Journal: Discover oncology

Article Title: Prognostic role of eIF2α protein in metastatic renal cell cancer patients.

doi: 10.1007/s12672-025-02916-2

Figure Lengend Snippet: Fig. 1 a: BCL2 expression: 80% (DABx200) b: Beclin-1 expression: 100% (DABx200) c–e: eIF2α expression with vary ing intensities: weak (+ 1) in (c), moderate (+ 2) in (d), and strong (+ 3) in (e). (DABx200)

Article Snippet: The following primary antibodies were used for immunohistochemical staining: monoclonal mouse anti-human BCL2 (clone 124, prediluted, Dako), monoclonal rabbit anti-human Beclin-1 (clone EP304, 1:100, Epitomics), monoclonal rabbit anti-human HIF-1 Alpha (clone EP118, 1:100, Epitomics), monoclonal rabbit anti-human eIF2α (clone D7D3, 1:800, Cell Signaling), and monoclonal rabbit anti-human LC3A (clone D50G8, 1:3000, Cell Signaling).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Licorice attenuates cisplatin-induced hepatotoxicity by alleviating endoplasmic reticulum stress and apoptosis

doi: 10.3389/fphar.2025.1557125

Figure Lengend Snippet: GC attenuated CP-induced apoptosis by modulating the ER-mediated PERK/ATF4/CHOP pathway. (A) The expressions of p-PERK, p-eIF2α, ATF4 and CHOP proteins were detected by WB analysis. (B) qRT-PCR was performed to detect the expression of CHOP and ATF4 mRNA. (C) Representative image and quantification of the immunofluorescence of CHOP and ATF4. (D) The expressions of cleaved caspase-3, cleaved caspase-8, and caspase-12 proteins were detected by WB analysis. (E) qRT-PCR was performed to detect the expression of caspase-8, and caspase-12 mRNA. All data are presented as the mean ± SD. ## p < 0.01 compared with control group; * p < 0.05, ** p < 0.01 compared with CP group.

Article Snippet: WB analysis was performed as previously described ( ) using specific primary antibodies against CHOP (1:1000, 15204-1-AP, Proteintech), ATF4 (1:1000, BM5179, Boster), ATF6 (1:1000, A00655, Boster), GRP78 (1:1000, BA 2042, Boster), p-IRE1α (1:1000, NB100-2323, Novus), p-eIF2α (1:1000, BM3942, Boster), Bcl-2 (1:1000, A00040-1, Boster), Bax (1:1000, A00183, Boster), cleaved caspase-3 (1:1000, #9664T, Cell Signaling Technology), caspase-12 (1:1000, BA3142, Boster), cleaved caspase-8 (1:5000, ab108333, Abcam), p-PERK (1:1000, abs137056, Absin) and β-actin (1:5000, 20536-1-AP, Proteintech).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Control

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: Rabbit anti-human eIF2α , Protein Tech, China , 11170-1-AP , 1:1000.

Techniques: Western Blot

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Primers of the genes used for qRT-PCR.

Article Snippet: Rabbit anti-human eIF2α , Protein Tech, China , 11170-1-AP , 1:1000.

Techniques: Sequencing

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Effects of BFA and TM on the levels of ER stress-related proteins (BiP, PERK, eIF2α, ATF4, and CHOP). HepG2 cells were treated with BFA (0.25 mg/L) and TM (1 mg/L), either individually or in combination, for 24 h. The protein levels of (A) BiP, (B) PERK, (C) phosphorylated PERK (p-PERK), (D) eIF2α, (E) phosphorylated eIF2α (p-eIF2α), (F) ATF4, and (G) CHOP were determined by Western blotting. Data are presented as means ± SD from three independent experiments performed in triplicate. ∗ P < 0.05, compared to the control group; # P < 0.05, compared to the DMSO (0.1% v/v, vehicle control) group; a P < 0.05, compared to the BFA (0.25 mg/L) group. Abbreviations: ATF4, activating transcription factor 4; BFA, brefeldin A; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; DMSO, dimethyl sulfoxide; eIF2α, eukaryotic translation initiation factor 2α; PERK, protein kinase R-like endoplasmic reticulum kinase; SD, standard deviation; TM, tunicamycin.

