mouse β tuj 1  (R&D Systems)

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: International Journal of Microbiology

Article Title: Redox‐Dependent Activation of Matrix Metalloproteinases Regulates Influenza A Virus Replication in Neuronal Cells

doi: 10.1155/ijm/7586794

Figure Lengend Snippet: NWS virus infects and replicates in differentiated SH‐SY5Y cells. (a) Phase‐contrast microscope images of undifferentiated (undiff., above) and differentiated (diff., below) SH‐SY5Y cells after 6 days of retinoic acid (RA) treatment. Differentiated cells exhibit neurite outgrowth and a neuronal‐like phenotype. (b) Relative mRNA expression of the neuronal marker β III‐tubulin (TUBB3) in undifferentiated and RA‐differentiated SH‐SY5Y cells, assessed by qRT‐PCR. Data are presented as mean ± SD of three independent experiments; p < 0.01 (Student′s t ‐test). (c) Western blot analysis of TUBB3 in undifferentiated (first lane) and RA‐differentiated, not infected (second lane) and NWS virus‐infected (third lane) SH‐SY5Y cells, 24‐h post‐infection (h p.i.). Actin was used as loading control. Densitometric analysis is reported above TUBB3 image, with values expressed as ratio TUBB3/actin. Blot was stripped and reprobed with anti‐influenza antibody which recognized viral proteins (HA, NP, and M1). Molecular weight of each protein is reported on the left of the blot. (d) Western blot analysis of viral proteins, in undifferentiated (second lane) and differentiated (third lane) SH‐SY5Y cells infected with NWS virus (MOI 0.1) at 24 h p.i, with anti‐influenza antibody. Actin served as loading control. (e) Viral load in supernatants from NWS‐infected undifferentiated and differentiated SH‐SY5Y cells 24 h p.i., measured by qRT‐PCR. Data are shown as mean ± SD ( n = 3).

Article Snippet: Proteins were visualized using the following primary and HRP‐conjugated secondary antibodies: anti‐ β III‐tubulin (ABclonal, A17913, diluted 1:1000); anti‐Influenza (Merck Millipore, AB1074, diluted 1:1000); anti‐Grx1, anti‐MMP‐9, anti‐M2, anti‐GAPDH (Santa Cruz Biotechnology, sc‐293250, sc‐393859, sc‐32238, sc‐47724, respectively, all diluted 1:1000); anti‐GSH (Virogen, 101‐A, diluted 1:500); anti‐Prdx1 (Abcam, 41906, diluted 1:1000); anti‐MMP‐2 (Bioss Antibodies, bs‐4599R, diluted 1:1000); anti‐Actin (Sigma‐Aldrich, A2066, diluted 1:1000); anti‐goat, anti‐mouse, anti‐rabbit (Bethyl Laboratories, A50‐101P, A120‐101P, A90‐116P, respectively, all diluted 1:10000).

Techniques: Virus, Microscopy, Expressing, Marker, Quantitative RT-PCR, Western Blot, Infection, Control, Molecular Weight