Journal: Arthritis Research & Therapy

Article Title: Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

doi: 10.1186/s13075-022-02817-7

Figure Lengend Snippet: Nrp2 expression in activated macrophages during joint inflammation. A Nrp2 expression in immune cells in the CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow-cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed as the means ± SD. N = 3 from three independent experiments. B The characteristics of Nrp2-high macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from two independent experiments. C Nrp2 expression on BMMs. BMMs were cultured under the indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from three independent experiments. D NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data were reanalyzed, and several cell types were defined by UMAP. NRP2 expression is denoted by purple dots. The statistical analyses were performed using an unpaired t-test

Article Snippet: Anti-neuropilin2 (Nrp2) polyclonal antibodies conjugated with FITC were purchased from Alomone Labs (#ANR-062).

Techniques: Expressing, Staining, Cell Culture, Flow Cytometry, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

doi: 10.1101/2022.02.12.480222

Figure Lengend Snippet: A. Nrp2 expression on immune cells in CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed means ± SD. N = 3 from 3 independent experiments. B. The characteristics of Nrp2-positive macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from 2 independent experiments. C. The expression of Nrp2 on BMMs. BMMs were cultured under indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from 3 independent experiments. D. NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data was re-analyzed, and several cell types were defined by UMAP. Expression of NRP2 is indicated with purple dots. The statistical analyses were performed using an unpaired t-test.

Article Snippet: Anti-Neuropilin2 (Nrp2) polyclonal antibody was from Alomone Labs. Antibodies against CD45 (104), CD3ε (145-2C11), CD4 (GK1.5), B220 (RA3-6B2), CD11b (M1/70), F4/80 (BM8), CD86 (GL-1), and MHC-II (M5/114.15.2), and Zombie Aqua were from BioLegend.

Techniques: Expressing, Staining, Cell Culture, Flow Cytometry, RNA Sequencing Assay

Journal: The Journal of Neuroscience

Article Title: miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non–Cell-Autonomous Mechanism in ALS

doi: 10.1523/JNEUROSCI.3037-17.2018

Figure Lengend Snippet: miR126-5p is depleted in SOD1 G93A muscles and regulates Sema3 and NRP expression. A , NanoString chip screen heat map of significantly altered miRs in P60 muscles of SOD1 G93A compared with LM mice (extended table under ). Red and green represent a high or low abundance of miRs, respectively. * p < 0.05 (Student's t test; n = 3). B , miR126-5p was the most significantly downregulated miRNA in SOD1 G93A muscles (Student's t test; n = 3, ** p = 0.003). C , qPCR analysis of P60 GC muscle extracts further validates the decrease in miR 126-5p in SOD1 G93A ( n = 3). D–G , qPCR analysis of Sema3A, NRP1, Sema3B, and NRP2 transcript levels in HeLa cells overexpressing either miR126-5p or miR142 demonstrates a reduction in their expression levels specifically under miR126-5p overexpression (Student's t test; n = 3, * p = 0.0438, * p = 0.034, * p = 0.031, and * p = 0.0434, respectively). H , Representative TIRF images of U87MG cells reveal a detachment of the cell membrane from the culture dish surface after Sema3A is added to the culture medium. Scale bar, 10 μm. I , Impedance recording of live cells over time shows that U87MG cells overexpressing miR126-5p are unresponsive to Sema3A added to the culture medium because their impedance continuously increases, whereas the impedance of U87MG cells overexpressing miR142 decreases after treatment .

Article Snippet: Antibodies were used at the following concentrations: anti-Neurofilament Heavy Chain 1:500 (NFH, Abcam, ab72996; 1:1000), synaptophysin (Millipore, MAB5258; 1:300), synaptotagmin (Alomone Labs, ant-003; 1:300), anti-NRP1 (1:100), anti-Sema3A (1:100), anti-NRP2 (1:100), and anti-Sema3B (1:100).

Techniques: Expressing, Over Expression