Article Snippet: Rabbit anti-human eIF2α , Protein Tech, China , 11170-1-AP , 1:1000.

Techniques: Western Blot, Control, Binding Assay, Standard Deviation

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Effects of BFA and TM on the mRNA levels of ER stress-related genes ( BiP, PERK, eIF2α, ATF4, and CHOP ). HepG2 cells were treated with BFA (0.25 mg/L) and TM (1 mg/L), either individually or in combination, for 24 h. The relative mRNA levels of (A) BiP , (B) PERK , (C) eIF2α, (D) ATF4 , and (E) CHOP were determined by qRT-PCR. Data are presented as means ± SD from three independent experiments performed in triplicate. ∗ P < 0.05, compared to the control group; # P < 0.05, compared to the DMSO (0.1% v/v, vehicle control) group; a P < 0.05, compared to the BFA (0.25 mg/L) group. Abbreviations: ATF4, activating transcription factor 4; BFA, brefeldin A; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; DMSO, dimethyl sulfoxide; eIF2α, eukaryotic translation initiation factor 2α; PERK, protein kinase R-like endoplasmic reticulum kinase; SD, standard deviation; TM, tunicamycin.

Article Snippet: Rabbit anti-human eIF2α , Protein Tech, China , 11170-1-AP , 1:1000.

Techniques: Quantitative RT-PCR, Control, Binding Assay, Standard Deviation

Journal: Liver Research

Article Title: Combination of brefeldin A and tunicamycin induces apoptosis in HepG2 cells through the endoplasmic reticulum stress-activated PERK-eIF2α-ATF4-CHOP signaling pathway

doi: 10.1016/j.livres.2025.01.004

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: Rabbit anti-human Phospho-eIF2α , CST, USA , 3398T , 1:1000.

Techniques: Western Blot

Journal: iScience

Article Title: The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress

doi: 10.1016/j.isci.2024.110196

Figure Lengend Snippet: ER stress promotes the heat shock-induced SG assembly while inhibiting disassembly In (F–I), upper and lower panels are representative images which were taken using microscope and their corresponding quantifications and statistical analysis, respectively. n = 3 replicates with >450 cells per condition. (A) U2OS cells were transfected with siHRD1 or siSEL1L oligos for 48 h and collected for immunoblotting using antibodies of the ER stress-related marker proteins. Tunicamycin-treated samples were employed as positive controls. (B) U2OS cells were incubated at 43°C for 30 min, or followed by 37°C for 8 min. Cells were then collected for immunoblotting using antibodies of the ER stress-related marker proteins. Tunicamycin-treated samples were employed as positive controls. (C and D) U2OS cells were transfected with siHRD1 (C) or siSEL1L (D) oligos for 48 h, and then maintained at 43°C for 30 min, or followed by 37°C for 8 min. Cells were collected for immunoblotting using antibodies of the ER stress-related marker proteins. (E) U2OS cells were pretreated with TAK243 for 15 min, and then incubated at 43°C for 30 min. Cells were then collected for immunoblotting using antibodies of the ER stress-related marker proteins. Tunicamycin-treated samples were employed as positive controls. Phosphorylation of PERK or eIF2α was confirmed using phos-tag gels. (F and G) The U2OS-EGFP-G3BP1 cells were pretreated with tunicamycin for 15 min, and then cultured at 43°C for 10 min (F). Alternatively, cells were pretreated with tunicamycin for 15 min and then incubated at 43°C for 30 min, or cells were maintained at 43°C with tunicamycin for 30 min followed by 37°C for 8 min (G). Data are shown as mean ± SD. Unpaired two-tailed Student’s t tests (F); two-way ANOVA with Sidak’s multiple comparisons test (G); ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (H and I) The U2OS-EGFP-G3BP1 cells were transfected with siBiP oligos for 48 h, and then incubated at 43°C for 10 min (H). Alternatively, cells were cultured at 43°C for 30 min, or followed by 37°C for 8 min (I). Data are shown as mean ± SD. Unpaired two-tailed Student’s t tests (H); two-way ANOVA with Sidak’s multiple comparisons test (I); ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (J) Same as (C) except that cells were transfected with siBiP oligos. (K) The U2OS-EGFP-G3BP1 cells were labeled with the ER tracker and incubated at 43°C for 30 min. White arrows indicate the representative ER tubules. Scale bar: 10 μm. (L) The U2OS-EGFP-G3BP1 cells were transfected with mCherry-Sec61B for 24 h, and maintained at 43°C for 30 min. White arrows indicate the representative ER tubules. Scale bar: 10 μm. See also Figure S5 .

