huh7 YFP-TIA1 cells, in the absence and presence of IFN-α. (B) Representative quantitative Western blot analysis of eIF2α and PKR. (C) Estimated eIF2α and PKR mean molecule number per cell (±SD). Number of repeats ( n ) and statistical significance compared to untreated cells (untr.) are indicated; **** P < 0.0001. ( D and E ) Determination of protein half-lives by CHX pulse experiments and Western blot analyses. (D) PKR and eIF2α half-lives ( n = 3). (E) GADD34 ( n = 4). Black line, best nonlinear fit.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activation Assay, Binding Assay, De-Phosphorylation Assay, Western Blot

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A to C ) Activation of PKR in Huh7 YFP-TIA1 cells transfected with increasing amounts of 200-bp dsRNA ( n = 3). (A) Representative Western blot analysis of p-PKR and p-eIF2α expression levels. Expression levels of β-actin served as loading control. The percentage of p-eIF2α was analyzed by Phos-tag polyacrylamide gel. (B) Shown are quantifications of mean p-PKR expression levels (±SD) normalized to the loading control and relative to untreated cells (top) and quantifications of the mean p-eIF2α percentage (±SD). Statistical significance is indicated compared to untreated cells. (C) The presence of SGs in transfected cells was analyzed by fluorescence microscopy (for each condition, n > 100). Shown are mean percentages ± SD. Statistical significance is indicated compared to untreated cells. * P < 0.05, ** P < 0.01. ( D to F ) Induction of oxidative stress in Huh7 YFP-TIA1 cells by treatment with increasing concentrations of arsenite for 45 min ( n = 3). (D) Representative Western blot and Phos-tag analyses. Shown are mean percentages ± SD of p-eIF2α (E) and SG-positive cells (for each condition, n > 100) (F). Statistical significance is indicated compared to untreated cells; **** P < 0.0001. ( G ) Dose-response analysis and determination of the p-eIF2α level that results in formation of SGs in 50% of cells upon treatment with arsenite [related to (F); n = 3] and thapsigargin (related to fig. S4, E to H; n = 3).

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Control, Fluorescence, Microscopy

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) In vitro PKR kinase assay. His-tagged PKR and His-tagged eIF2α were incubated with increasing molarities of 200-bp dsRNA ( n = 3). The top panels show representative Western blot analyses of p-PKR and p-eIF2α levels. Silver staining of proteins in the gel served as loading control. Quantifications of mean levels relative to untreated control ± SD are shown in the bottom panels. Statistical significance is indicated compared to untreated; * P < 0.05, ** P < 0.01. ( B to E ) Computational prediction of PKR activation by dsRNA. (B) Overview of the different steps tested in PKR model development. (C) Differences in the chi-square (Δχ 2 ) and corrected Akaike information criterion (ΔAICc) to the optimal PKR activation model variant (variant 3, see figs. S6 and S7). The model describing PKR dimerization upon binding to dsRNA was significantly improved by considering PKR cooperative binding to dsRNA (ΔAICc > 200). Cis and trans reactions did not improve the cooperativity model (ΔAICc = 18.4). (D) Overview of the optimal model variant. PKR monomers reversibly bind to PKR on dsRNA in a cooperative manner and form active PKR oligomers (dsR:PKRoligo). (E) Best model fits of p-PKR levels to in-cell (related to ) and in vitro kinase assays ( n = 500 multistart optimization runs). a.u., arbitrary units.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: In Vitro, Kinase Assay, Incubation, Western Blot, Silver Staining, Control, Activation Assay, Variant Assay, Binding Assay

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) FISH analysis. Top: Representative still images of uninfected and HCV-infected cells treated with IFN-α. HCV (+) ssRNA genomes, GADD34 transcripts, and total polyA-tailed mRNAs were detected by FISH. Outlined in red, SG-positive cell; outlined in white, unstressed cell. White circles indicate single transcripts. Scale bars, 20 μm. Bottom: GADD34 mean transcript levels ± SD. Statistical significance and the number of analyzed cells ( n ) are indicated at the top of the graph; ** P < 0.001, **** P < 0.0001. ( B ) Model best fits ( n = 2500 multistart optimization runs) to the percentage of SG-positive cells experimentally measured in arsenite or thapsigargin titration. ( C ) Computational simulations of dose-response curves for p-eIF2α, GADD34 mRNA, and protein and SG-positive cells in the population at steady state. Shown are percentages of maximal values as a function of different kinase activities. The reference kinase activity (10 0 ) results in 50% SG-positive cells (intermediate stress). Kinase activities <10 −1 , low to moderate stress; >2, high stress. ( D ) Model prediction: behavior of the GADD34 negative feedback loop parameters (promoter activity, mRNA, and protein) after stress release. The percentage of their maximum response over time is shown. Estimated decay processes { t 1/2 , Prom. ≈ 256 min; mRNA ≈ 200 min [from ]; protein ≈ 37 min}. ( E ) Mean expression levels ± SD of GADD34 pre-mRNA and mature mRNA upon thapsigargin treatment ( n = 3). ( F ) Model prediction: behavior of p-eIF2α levels and number of SG-positive cells after a second 1-hour stress pulse applied at different times after stress release. Phases I to III, levels of cell protection against a second stress pulse.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Infection, Titration, Activity Assay, Expressing

