Journal: Neural Regeneration Research

Article Title: Inhibition of the NLRP3 inflammasome attenuates spiral ganglion neuron degeneration in aminoglycoside-induced hearing loss

doi: 10.4103/NRR.NRR-D-23-01879

Figure Lengend Snippet: Increased release of IL-1β and IL-18 after kanamycin treatment. (A) mRNA levels of IL-1 β and IL-18 in SGN regions after kanamycin injection ( n = 3). (B) Immunohistochemical staining of IL-1β and IL-18 in the middle turn of Rosenthal’s canal from the Con, 1 D, and 15 D groups. IL-1β and IL-18 were increased at 1 D and 15 D. Scale bar: 25 µm. (C) Enzyme-linked immunosorbent assay for IL-1β and IL-18 after kanamycin injection ( n = 3). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Dunnett’s post hoc test). Con: Control; D: day(s); IL: interleukin; SGN: spiral ganglion neuron.

Article Snippet: Commercial mouse enzyme-linked immunosorbent assay kits (Cloud-Clone Corp., Wuhan, China) were used to quantify IL-1β and IL-18 expression in the cochlear SGNs and cultured organ explants according to the manufacturer’s instructions.

Techniques: Injection, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Neural Regeneration Research

Article Title: Inhibition of the NLRP3 inflammasome attenuates spiral ganglion neuron degeneration in aminoglycoside-induced hearing loss

doi: 10.4103/NRR.NRR-D-23-01879

Figure Lengend Snippet: SGN protection and reduced expression of pyroptosis-related molecules after NLRP3 inflammasome inhibition. (A) SGN fibers (beta-III tubulin, green, Alexa Fluor 488) in the apical, middle, and basal turns. The density and morphology of the SGN fibers were improved by Mcc950 after kanamycin treatment in the apical, middle, and basal turns. (B) SGN somas in the apical, middle, and basal turns. The density and morphology of the SGN somas were improved by Mcc950 after kanamycin treatment in the apical, middle, and basal turns. Scale bars: 20 µm. (C) Quantification of SGN fibers in the four groups ( n = 3). (D) Quantification of SGNs in the four groups ( n = 3). (E) Relative mRNA expression levels of Nlrp3 , ASC , Caspase-1 , and Gsdmd ( n = 3). (F) Enzyme-linked immunosorbent assay for IL-1β and IL-18 ( n = 3). Data are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs . kanamycin group (one-way analysis of variance followed by Dunnett’s post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; C: Con group; Gsdmd: Gasdermin D; IL: interleukin; K: kanamycin group; K+M: kanamycin plus Mcc950 group; M: Mcc950 group; Nlrp3: NOD-like receptor family pyrin domain containing 3; SGN: spiral ganglion neuron.

Article Snippet: Commercial mouse enzyme-linked immunosorbent assay kits (Cloud-Clone Corp., Wuhan, China) were used to quantify IL-1β and IL-18 expression in the cochlear SGNs and cultured organ explants according to the manufacturer’s instructions.

Techniques: Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Control

Journal: Neural Regeneration Research

Article Title: Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01933

Figure Lengend Snippet: Pla2g7 is essential for mediating the regulatory effects of CerS5 on pyroptosis. (A) Ceramide concentrations in the spinal cord in the SCI and Sham groups were detected by ELISA, and the data were normalized to the Shame group ( n = 3). (B) Western blot analysis of Pla2g7, p50, GSDMD, and GSDMD-N expression levels in BV2 cells after treatment with Darapladib (Pla2g7 inhibitor) ( n = 3). ** P < 0.01, vs . C16 ceramide. (C) Representative examples of immunofluorescence staining images used to quantify GSDMD + Iba1 + cells after treatment with Darapladib ( n = 5). There were significantly fewer GSDMD + Iba1 + cells in the C16 ceramide + Darapladib group than in the C16 ceramide group. Iba1, green, CoraLite 488; GSDMD, red, CoraLite 594; DAPI, blue. Scale bar: 100 μm. (D) Western blot analysis of the expression levels of proteins associated with the classical NLRP3 pyroptosis pathway in BV2 cells after treatment with siCerS5 or Darapladib ( n = 3). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). ASC: Apoptosis associated speck like protein containing CARD; CerS: ceramide synthase; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; GSDMD-N: N-terminal GSDMD; Iba1: ionized calcium binding adaptor molecule 1; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury; si.CerS5: CerS5 siRNA.

