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 dose modulates NSCs lineage commitment, viability, oxidative stress, and mitochondrial membrane potential at day 5. (A) Representative immunofluorescence images of TUJ1 with Nestin in NSCs cultured within the cell-laden MT/BEM matrix and exposed to melatonin (0, 25, 50, 75, 100 μM) for 5 days (scale bar, 50 μm). (B) Representative images of GFAP with Nestin under the same conditions (scale bar, 50 μm). (C) Representative images of Olig2 with Nestin (scale bar, 50 μm). (D) Percentages of TUJ1(+), GFAP (+), and Olig2(+) cells relative to total nuclei (DAPI) (n = 10 fields/group). (E) RT-qPCR of TUJ1, GFAP, and Olig2 normalized to GAPDH and expressed as fold change versus control (ΔΔCt) (n = 6). (F) Live/Dead staining (Calcein AM/EthD-1) at day 5. (G) Western blots of TUJ1 and GFAP with GAPDH loading control. (H) Densitometry of TUJ1/GAPDH and GFAP/GAPDH (n = 3 independent experiments). (I) ROS staining by DCFH-DA with Rosup as the positive control. (J) Quantification of ROS fluorescence intensity (n = 10 fields/group, compared to control). (K) JC-1 staining of mitochondrial membrane potential (ΔΨm) with CCCP as the positive control for depolarization. (L) JC-1 red/green ratio (n = 10 fields/group, compared to control). Statistical analysis: Data are presented as mean ± SD. one-way ANOVA with Holm–Sidak's multiple comparisons for multi-group datasets (D, E, J, L); unpaired two-tailed t -test for the two-group comparison (H). Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

Techniques: Membrane, Immunofluorescence, Cell Culture, Quantitative RT-PCR, Control, Staining, Western Blot, Positive Control, Fluorescence, Two Tailed Test, Comparison

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: Three-dimensional immunofluorescence and transcriptomic profiling of melatonin-treated NSCs. (A) Representative 3D confocal reconstructions of NSCs networks cultured for 5 days in BEM or MT/BEM hydrogels, immunostained for Nestin, TUJ1, GFAP and OLIG2 with DAPI nuclear counterstain. Scale bar: 50 μm. (B-C) Quantification of neurite outgrowth showing total neurite length (μm) (B) and neurite filament area (μm 2 ) (C) per field of view. (D) Quantification of astroglial differentiation expressed as GFAP + area (% of ROI). (E) Quantification of oligodendroglial lineage commitment expressed as OLIG2 + cells (% of DAPI + nuclei). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired two-tailed t -test; ∗p < 0.05, ∗∗p < 0.01 versus NSCs@BEM.

Article Snippet: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

Techniques: Immunofluorescence, Cell Culture, Two Tailed Test

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: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

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

Journal: PLOS One

Article Title: Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress

doi: 10.1371/journal.pone.0345890

Figure Lengend Snippet: (A) Schematic of the neuron-astrocyte coculture . (B) Representative images of the neuron-astrocyte coculture. Coculture of GFP-LC3 transgenic astrocytes and non-transgenic neurons were fixed on DIV7 of coculture (neurons were a total of DIV11) and immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), AQP4 (astrocyte marker) and β3-tubulin (neuron marker); nuclei were labeled with Hoechst (shown in blue in the merged image). Shown are maximum projections of z-stacks. Dashed box indicates location of the zoom-in. Scale bars, 10 µm. (C) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and LAMP1. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for GFP(LC3), p62, and LAMP1 are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate puncta co-positive for GFP(LC3) and LAMP1, or p62 and LAMP1. Empty yellow arrowheads indicate GFP(LC3) or p62 puncta that are negative for LAMP1. Scale bars, 10 µm. ( C’ ) Representative line scans of p62 and LAMP1-positive puncta in the soma of neurons and astrocytes. Asterisks denote peaks co-positive for p62 and LAMP1. (D) Cocultured neurons and astrocytes were treated for 1 hr with 1 mM LLOMe to induce lysosomal damage or an equivalent volume of DMSO solvent as a control. Cocultures were immunostained for GFP (LC3; labels GFP-LC3 transgenic astrocytes), p62, and ubiquitin; nuclei (Nuc) were labeled with Hoechst. Shown are maximum projections of z-stacks. Individual images are shown in grayscale; images for p62 and ubiquitin are grayscale-matched within a protein marker, across treatment conditions. Filled yellow arrowheads indicate colocalization between p62 puncta and ubiquitin puncta. Empty yellow arrowheads indicate p62 puncta with no ubiquitin puncta correlate. Scale bar, 10 µm. (E-E’) Quantification of the total area occupied by p62-positive puncta normalized to soma area for neurons (E) or astrocytes (E’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 37-40 neurons and N = 27-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (F-F’) Quantification of the total area occupied by ubiquitin-positive puncta normalized to soma area for neurons (F) or astrocytes (F’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 35-36 neurons and N = 29-33 astrocytes from 3 independent experiments; 7-8 DIV of coculture. (G-G’) Quantification of the percentage of overlapping area between p62-positive puncta and ubiquitin-positive puncta normalized to soma area for neurons (G) or astrocytes (G’) . Horizontal bars represent the means of the biological replicates ± SEM; shown are p-values from a LME model; N = 32-33 neurons and N = 23-30 astrocytes from 3 independent experiments; 7-8 DIV of coculture. Throughout the figure, neurons are circled in orange and astrocytes are circled in purple. For all graphs in all figures, small circles indicate the measurements from individual cells (e.g., the technical replicates) from each of the independent experiments; large triangles indicate the corresponding biological means from each of the independent experiments (e.g., the biological replicates); independent experiments are color-coded.

Article Snippet: Primary antibodies for immunofluorescence include mouse anti-βIII Tubulin (R&D Systems, MAB1195), rabbit anti-AQP4 (Millipore Sigma, HPA014784), chicken anti-GFP (Aves Labs, Inc., GFP-1020), rabbit anti-p62 (Abcam, Ab109012 ), mouse anti-Ubiquitin (Enzo Life Sciences, ENZ-ABS840; − and ), mouse anti-Ubiquitin (Enzo Life Sciences, BML-PW8810; , and ), and rat anti-LAMP1 (Abcam, ab25245).

Techniques: Transgenic Assay, Marker, Labeling, Solvent, Control, Ubiquitin Proteomics