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: ERSand MS mediated the IP3R1–GRP75–VDAC1 Ca2 + channeling complex in the first 72 h following SAH in the temporal cortex of mice in vivo. A – D Representative Western blot band and densitometric quantification of the time-dependent expression of IP3R1, GRP75, and VDAC1 in the temporal cortex of SAH mice models in the initial 72 h. The expression of the IP3R1–GRP75–VDAC1 complex was upregulated. 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. E – F 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. G – I The outline and the proportion of surviving neurons induced by SAH in the bilateral temporal cortex and CA3 region. Scale bar = 50 μm, N = 4 per group. J The TEM showed dilated rough ER fragments and swollen mitochondria in the bilateral temporal cortex in each group. Images were acquired at a magnification of 20,000 × , Scale bar = 250 nm. 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, 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 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: 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: Metformin ameliorates early brain injury after subarachnoid hemorrhage via improving ERS and MS-mediated Ca 2+ imbalance. The IP3R1–GRP75–VDAC1 complex regulates MAM and plays an imperative role in EBI. The treatment of Met ameliorates ER stress, MS, and Ca 2+ overload, further improving neuroapoptosis and alleviating the EBI of SAH

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:

Journal: International Journal of Oncology

Article Title: Targeting CALR reduces energy metabolism of esophageal cancer cells and inhibits tumor-associated fibroblast infiltration

doi: 10.3892/ijo.2025.5755

Figure Lengend Snippet: CALR regulates IP3R calcium ion release channels and ER stress. (A) Protein expression of (B) CALR, (C) CANX and (D) PDIA3 in KYSE150 after treatment with ER stress agonist thapsigargin. (E) Protein expression of (F) CALR, (G) CANX and (H) PDIA3 in KYSE410 cells following treatment with ER stress agonist thapsigargin. (I) In intracellular GRP78 expression after overexpression or knockdown of CALR in KYSE150. (J) Protein expression of GRP78 in KYSE150. (K) In intracellular GRP78 expression after overexpression or knockdown of CALR in KYSE410. (L) Protein expression of GRP78 in KYSE410. (M) Co-IP revealed the interaction of GRP75 and VDAC1 and IP3R1 in KYSE150. (N) Co-IP revealed the interaction of GRP75 and VDAC1 and IP3R1 in KYSE410. †† P<0.01, ††† P<0.001 vs. DMSO, ** P<0.01, *** P<0.001 vs. OE-NC, ### P<0.001 vs. sh-NC. CALR, Calreticulin; IP3R, inositol 1,4,5-Trisphosphate Receptor; ER, endoplasmic reticulum; CANX, calnexin; PDIA3, protein disulfide isomerase A3; ESCC, esophageal squamous cell carcinoma; GRP, glucose regulatory protein; VDAC, voltage-dependent anion channel; IB, immunoblotting; IP, immunoprecipitation; OE, overexpression; NC, negative control; sh, short hairpin.

Article Snippet: A total of 500 μ l VDAC1 (cat. no. 600-401-882) or IP3R1 antibody (both 1:2,000, cat. no. PA1-901; both Thermo Fisher Scientific, Inc.) or normal IgG working solution was added to 20 μ l Protein A + G magnetic beads for incubation at room temperature for 2 h. Sample protein and magnetic beads bound with antibodies or normal IgG were added at a volume ratio of 25:1 and incubated at 4°C overnight.

Techniques: Expressing, Over Expression, Knockdown, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Negative Control