Journal: Science Advances

Article Title: Temporal control of the integrated stress response by a stochastic molecular switch

doi: 10.1126/sciadv.abk2022

Figure Lengend Snippet: ( A ) Computational simulations of two consecutive 1-hour stress pulses interspaced by a 5-hour recovery period. Shown is a range of stress kinase activities (stress intensity) varying between 10-fold lower (min) and 10-fold higher (max) than the reference kinase activity leading to 50% SG-positive cells. Color plots show the behavior of ppp1r15a promoter activity, concentrations of GADD34 mRNA and protein, and percentages of p-eIF2α and SG-positive cells. Graphs at the bottom reflect the behavior of the abovementioned components for one chosen stress intensity (black line, moderate stress). ( B ) Experimental validation of the predictions shown in (A). Huh7 cells were subjected to a first heat shock (HS1) at 42°C for 1 hour and immediately transferred at 37°C for recovery. Cells were harvested at the indicated time points after the first or the second heat shock (HS2) ( n = 2). Cells treated with 2 μM thapsigargin for 1 and 6 hours served as reference. Shown are representative Western blot and Phos-tag gel analyses. Bottom panels show the quantification of GADD34 expression levels normalized to the loading control and relative to untreated cells as well as the percentage of p-eIF2α. ( C ) Model prediction: behavior of the SG response and GADD34 negative feedback loop components over a 24-hour time period upon continuous stress. The color of the curves reflects different kinase activity levels. ( D ) During the adaptation to a stress stimulus, depending on PKR activity, the expression of GADD34 will result in dose-response curve shifts (hysteresis) in the percentage of p-eIF2α (top) and of SG-positive cells (bottom). The shift of the dose-response curve depends on the stress duration and intensity and is reverted after stress relief. Blue and red shared areas indicate 1σ confidence intervals of estimated PKR activities in HCV and HCV + IFN-α experiments.

Article Snippet: Lysates ( n = 8) containing 20 μg of total protein were spiked with different amounts of recombinant GST-tagged eIF2α (Abnova, 5/10/15/22/33/50/75 ng), recombinant GST-tagged PKR kinase domain (Abcam, 1/2/3/4.4/6.7/10/12.5 ng), or protein lysis buffer.

Techniques: Activity Assay, Western Blot, Expressing, Control

Journal: Drug Design, Development and Therapy

Article Title: eIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats

doi: 10.2147/DDDT.S213817

Figure Lengend Snippet: Expression changes of p-eIF2α, eIF2α, and LC3B, P62 in pulmonary arterial tissue of MCT-induced PAH rats. ( A ) Expression of eIF2α mRNA in pulmonary artery tissue. ( B ) Protein expression of p-eIF2α, eIF2α, LC3B, and p62 in pulmonary artery tissue in MCT-induced PAH rats. ( C ) The ratio of p-eIF2α/total eIF2α. ( D ) The ratio of LC3B-II/LC3B-I. ( E ) Statistical analysis of p62 gray value. The data are expressed as mean±standard error, n=8, * P <0.05 vs Control.

Article Snippet: The eIF2α siRNA were purchased from Ribobio (Guangzhou, People’s Republic of China).

Techniques: Expressing

Journal: Drug Design, Development and Therapy

Article Title: eIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats

doi: 10.2147/DDDT.S213817

Figure Lengend Snippet: The expression changes of eIF2α, p-eIF2α, LC3B, and P62 in the PDGF-induced PASMCs proliferation and the effect of eIF2α siRNA on autophagy. ( A ) Expression of eIF2α, p-eIF2α, LC3B, and p62 proteins in the different groups. ( B ) Statistical analysis of gray values of different groups of total eIF2α. ( C ) The ratio of LC3B-II/LC3B-I. ( D ) Analysis of gray value of p62. Control: control group. PDGF: cell proliferation group. +Ng: PDGF+negative group.+eIF2α siRNA (50 nM): after transfected with eIF2α siRNA (50 nM) for 4–6 hrs, the medium was replaced and PDGF was induced for 48 hrs. The data are expressed as mean±standard error, n=3, * P <0.05 vs Control. # P <0.05 vs PDGF.

