Journal: Journal of Cellular and Molecular Medicine

Article Title: EIF2α – ATF4 – CHAC1 Signalling Links ER Stress to Ferroptosis in Human Aortic Smooth Muscle Cells: Mechanistic Insights and Therapeutic Implications

doi: 10.1111/jcmm.71104

Figure Lengend Snippet: Effects of the selective eIF2α dephosphorylation inhibitor salubrinal on ferroptosis‐related protein expression in HASMCs and AoSMCs. (A) Alterations in Ferroptosis‐Related Protein Expression Following Treatment with the eIF2α Dephosphorylation Inhibitor Salubrinal in HASMCs and AoSMCs. (B) Glucose‐regulated protein 78 (GRP78) expression, reflecting the activation status of ER chaperone machinery under conditions of altered eIF2α signalling. (C) Western blot analysis of phosphorylated protein kinase RNA‐like ER kinase (p‐PERK) in HASMCs and AoSMCs after 24 h treatment with salubrinal (25 μM), showing modulation of the ER stress response via inhibition of eIF2α dephosphorylation. (D) Phosphorylated eukaryotic initiation factor 2α (p‐EIF2α) protein levels, demonstrating sustained phosphorylation in both cell types following salubrinal treatment. (E) Activating transcription factor 4 (ATF4) protein levels, upregulated in response to persistent eIF2α phosphorylation, consistent with activation of downstream stress signalling. (F) ChaC glutathione‐specific γ‐glutamylcyclotransferase 1 (CHAC1) protein expression, indicating potential enhancement of glutathione degradation following eIF2α pathway activation. (G) Glutathione peroxidase 4 (GPX4) protein levels, showing a trend toward reduction, suggestive of compromised antioxidant defence and potential promotion of ferroptotic processes. Densitometric quantification of protein bands from panels (A–F), normalized to β‐Actin and expressed as fold change relative to control, confirming significant alterations in ferroptosis‐related proteins in response to salubrinal treatment. Data are presented as mean ± SD ( n = 3 independent experiments). * p < 0.05, ** p < 0.01 vs. Control.

Article Snippet: Erastin (Targetmol, Cat. No. T1765, China); BSO (Targetmol, Cat. No. T5371, China); Salubrinal (Targetmol, Cat. No. 405060‐95‐9, China); Ferrostatin‐1 (Fer‐1) (Targetmol, Cat. No. T6500, China); TRIzol reagent (Invitrogen, Cat. No. 15596026, USA); CCK‐8 kit (Abbkine, Cat. No. BMU106‐CN, China); BCA Protein Assay Kit (Thermo Fisher Scientifi, Cat. No. 23225, USA); C11‐BODIPY 581/591 (Beyotime, Cat. No. S0043S, China); FerroOrange (Maokangbio, Cat. No. MX4559, China); MDA Assay Kit (Abbkine, Cat. No. KTB1050, China); TUNEL Apoptosis Detection Kit (Abbkine, Cat. No. KTA2010, China).

Techniques: De-Phosphorylation Assay, Expressing, Activation Assay, Western Blot, Inhibition, Phospho-proteomics, Control

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Article Snippet: The ER stress inhibitors sodium 4-phenylbutyrate (4-PBA, 50 and 100 μM, Selleck, Houston, TX, USA) and salubrinal (25 and 100 μM, Selleck) were added for 1 hour before OGD or after reperfusion.

Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro