nicd  (Cell Signaling Technology Inc)

Journal: Research

Article Title: Low-Intensity Pulsed Ultrasound Promotes Oligodendrocyte Maturation and Remyelination by Down-regulating the Interleukin-17A/Notch1 Signaling Pathway in Mice with Ischemic Stroke

doi: 10.34133/research.0676

Figure Lengend Snippet: LIPUS down-regulates IL-17A expression and Notch1 activation after ischemic stroke. (A to C) RNA-seq analysis was performed on ischemic penumbra brain tissue from dMCAO nonintervention and LIPUS-treated mice at postoperative day 14, n = 4 mice/group. (A) The results of hierarchical cluster analysis for differential genes between the 2 groups were displayed using a heatmap. (B) A volcano plot was employed to visualize the changes in gene expression (up-regulation in red and down-regulation in blue) detected by RNA-seq between LIPUS-treated mice and nonintervention MCAO mice. (C) KEGG pathway analysis revealed alterations in several important pathways within the ischemic penumbral brain tissue following LIPUS intervention. (D) Differences in IL-17A protein levels within the peri-infarct brain regions of Sham, MCAO, and LIPUS group mice are shown with representative images in (i) and quantitative plots in (i). β-Actin serves as a loading control. n = 6 mice/group. (E) Expression of IL-17A in brain tissue homogenates from 3 groups of mice by ELISA. n = 6 mice/group. (F) Representative maps of double immunofluorescence staining for Notch1 (red) and PDGFR-α (green) in peri-infarct cortex. (G) RT-qPCR detection of Notch1 expression. Experiments were repeated 3 to 5 times. n = 6 mice/group. (H) Western blot analysis of protein levels of Notch1 and its activated state (Cleaved-Notch1) in semi-dark band brain tissues. (i) Representative images and (ii and iii) quantitative statistical plots. n = 6 mice/group. Data are reported as means ± SD. Statistical analysis for comparisons between groups (D, E, and H) was conducted using one-way ANOVA followed by Tukey’s multiple comparisons test, while an unpaired 2-tailed Student’s t test was employed for the analysis of group (G). * P < 0.05, ** P < 0.01, * * P < 0.001, ns = no statistical difference.

Article Snippet: Primary antibodies employed were as follows: rabbit anti-β-actin (1:10,000, AB0035, Abways, China), rabbit anti-MBP (1:1,000, ab218011, Abcam, UK), rabbit anti-APC (1:2,000, ab40778, Abcam, UK), rabbit anti-IL-17A (1:500, A00421-2, Boster, China), rabbit anti-Notch1 (1:1,000,4380,Cell Signaling Technology, USA), and rabbit anti-cleaved Notch1 (1:1,000,4147, Cell Signaling Technology, USA).

Techniques: Expressing, Activation Assay, RNA Sequencing, Gene Expression, Control, Enzyme-linked Immunosorbent Assay, Double Immunofluorescence Staining, Quantitative RT-PCR, Western Blot

Journal: Research

Article Title: Low-Intensity Pulsed Ultrasound Promotes Oligodendrocyte Maturation and Remyelination by Down-regulating the Interleukin-17A/Notch1 Signaling Pathway in Mice with Ischemic Stroke

doi: 10.34133/research.0676

Figure Lengend Snippet: LIPUS promotes differentiation of OPCs and improves cerebral WM integrity after ischemic stroke by inhibiting the IL-17A/Notch1 pathway. (A) Western blot analysis of the protein levels of IL-17A in 4 groups of penumbra brain tissues, with representative images (i) and quantitative analysis plots (ii), n = 6 mice/group. (B) Western blot analysis of the protein levels of Notch1 and Cleaved-Notch1 in 4 groups of penumbra brain tissues, with representative images (i) and quantitative analysis plots of Notch1 (ii) and Cleaved-Notch1 (iii), n = 6 mice/group. (C) Representative photographs of TTC staining (i) and quantitative analysis of infarct volume (ii), n = 6 mice/group. (D) Representative images depicting that the Western blot analysis of MBP was obtained for each group (i). The expression levels of the target proteins were normalized relative to β-actin (ii), n = 6 mice/group. (E) Representative images depicting that the Western blot analysis of APC was obtained for each group (i). The expression levels of the target proteins were normalized relative to β-actin (ii), n = 6 mice/group. (F) Double-labeled immunofluorescence staining of MBP (green) and SMI32 (red) in 3 brain regions, CTX, EC, and STR, scale bar = 50μm, n = 6 mice/group. (G) is the quantification of MBP immunoreactivity in (F), and (H) is the SMI32/MBP intensity ratio in (F). (J) Representative images showing the colocalization of APC (green) and BrdU (red) through double immunofluorescence staining, scale bar = 50μm, n = 6 mice/group. (I) is a quantitative map of Brdu+APC+ cells in (J). Data are presented as means ± SD, (A) to (E) were performed by one-way ANOVA followed by Tukey’s multiple comparisons test. (G) to (I) were performed by a 2-way ANOVA followed by Tukey’s multiple comparisons test.* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = no statistical difference.

Article Snippet: Primary antibodies employed were as follows: rabbit anti-β-actin (1:10,000, AB0035, Abways, China), rabbit anti-MBP (1:1,000, ab218011, Abcam, UK), rabbit anti-APC (1:2,000, ab40778, Abcam, UK), rabbit anti-IL-17A (1:500, A00421-2, Boster, China), rabbit anti-Notch1 (1:1,000,4380,Cell Signaling Technology, USA), and rabbit anti-cleaved Notch1 (1:1,000,4147, Cell Signaling Technology, USA).

Techniques: Western Blot, Staining, Expressing, Labeling, Immunofluorescence, Double Immunofluorescence Staining

Journal: Research

Article Title: Low-Intensity Pulsed Ultrasound Promotes Oligodendrocyte Maturation and Remyelination by Down-regulating the Interleukin-17A/Notch1 Signaling Pathway in Mice with Ischemic Stroke

doi: 10.34133/research.0676

Figure Lengend Snippet: Primers for RT-qPCR

Article Snippet: Primary antibodies employed were as follows: rabbit anti-β-actin (1:10,000, AB0035, Abways, China), rabbit anti-MBP (1:1,000, ab218011, Abcam, UK), rabbit anti-APC (1:2,000, ab40778, Abcam, UK), rabbit anti-IL-17A (1:500, A00421-2, Boster, China), rabbit anti-Notch1 (1:1,000,4380,Cell Signaling Technology, USA), and rabbit anti-cleaved Notch1 (1:1,000,4147, Cell Signaling Technology, USA).

Techniques: