Journal: Neurotherapeutics

Article Title: CCR5 protects against permanent brain ischemia by suppressing neuronal ferroptosis through AMPK/ACC signaling

doi: 10.1016/j.neurot.2026.e01088

Figure Lengend Snippet: CCR5 acts as a positive upstream regulator of the AMPK/ACC signaling under hypoxia-ischemia-mimicking conditions. (A–B) SH-SY5Y cells were transfected with control scramble (Scr) or CCR5 shRNA and cultured under hypoxia-ischemia-mimicking conditions. Representative western blots (A) and quantitative analyses (B) of phosphorylated AMPK (p-AMPK), total AMPK, phosphorylated ACC (p-ACC), total ACC, and the corresponding phosphorylation ratios were shown. (C–D) SH-SY5Y cells were transfected with empty vector (EV) or a CCR5 overexpression plasmid and cultured under the indicated conditions. Representative western blots (C) and quantitative analyses (D) of p-AMPK, AMPK, p-ACC, ACC, and the corresponding phosphorylation ratios were shown. (E–F) SH-SY5Y cells were treated with MVC and cultured under normal conditions or hypoxia-ischemia-mimicking conditions. Representative western blots (E) and quantitative analyses (F) of p-AMPK, AMPK, p-ACC, ACC, and the corresponding phosphorylation ratios were shown. β-Tubulin was used as a loading control. Data are presented as mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Fisher's LSD post hoc tests. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Following electrophoretic separation, the proteins were transferred onto a nitrocellulose membrane, which was then blocked with 5% skimmed milk at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies against HIF-1α (ABclonal Technology, A24002), CCR5 (Affinity Biosciences LTD, AF6339), Phospho-AMPKα (Thr172) (Beyotime, AA393), AMPKα (Proteintech, 10929-2-AP), Phospho-ACC (Proteintech, 29119-1-AP), ACC (Proteintech, 21923-1-AP), or GPX4 (Abmart, T56959 ), each diluted to a ratio of 1:1000 in 5% bovine serum albumin (BSA).

Techniques: Transfection, Control, shRNA, Cell Culture, Western Blot, Phospho-proteomics, Plasmid Preparation, Over Expression

Journal: Neurotherapeutics

Article Title: CCR5 protects against permanent brain ischemia by suppressing neuronal ferroptosis through AMPK/ACC signaling

doi: 10.1016/j.neurot.2026.e01088

Figure Lengend Snippet: AMPK inhibition completely abolishes CCR5-mediated GPX4 maintenance and anti-ferroptotic protection. (A–B) SH-SY5Y cells were treated with the AMPK inhibitor Compound C (COC, 10 μM) and cultured under hypoxia-ischemia-mimicking conditions. Representative western blots (A) and quantitative analyses (B) of phosphorylated AMPK (p-AMPK), total AMPK, phosphorylated ACC (p-ACC), total ACC, and the corresponding phosphorylation ratios were shown. (C–E) Under the same experimental conditions, representative western blots (C) and quantitative analyses (D) of CCR5 and GPX4 protein expression, together with MDA levels (E), were shown. β-Tubulin or β-actin were used as a loading control. Data are presented as mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Fisher's LSD post hoc tests. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Following electrophoretic separation, the proteins were transferred onto a nitrocellulose membrane, which was then blocked with 5% skimmed milk at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies against HIF-1α (ABclonal Technology, A24002), CCR5 (Affinity Biosciences LTD, AF6339), Phospho-AMPKα (Thr172) (Beyotime, AA393), AMPKα (Proteintech, 10929-2-AP), Phospho-ACC (Proteintech, 29119-1-AP), ACC (Proteintech, 21923-1-AP), or GPX4 (Abmart, T56959 ), each diluted to a ratio of 1:1000 in 5% bovine serum albumin (BSA).

Techniques: Inhibition, Cell Culture, Western Blot, Phospho-proteomics, Expressing, Control

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Melatonin activates AMPK signaling and enhances mitochondrial function in vitro. (A) GO enrichment bar plot of differentially expressed genes (DEGs) between Control and Melatonin-treated NSCs. (B) KEGG pathway enrichment bar plot of DEGs between Control and Melatonin groups. (C) Heatmap of selected DEGs associated with neuronal differentiation and mitochondrial function. DEGs were defined as transcripts with FDR <0.05. (D) Representative Western blots showing phosphorylated AMPK (p-AMPK, Thr172) and phosphorylated ACC (p-ACC, Ser79) in Control, Melatonin, Inhibitor, and Melatonin + Inhibitor groups. (E) Densitometric analysis of p-AMPK/total AMPK and p-ACC/GAPDH ratios. (F) RT-qPCR analysis of Ppargc1a and Tfam expression, normalized to GAPDH and presented as fold change relative to the Control group. (G) Representative Western blots of mitochondrial oxidative phosphorylation (OXPHOS) complexes I-V. (H) Densitometric quantification of OXPHOS complexes I-V, normalized to GAPDH (or the corresponding loading control). (I) Representative JC-1 fluorescence images indicating mitochondrial membrane potential (ΔΨm). (J) Quantification of the red/green JC-1 fluorescence ratio from (I). (K) Schematic representation of the proposed melatonin-AMPK-ACC-PGC-1α-NRF1/TFAM signaling axis driving mitochondrial biogenesis in NSCs. All quantitative data (E, F, H, J) are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Holm–Sidak's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. K created with BioRender.com .

Article Snippet: For AMPK inhibition experiments, BAY-3827 (HY-112083, MedChemExpress, USA), a selective AMPK inhibitor, was used at a final concentration of 2 μM for 24 h. The mitochondrial membrane potential was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2003S, Beyotime Biotechnology, China).

Techniques: In Vitro, Control, Western Blot, Quantitative RT-PCR, Expressing, Phospho-proteomics, Fluorescence, Membrane

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Molecular validation of neural repair and mechanism activation in spinal cord tissue. Western blot and qPCR analyses of spinal cord tissue lysates from Sham, SCI, BEM, NSCs@BEM, and NSCs@MT/BEM groups. (A) Representative Western blots for the neuronal marker TUJ1 and the glial scar marker GFAP. (B) Representative Western blots for phosphorylated AMPK (p-AMPK), phosphorylated ACC (p-ACC), and their respective total proteins. (C) Representative Western blots for the five oxidative phosphorylation (OXPHOS) complex subunits. (D) Densitometric quantification of TUJ1 and GFAP protein levels. (E) Densitometric quantification of the p-AMPK/total AMPK and p-ACC/total ACC ratios. (F) Densitometric quantification of OXPHOS complex protein levels. (G) Relative mRNA expression of neural markers (TUJ1, GFAP, Olig2) and key mitochondrial biogenesis regulators (Ppargc1a, Tfam) determined by qPCR. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Holm–Sidak's multiple comparisons test. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: For AMPK inhibition experiments, BAY-3827 (HY-112083, MedChemExpress, USA), a selective AMPK inhibitor, was used at a final concentration of 2 μM for 24 h. The mitochondrial membrane potential was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2003S, Beyotime Biotechnology, China).

Techniques: Biomarker Discovery, Activation Assay, Western Blot, Marker, Phospho-proteomics, Expressing