Article Snippet: Gateway entry clones containing CDS of human HRD1 and eIF2α were obtained from Ultimate™ ORF LITE Clones (Human collection, Invitrogen), and maintained by the Core Facility of Life Sciences Institute, Zhejiang University.

Techniques: Microscopy, Transfection, Western Blot, Marker, Incubation, Cell Culture, Two Tailed Test, Labeling

Journal: iScience

Article Title: The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress

doi: 10.1016/j.isci.2024.110196

Figure Lengend Snippet: ER stress couples with the PERK-eIF2α axis to promote the heat shock-induced SG genesis In all figures, left and right panels are representative images which were taken using microscope and their corresponding quantifications and statistical analysis, respectively. n = 3 replicates with >450 cells per condition. (A) The U2OS-EGFP-G3BP1 cells were transfected with siPERK oligos for 48 h, and then incubated at 43°C for 30 min, or followed by 37°C for 5 min. Data are shown as mean ± SD, two-way ANOVA with Sidak’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (B) The U2OS-EGFP-G3BP1 cells were pretreated with PERK inhibitor for 15 min and then incubated at 43°C for 30 min, or cells were cultured at 43°C with PERKi for 30 min followed by incubation at 37°C for 5 min. Data are shown as mean ± SD, two-way ANOVA with Sidak’s multiple comparisons test; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (C) Same as in (A) except that cells were transfected with sieIF2α oligos. Data are shown as mean ± SD, two-way ANOVA with Sidak’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (D) The U2OS-EGFP-G3BP1 cells stably expressing either the wildtype or the S51A mutant of eIF2α rescue cDNAs were transfected with sieIF2α oligos for 48 h, and then incubated at 43°C for 30 min. Data are shown as mean ± SD, one-way ANOVA with Tukey’s multiple comparisons test; ∗ p ≤ 0.05; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (E) Same as in (B) except that cells were treated with Salubrinal and recovered at 37°C for 8 min. Data are shown as mean ± SD, two-way ANOVA with Sidak’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. (F) The U2OS-EGFP-G3BP1 cells were incubated at 43°C with Tunicamycin, PERKi, or both for 30 min, followed by 37°C for 8 min. Data are shown as mean ± SD, one-way ANOVA with Tukey’s multiple comparisons test; ∗∗∗∗ p ≤ 0.0001. Scale bar: 10 μm. See also Figure S6 .

Article Snippet: Gateway entry clones containing CDS of human HRD1 and eIF2α were obtained from Ultimate™ ORF LITE Clones (Human collection, Invitrogen), and maintained by the Core Facility of Life Sciences Institute, Zhejiang University.

