Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Nox4 Improves AVIC Survival During Calcification via Activation of elF2α/ATF4 Signaling (A) The effects of down-regulation of endogenous Nox4 with shNox4 adenovirus, or (B) overexpression of Nox4 with adNox4 adenovirus on protein levels of integrated stress response (ISR) markers elF2α phosphorylation and ATF4, ER chaperone KDEL, and cell survival markers CHOP and cleaved caspase-12 in cultured AVIC with or without OGM treatment. AVIC transfected with green fluorescent protein (GFP) virus or β-galactosidase (β-gal) virus as respective control cells. Mean data shown at the right. n = 3/group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), # P < 0.05, ## P < 0.01, compared with OGM-stimulated AVIC transfected with GFP or β-gal virus. 2-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. CHOP = C/EBP homologous protein; elF2α = eukaryotic Initiation Factor 2; KDEL = peptide sequence Lys-Asp-Glu-Leu; other abbreviations as in .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Cell Culture, Transfection, Virus, Control, Sequencing

Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Guanabenz Attenuates AVIC Calcification Through Activation of elF2α/ATF4 Pathway (A) Calcium deposition by Alizarin Red staining and calcium concentration with or without Guanabenz (Gbz, 5 μmol/l, 14 days) treatment. Scale bar: 100 μm. Mean data shown at the right. (B) Immunoblots of calcification markers OPN and Runx2. (C) Western blots showing the effects of Gbz on expression of Nox4, p-eIF2α/ATF4 stress signaling, KDEL, and survival markers CHOP and cleaved caspase-12 in AVIC. Mean data shown at the right. n = 3-5/group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), † P < 0.05, †† P < 0.01, compared with control cells without Gbz treatment, # P < 0.05, ## P < 0.01, compared with OGM without Gbz treatment. Two-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. Abbreviations as in , , .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Activation Assay, Staining, Concentration Assay, Western Blot, Expressing, Control

Journal: JACC: Basic to Translational Science

Article Title: Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease

doi: 10.1016/j.jacbts.2025.101433

Figure Lengend Snippet: Sephin1 Diminishes AVIC Calcification Through the Increase in elF2α Phosphorylation (A) Calcium deposition by Alizarin Red staining and calcium concentration with or without Sephin1 (0.5 μmol/L, 14 days) treatment. Scale bar: 100 μm. Mean data shown at the right. (B) Immunoblots of calcification markers OPN and Runx2 with Sephin1 treatment. (C) Western blots showing the activation of p-eIF2α/ATF4/KDEL stress signaling and Nox4 protein levels, and the decreases in apoptotic markers CHOP and cleaved caspase-12 in AVIC treated with Sephin1. Mean data shown at the right. n = 3-4/group, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared with respective control cells (Con), # P < 0.05, ## P < 0.01, ### P < 0.001, compared with OGM without Sephin1 treatment. Two-way analysis of variance with a post hoc Tukey’s test. All data are mean ± SEM. Abbreviations as in , , .

Article Snippet: The primary antibodies used were: Nox4 (ab109225, Abcam); Runx2 (8486, Cell Signaling); Osteopontin (ab8448, Abcam); p-eIF2α (ab32157, Abcam); eIF2α (11170-1-AP, Proteintech); ATF4 (ab1371, Abcam); KDEL (ab69659, Abcam); Cleaved caspase-12 (2202, Cell Signaling); Caspase-12 (55238-1-AP, Proteintech); CHOP (ab11419, Abcam); GAPDH (10494-1-AP, Proteintech).

Techniques: Phospho-proteomics, Staining, Concentration Assay, Western Blot, Activation Assay, Control

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Severity of ERS, MS, and Ca2 + imbalance in the initial 72 h following SAH in the temporal cortex of mice in vivo. A – C Representative Western blot band and densitometric quantification of the time-dependent expression of CHOP and ATF4 in the temporal cortex of SAH mice models in the initial 72 h. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. D The arterial blood glucose in the SAH model groups. ns P > 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). E – L Representative Western blot band and densitometric quantification of the time-dependent expression of PTP1B, p-AKT, AKTp-eIF2a, eIF2a, Bcl-2, Bax, and CC3 in the temporal cortex of SAH mice models in the initial 72 h. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). M The concentration of Ca 2+ in the temporal cortex of SAH groups. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). N – Q Immunofluorescence revealed the number of TUNEL-positive cells in the initial 72 h following SAH in the bilateral temporal cortex and CA3, N = 4 per group, scale bar = 50 μm. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vivo, Western Blot, Expressing, Derivative Assay, Control, Concentration Assay, Immunofluorescence, TUNEL Assay