Article Snippet: The ceramide concentration in cell culture supernatants was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Abmart, Shanghai, China, AB5960) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Binding Assay

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: Primers for quantitative reverse transcription-polymerase chain reaction

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques:

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: Differential expressed proteins in plasma exosomes derived from healthy and Parkinson’s disease patients

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Derivative Assay

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: Characteristics of PD patients and healthy controls for exosomes ELISA testing

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: NAGLU expression in exosomes derived from patients with PD and from MPP + -treated cells. (A) Immunoblot analysis of NAGLU and NT5E expression in plasma exosomes from an independent cohort comprising seven patients with PD and seven healthy controls. In line with the MS result, compared with healthy controls, NAGLU expression was reduced, while NT5E expression was significantly increased, in exosomes derived from patients with PD. Flotillin-1 was used as a loading control. (B) Relative protein expression levels for each treatment group are shown as the mean ±SEM ( n = 7). (C) Immunoblot analysis of NAGLU expression in exosomes derived from MPP + -treated N2A cells. β-Actin was used as a loading control. (D) Relative protein expression levels in the MPP + -treated and untreated groups are shown as the mean ± SEM ( n = 3). (E) Immunoblot analysis of NAGLU expression in exosomes derived from MPP + -treated SY5Y cells. Flotillin-1 was used as a loading control. (F) Relative protein expression levels in the MPP + -treated and untreated groups are shown as the mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001 (unpaired t -test). HC: Healthy control; MPP + : methyl-4-phenylpyridinium; NAGLU: N-acetyl-alpha-glucosaminidase; NT5E: 5′-Nucleotidase Ecto; PD: Parkinson’s disease.

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: NAGLU and α-syn expression in MPP + -treated N2A cells and α-syn-overexpressing SH-SY5Y cells. (A) Immunoblot analysis of NAGLU and α-syn expression in untreated and MPP + -treated N2A cells. β-actin was used as a loading control. (B, C) Relative protein (B) and mRNA expression (C) of NAGLU and α-syn. (D) Immunoblot analysis of NAGLU and α-syn expression in untransfected cells (control), cells transfected with a control vector (vector), and cells transfected with plasmids encoding α-syn WT (WT) or α-syn A53T (A53T). β-Actin was used as a loading control. (E) Relative expression of NAGLU and α-syn. (G) Double-immunostaining for NAGLU (green) and α-syn (red) in untransfected SH-SY5Y cells (control), SH-SY5Y cells transfected with a control vector (vector), and SH-SY5Y cells transfected with plasmids encoding α-syn WT (WT) or α-syn A53T (A53T). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (H) NAGLU and α-syn fluorescence intensity. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test for B, C, and H; one-way analysis of variance followed by Tukey’s post hoc test for E, F). Data are shown as the mean ± SEM ( n = 3). DAPI: 4′,6-Diamidino-2-phenylindole; MPP + : methyl-4-phenylpyridinium; NAGLU: N-acetyl-alpha-glucosaminidase; N.S.: not significant; WB: western blot; WT: wild-type; α-syn: α-synuclein.