Article Snippet: The eIF2α siRNA were purchased from Ribobio (Guangzhou, People’s Republic of China).

Techniques: Expressing, Transfection

Journal: Drug Design, Development and Therapy

Article Title: eIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats

doi: 10.2147/DDDT.S213817

Figure Lengend Snippet: Effect of eIF2α siRNA on proliferation of PDGF-induced PASMCs. ( A ) Effect of eIF2α siRNA on PDGF-induced PASMCs proliferation. ( B ) Effect of eIF2α siRNA on Ki-67 mRNA expression. ( C ) Effect of eIF2α siRNA on PCNA mRNA expression. +Ng: PDGF+negative group.+eIF2α siRNA (50 nM): after transfected with eIF2α siRNA (50 nM) for 4–6 hrs, the medium was replaced and treated with PDGF for 48 hrs. The data are expressed as mean±standard error; n=3, * P <0.05 vs Control. # P <0.05 vs PDGF. Abbreviations: Control, control group; PDGF, cell proliferation group.

Article Snippet: The eIF2α siRNA were purchased from Ribobio (Guangzhou, People’s Republic of China).

Techniques: Expressing, Transfection

Journal: Drug Design, Development and Therapy

Article Title: eIF2α promotes vascular remodeling via autophagy in monocrotaline-induced pulmonary arterial hypertension rats

doi: 10.2147/DDDT.S213817

Figure Lengend Snippet: The proposed pathway of eIF2α mediation contributes to pulmonary vascular remodeling. eIF2α can promote PASMCs proliferation and pulmonary vascular remodeling by activating autophagy in MCT-induced PAH rats and PDGF-induced PASMCs proliferation; and eIF2α siRNA can inhibit autophagy activation and inhibit the proliferation of PASMCs; chloroquine inhibits the proliferation of PASMCs by inhibiting autophagy.

Article Snippet: The eIF2α siRNA were purchased from Ribobio (Guangzhou, People’s Republic of China).

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) Is a Critical Mediator of Endoplasmic Reticulum (ER) Stress Signaling

doi: 10.1371/journal.pone.0064256

Figure Lengend Snippet: ( a ) Irak2 +/+ and Irak2 −/− mice were administered 1 µg/gram body weight of Tunicamycin. At 10 hrs post injection, animals were sacrificed and their livers collected. RNA was extracted and mRNA levels of CHOP, BiP, spliced XBP1 (relative to total XBP1), IRE1, and ATF6 were analyzed by qRT-PCR, normalizing results relative to housekeeping gene, cyclophilin (mean±SD; n = 3 mice per group). ( b ) Proteins extracted from livers of Irak2 +/+ and Irak2 −/− mice (50 or 100 µg) were analyzed by immunoblotting using antibodies specific for phospho-c-JNK1, c-JNK1, phospho-eIF2α, eIF2α, DR5, and ATF6. Actin served as a loading control. Immunoblot data were quantified, normalized for Actin, and data compared relative to untreated wild-type mice. For P-JNK1 and P-eIF2α, results were additionally normalized relative to JNK1 and eIF2α protein levels, respectively. For ATF6, relative levels of the cleaved protein are reported. *: p<0.05. ( c ) Irak2 −/− and Irak2 +/+ MEFs were cultured for various times with 5 µM TG. Cell lysates were normalized for total protein content and analyzed by SDS-PAGE/immunoblotting using antibodies specific for murine IRAK2, JNK, p38MAPK, IRE1, β-Actin, and antibodies for phospho-JNK (p-JNK) and phospho-p38MAPK (p-p38MAPK). Note that while p38MAPK levels are lower in IRAK2-deficient MEFs, the ratio of p-p38MAPK to p38MAPK protein nevertheless reveals a relative diminution in p38MAPK phosphorylation in Irak2 −/− relative to Irak2 +/+ cells.

Article Snippet: Samples were subsequently boiled for 5 minutes, run on 4–20% gradient gels (Life Technologies), transferred to nitrocellulose membranes using the Trans-Blot Turbo system (Bio-Rad) and immunoblotted using various antibodies that included Phospho-JNK, JNK, ATF6, DR5 from Cell Signaling (USA) and Phospho-eIF2α from Enzo Life Sciences (USA).

Techniques: Injection, Quantitative RT-PCR, Western Blot, Cell Culture, SDS Page