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Met significantly suppressed Ca2 + transfer from the ER to the mitochondria through the PTP1B/AKT following SAH 24 h in the temporal cortex of mice in vivo. A – G To determine the regulatory role of Met in ER stress and mitochondrial signaling, we employed lentivirus-mediated modulation in a 24-h SAH model and confirmed the effects by Western blot analysis. Representative Western blot band and densitometric quantification of the time-dependent expression of CHOP, ATF4, PTP1B, p-eIF2a, eIF2a, p-AKT, and AKT following SAH 24 h in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. H The effect of Met on the concentration of Ca 2+ in the temporal cortex of SAH groups. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). I – L Representative Western blot band and densitometric quantification of the time-dependent expression of IP3R1, GRP75, and VDAC1 in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). M , N The effect of Met on the conventional immunofluorescence of PTP1B, calnexin, VDAC1, and DAPI in the bilateral temporal cortex and CA3. The subsequent MAM junction variation was detected by the conventional immunofluorescence colocalization of calnexin and VDAC1 in vivo. Scale bar = 50 μm, N = 4 per group. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vivo, Western Blot, Expressing, Derivative Assay, Control, Concentration Assay, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Metformin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage Via Improving Endoplasmic Reticulum Stress and Mitochondrial Stress-Mediated Ca 2+ Imbalance

doi: 10.1007/s12035-025-05558-1

Figure Lengend Snippet: Met significantly suppressed Ca 2+ transfer from the ER to the mitochondria through the PTP1B/AKT following SAH 24 h in vitro. A – K Our vitro data demonstrate that PTP1B plays a critical role in integrating ER stress with mitochondrial dysfunction following SAH 24 h. Representative Western blot band and densitometric quantification of PTP1B, p-AKT, AKT, p-eIF2a, eIF2a, IP3R1, GRP75, VDAC1, ATF4, and CHOP in primary neurons induced by the OxyHb. The expression of the IP3R1–GRP75–VDAC1 complex was upregulated accordingly. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Samples derived from the same experiment, and the gels were processed in parallel. Protein expression levels were normalized to GAPDH as an internal control. Individual gels were run for the separate proteins when the loading protein amount was consistent. L Conventional immunofluorescence confirmed the colocalization of NEUN, MAP-2, and DAPI in primary neurons. Scale bar = 50 μm. M – R The effect of Met on the apoptosis of SAH in vitro. Representative Western blot bands of Bcl-2, Bax, CC3, and flow cytometry were used to evaluate the effect of Met on apoptosis in vivo. ns P > 0.05, * P < 0.05. Values were presented as the mean ± SD from four independent experiments ( N = 4). Protein expression levels were normalized to GAPDH as an internal control. S The conventional immunofluorescence staining showed that the colocalization of intracellular Ca 2+ concentration increased significantly in the OxyHb24H group in primary neurons. Scale bar = 50 μm. T The conventional immunofluorescence confirmed the colocalization of PTP1B, calnexin, VDAC1, and DAPI in primary neurons after the intervention of lentivirus and inhibitors. To assess the effect of Met on MAM integrity, we performed immunofluorescence co-localization analysis of the ER marker calnexin and the mitochondrial marker VDAC1 in treated primary neurons. Scale bar = 50 μm. An unpaired Student’s t test was used for comparisons between two groups, whereas ANOVA followed by Tukey’s post hoc test was used for comparisons among multiple groups

Article Snippet: The PVDF membrane was blocked in 5% skim milk for 90 min at room temperature, followed by incubation with the primary antibodies against PTP1B (1:2000 Proteintech, catalog number 11334–1-AP), IP3R1 (1:2000, Abcam, catalog number ab264281), CHOP (1:1000, Proteintech, catalog number 15204–1-AP), Bcl-2 (1:2000, Affinity, lot# 70g9181), Bax (1:2000, Proteintech, catalog number 50599–2-2 g), VDAC1 (1:2000, Proteintech, catalog number 10866–1-AP), AKT (1:1000 Proteintech, catalog number 10176–2-AP), p-AKT (1:1000 Proteintech, catalog number 80455–1-RR), GRP75 (1:1000, Bio-Techne, catalog number MAB3584, ATF4 (1:2000, Proteintech, catalog number 10835–1-AP, p- eIF2a (1:500, Affinity, catalog number AF3087), eIF2a (1:1000, Proteintech, catalog number 11170–1-AP), and CC3 (1:1000, Bioss, Boston, MA, USA lot: BJ03319208), respectively.

Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Control, Immunofluorescence, Flow Cytometry, In Vivo, Staining, Concentration Assay, Marker

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Antibodies used for western blotting

Article Snippet: rabbit anti-eIF2α , 1: 1000 , Proteintech , 11170-1-AP , AB_2096489.

Techniques: Western Blot