Techniques: Microscopy, Transfection, Incubation, Cell Culture, Stable Transfection, Expressing, Mutagenesis

Journal: iScience

Article Title: The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress

doi: 10.1016/j.isci.2024.110196

Figure Lengend Snippet: A proposed working model Extracellular heat stress results in formation of SGs and concomitant overload of mis/unfolded protein in the ER. These overloaded lesions are detected by the UPR governing protein BiP, which then dissociates from the UPR transducers PERK, IRE1α, and ATF6, thereby releasing their activities. PERK then phosphorylates eIF2α at its S51 site, leading to a global translation arrest and ultimately promoting SG assembly. In response to heat shock, the HRD1-SEL1L ubiquitin ligase appears to relieve ER stress by ubiquitylating mis/unfolded proteins in the ER and then sending those ubiquitylated proteins to the proteasome in coordination with p97 for turnover. The HRD1-SEL1L ubiquitin ligase regulates the SG homeostasis via the BiP-PERK-eIF2α axis. ATF6 seems to regulate the SG formation via a negative feedback mechanism to counteract SG assembly by suppressing PERK and IRE1α signaling branches during this process, although how IRE1α showed synergistic effect with PERK remain unknown.

Article Snippet: Gateway entry clones containing CDS of human HRD1 and eIF2α were obtained from Ultimate™ ORF LITE Clones (Human collection, Invitrogen), and maintained by the Core Facility of Life Sciences Institute, Zhejiang University.

Techniques:

Journal: iScience

Article Title: The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress

doi: 10.1016/j.isci.2024.110196

Figure Lengend Snippet:

Article Snippet: Gateway entry clones containing CDS of human HRD1 and eIF2α were obtained from Ultimate™ ORF LITE Clones (Human collection, Invitrogen), and maintained by the Core Facility of Life Sciences Institute, Zhejiang University.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: bioRxiv

Article Title: Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2

doi: 10.1101/2024.04.24.591037

Figure Lengend Snippet: A) Western blot for total eIF2α and phosphorylated eIF2α levels from in vitro kinase assays. eIF2α only lane: kinase assay with only eIF2α substrate present. GCN2 only lane: kinase assay with GCN2 Kinase/HRSL eluted from pulldown only. No eIF2α substrate present. Naïve only lane: cell lysate from naïve cells (cells were not transfected with plasmid bearing GCN2 Kinase/HRSL) underwent a mock pull-down and elution with flag peptide similar to GCN2 Kinase/HRSL pulldown. Performed kinase assay with naïve cell eluate only (No eIF2α or GCN2 Kinase/HRSL eluate present) (B) replicate. (C) Replicate with western blot for GCN2 Kinase/HRSL using anti-flag antibody.

Article Snippet: Following 15 minutes incubation with blocking buffer [5% (w/v) nonfat dry milk in TRIS-Buffered Saline and Tween-20 (standard TBST)], the membranes were probed with the following primary antibodies: 1) mouse anti-human total eIF2α (Cell Signaling Technology) at 1:1,000, 2) rabbit anti-human eIF2α-phosphoS51 (Abcam) at 1:1,000 3) mouse anti-Flag M2 (Sigma) at 1:2,000.

Techniques: Western Blot, In Vitro, Kinase Assay, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2

doi: 10.1101/2024.04.24.591037

Figure Lengend Snippet: A) Western blot for eIF2α phosphorylation from in vitro kinase assay with GCN2 Kinase/HRSL +/- RNase A treatment (350 nM final concentration). Western blot for flag-tagged GCN2 Kinase/HRSL controls for equal loading. (B-C) Biological replicates related to A . D) Results for three replicates (A-C) quantified and normalized to total eIF2α.

Article Snippet: Following 15 minutes incubation with blocking buffer [5% (w/v) nonfat dry milk in TRIS-Buffered Saline and Tween-20 (standard TBST)], the membranes were probed with the following primary antibodies: 1) mouse anti-human total eIF2α (Cell Signaling Technology) at 1:1,000, 2) rabbit anti-human eIF2α-phosphoS51 (Abcam) at 1:1,000 3) mouse anti-Flag M2 (Sigma) at 1:2,000.

Techniques: Western Blot, Phospho-proteomics, In Vitro, Kinase Assay, Concentration Assay