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Double Immunostaining, Staining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: NAGLU overexpression protects N2A cells against the effects of MPP + in vitro. (A) Western blot showing that NAGLU expression was significantly elevated in cells transfected with an NAGLU overexpression plasmid compared with untransfected cells and cells transfected with a control vector. β-Actin was used as a loading control. (B) Relative NAGLU expression. (C) Relative NAGLU mRNA expression normalized to the control. (D) Cells were transfected with an NAGLU overexpression plasmid or a control vector and treated with MPP + (500 μM) for 24 hours, and cell survival was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. (E) NAGLU and α-syn expression in each group. β-Actin was used as a loading control. (F, G) NAGLU and α-syn expression. (H) Double-immunostaining for NAGLU (green) and α-syn (red) in N2A cells. Nuclei (blue) were counterstained with DAPI. Scale bar: 50 μm. (I) NAGLU and α-syn fluorescence intensities are shown. Data are presented as the mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; MPP + : methyl-4-phenylpyridinium; NAGLU: N-acetyl-alpha-glucosaminidase; N.S.: not significant; OE: overexpression; α-syn: α-synuclein.

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Over Expression, In Vitro, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, MTT Assay, Double Immunostaining, Fluorescence

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: NAGLU expression was reduced in the brains of MPTP-treated mice. (A) Representative immunofluorescence staining images of the substantia nigra pars compacta showing that the number of TH+ cells (red) was decreased in MPTP-treated mice. Nuclei (blue) were counterstained with DAPI. (B, D) TH, NAGLU, and α-syn fluorescence intensities. (C, E) α-syn (red) (C) and NAGLU (red) (E) immunofluorescence in the midbrain. Scale bar: 200 μm. (F) Relative NAGLU and α-syn mRNA expression (qRT-PCR). Results are presented as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01 (unpaired t -test). DAPI: 4′,6-Diamidino-2-phenylindole; TH: tyrosine hydroxylase; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NAGLU: N-acetyl-alpha-glucosaminidase; α-syn: α-synuclein.

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR

Journal: Neural Regeneration Research

Article Title: Comparative proteomic analysis of plasma exosomes reveals the functional contribution of N-acetyl-alpha-glucosaminidase to Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01500

Figure Lengend Snippet: Evaluation of the diagnostic value of exosomal NAGLU in PD. (A) Plasma exosome NAGLU levels were significantly lower in patients with PD ( n = 63) than in healthy controls ( n = 66), as determined by ELISA (Student’s t -test). (B) Receiver operating characteristic curves for exosome NAGLU expression levels between patients with PD and healthy controls. (C) Exosomal NAGLU levels at different H–Y stages (one-way analysis of variance followed by Tukey’s post hoc test). (D) Correlation analysis of H–Y stages and NAGLU level. Correlations between exosome α-syn and NAGLU expression in patients with PD (E) and controls (F). * P < 0.05, ** P < 0.01, *** P < 0.001. Pearson correlation analysis was used in D, E, and F. H–Y: Hoehn and Yahr stage; NAGLU: N-acetyl-alpha-glucosaminidase; N.S.: not significant; PD: Parkinson’s disease.

Article Snippet: Exosomal NAGLU and α-syn concentrations were measured using commercial NAGLU enzyme-linked immunosorbent assay (ELISA) kits (Uscn Life Science, Wuhan, China) and α-syn ELISA kits (AS-55550-H, AnaSpec Inc., Fremont, CA, USA) according to the manufacturers’ protocols (Shi et al., 2017).

Techniques: Diagnostic Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: CR2-Crry counteracts the effects of CHI mouse serum-induced NF-κB activation in microglia. (A, B) quantitative reverse transcription-polymerase chain reaction results showing the levels of Tnf-α (A) and Igf-1 (B) in microglia among the PBS (microglia treated with PBS), CHI (microglia treated with CHI mouse serum), and CHI + CR2-Crry (microglia treated with CHI mouse serum and CR2-Crry) groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (C) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (D–F) Histograms depicting the relative levels of C9 (D), TNF-α (E), and IGF-1 (F) in microglia among the three groups ( n = 6/group; Student’s t -test and one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). (G) Representative immunoblots illustrating the levels of phospho-p65 and p65 in the nuclei of microglia among the three groups (full-length blots are presented in Additional Figure 13 ). (H) Histograms depicting the relative levels of phospho-p65/p65 in the nuclei of microglia among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–K) Histograms showing the levels of CXCL12 (I), TNF-α (J), and IGF-1 (K) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the three groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). CHI: Closed head injury; CR2-Crry: complement receptor 2-conjugated Crry; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; Igf-1/IGF-1: insulin-like growth factor-1; NF-κB: nuclear factor-κB; Tnf-α/TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: CXCR4 knockdown reduces the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and CXCR4-specific siRNA-transfected (iNSCs treated with CXCR4-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 14 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 15 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and CXCR4-specific siRNA-transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Knockdown, Activation Assay, Western Blot, Control, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: Akt knockdown diminishes the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 in microglia among the control, untransfected (iNSCs not treated with any siRNA) coculture, control siRNA-transfected (iNSCs treated with control siRNA) coculture, and Akt-specific siRNA–transfected (iNSCs treated with Akt-specific siRNA) coculture groups (full-length blots are presented in Additional Figure 16 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt expression in iNSCs among the four groups (full-length blots are presented in Additional Figure 17 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) expression in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) expression in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (M, O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific siRNA–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N, P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Knockdown, Activation Assay, Western Blot, Control, Transfection, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

doi: 10.4103/NRR.NRR-D-23-01495

Figure Lengend Snippet: Akt activation enhances the immunoregulatory effects of iNSCs on closed head injury mouse serum–induced NF-κB activation in microglia. (A) Representative immunoblots illustrating the levels of C9, TNF-α, and IGF-1 expression in microglia among the control, untransfected (iNSCs note treated with LACs) coculture, control LAC-transfected (iNSCs treated with control LACs) coculture, and Akt-specific LAC-transfected (iNSCs treated with Akt-specific LACs) coculture groups (full-length blots are presented in Additional Figure 18 ). (B–D) Histograms depicting the relative levels of C9 (B), TNF-α (C), and IGF-1 (D) expression in microglia among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (E) Representative immunoblots illustrating the levels of CXCR4, Crry, p-Akt, and Akt in iNSCs among the four groups (full-length blots are presented in Additional Figure 19 ). (F–H) Histograms depicting the relative levels of CXCR4 (F), Crry (G), and p-Akt/Akt (H) in iNSCs among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, * P < 0.05, ** P < 0.01, *** P < 0.001). (I–L) Histograms showing the levels of CXCL12 (I), TNF-α (J), IGF-1 (K), and soluble Crry (sCrry) (L) in cell culture supernatants, as measured by enzyme-linked immunosorbent assay, among the four groups ( n = 6/group; one-way analysis of variance with Tukey’s post hoc test, *** P < 0.001). (M and O) Representative flow cytometric analysis of CXCR4 (M) and Crry (O) expression in iNSCs among the control (green line), untransfected coculture (red line), and Akt-specific LAC–transfected coculture (blue line) groups. Isotype antibodies were used as controls (black line). (N and P) Histograms showing the median fluorescence intensity (MFI) values of CXCR4 (N) and Crry (P) expression in iNSCs among the three groups ( n = 3/group; one-way analysis of variance with Tukey’s post hoc test, *P < 0.05, ** P < 0.01, *** P < 0.001). Data are presented as mean ± standard deviation (SD). GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; IGF-1: insulin-like growth factor-1; iNSCs: induced neural stem cells; LACs: lentiviral activation particles; NF-κB: nuclear factor-κB; TNF-α: tumor necrosis factor-α.

Article Snippet: CXCL12, TNF-α, IGF-1, and soluble Crry (sCrry) levels in the supernatants were measured using enzyme linked immunosorbent assay (ELISA) kits (CXCL12, TNF-α, and IGF-1 ELISA kits purchased from R&D Systems, Minneapolis, MN, USA; sCrry ELISA kit purchased from Abcam, Cambridge, MA, USA) according to the manufacturers’ protocols.

Techniques: Activation Assay, Western Blot, Expressing, Control, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Fluorescence, Standard Deviation