Journal: Biochemical and biophysical research communications

Article Title: Deficiency of BMAL1 promotes ROS generation and enhances IgE-dependent degranulation in mast cells.

doi: 10.1016/j.bbrc.2023.149295

Figure Lengend Snippet: Fig. 1. Bmal1 deficiency does not affect differentiation and morphology of BMMCs. (A) Representative dot-plot data (Left) and percentage of c-kit+FcεRIα+ cells (Right, n¼6) of WT or Bmal1-KO BMMCs cultured in the presence or absence of 2-ME after 6–8 weeks detected by flowcytometric analysis. (B) Flowcytometric analysis of mean fluorescence intensity (MFI) of c-kit (Left) or FceR1a levels (Right) of WT or Bmal1-KO BMMCs cultured in the presence or absence of 2-ME after 6–8 weeks (n = 6). (C-E) Representative pictures of toluidine blue (C), Diff-Quik (D) or esterase (E) staining of WT or Bmal1-KO BMMCs cultured in the presence of 2- ME after 6–8 weeks. (F) Expression of mast cell differentiation marker and functional genes, determined by qPCR in WT or Bmal1-KO BMMCs cultured in the presence or absence of 2-ME for 6–8 weeks (n = 3). Mean ± SD is shown. Statistical differences were determined by two-way ANOVA with Tukey’s post hoc test, *P < 0.05. ns: non-specific difference. (For inter pretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The cDNA) was synthesized from RNA samples by the ReverTra AceTM qPCR RT Master Mix with gDNA remover (TOYOBO, Japan).

Techniques: Cell Culture, Fluorescence, Diff-Quik, Staining, Expressing, Cell Differentiation, Marker, Functional Assay

Journal: NAR Genomics and Bioinformatics

Article Title: Comparative analysis and classification of highly divergent mouse rDNA units based on their intergenic spacer (IGS) variability

doi: 10.1093/nargab/lqae070

Figure Lengend Snippet: Schematic representation of mouse rDNA isolation. (A) Step 1: Isolation of genomic DNA from the 4T1 cells. (B) Scheme of the mouse rDNA repeat. The rDNA repeat is composed of the ∼13 kb transcribed region encoding 45S rRNA (5′ ETS, 18S, ITS1, 5.8S, ITS2, 28S and 3′ ETS) and a ∼22 to 32 kb intergenic spacer (IGS). The pJY-mrDNA TAR vector contains 5′-84 bp and 3′-78 bp targeting hooks derived from the mouse 18S rDNA sequence. To clone the mouse rDNA repeat, TAR vector containing 5′ hook (red) and 3′ hook (blue) is linearized to expose targeting sequences. (C) TAR cloning of the rDNA repeats. Homologous recombination between the 5′ and 3′ targeting hook sequences and the homologous targeted sequences in the rDNA repeat leads to rescue of mouse rDNA repeats as circular YAC/BAC molecules in yeast Saccharomyces cerevisiae. (D) Southern blot analysis of rDNA in mouse 4T1 and MEFs cell lines. After I-PpoI digestion of DNA isolated from each cell line, rDNA repeats were detected with a probe specific to 28S. As a positive control, I-PpoI-digested DNA isolated from the human RPE1 cell line was used. (E) A copy number of the rDNA repeats was estimated by qPCR with a set of the 45S primers specific to 18S and 28S (Supplementary Table S1).

Article Snippet: Based on qPCR, 4T1 and MEFs cell lines contain 173 and 169 rDNA units, respectively, while a third sample, Promega mouse genomic DNA, contains 473 units (Figure ).

Techniques: Isolation, Plasmid Preparation, Derivative Assay, Sequencing, Cloning, Homologous Recombination, Southern Blot, Positive Control

Journal: eLife

Article Title: Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis

doi: 10.7554/eLife.97662

Figure Lengend Snippet: ( A ) Identification of candidate genes expressed in FAPs in response to glial cell line-derived neurotrophic factor (GDNF) that may contribute to nerve regeneration. Number of genes that fit into each criterion is indicated. ( B ) Expression of Bdnf in each scRNA-seq sample shown on uniform manifold approximation and projection (UMAP) plots. ( C ) Violin plot displaying the expression levels of Bdnf in the seven FAP clusters. ( D ) Scheme for sampling Schwann cells and FAPs at different time points post sciatic nerve crush (SNC) for gene expression analyses. ( E ) RT-qPCR results showing expression levels of Gdnf in Schwann cells (orange dot and line, left y-axis) and Bdnf in FAPs (blue dot and line, right y-axis) at different time points post-SNC. n=4, except for 0 and 2 dpi, where n=3. One-way ANOVA with Bonferroni’s post hoc test. *p<0.05, ***p<0.001, n.s., not significant. ( F ) Western blot results showing BDNF protein expression in PDGFRα + FAPs isolated from SNC-affected or uninjured contralateral muscles at 7 dpi. n=3. Mature form of BDNF is indicated with a red asterisk. Quantified values normalized to GAPDH is indicated below each protein. See for the experimental scheme. ( G ) Scheme for intramuscular injection of either PBS or recombinant mouse GDNF protein, with the time point for FAP isolation post-injection indicated. ( H ) RT-qPCR results show the expression level of Bdnf in FAPs 48 hr post intramuscular injection of either PBS (n=4) or GDNF (n=5). Unpaired t-test with Welch’s correction. **p<0.01. Figure 5—source data 1. The zip file contains raw western blot images, a marker image, and a marker-merged, labeled image obtained for .

Article Snippet: Tibialis anterior muscle and the two gastrocnemius muscles (GA, lateral, and medial) were each injected with 10 μg of either normal rabbit IgG (Sino Biological) or anti-GDNF antibodies (Alomone Labs) using 31-gauge insulin syringes 24 hr post-SNC.

Techniques: Derivative Assay, Expressing, Sampling, Gene Expression, Quantitative RT-PCR, Western Blot, Isolation, Muscles, Injection, Recombinant, Marker, Labeling

Journal: eLife

Article Title: Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis

doi: 10.7554/eLife.97662

Figure Lengend Snippet: ( A ) Experimental scheme depicting intramuscular injection of either anti-GDNF antibodies or IgG control after sciatic nerve crush (SNC), along with the experimental timeline. ( B ) RT-qPCR results show the expression levels of Bdnf in nerve injury-exposed FAPs affected by intramuscular injection of either IgG control or anti-GDNF antibodies. n=4. Unpaired t-test.

Article Snippet: Tibialis anterior muscle and the two gastrocnemius muscles (GA, lateral, and medial) were each injected with 10 μg of either normal rabbit IgG (Sino Biological) or anti-GDNF antibodies (Alomone Labs) using 31-gauge insulin syringes 24 hr post-SNC.

Techniques: Injection, Control, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis

doi: 10.7554/eLife.97662

Figure Lengend Snippet:

Article Snippet: Tibialis anterior muscle and the two gastrocnemius muscles (GA, lateral, and medial) were each injected with 10 μg of either normal rabbit IgG (Sino Biological) or anti-GDNF antibodies (Alomone Labs) using 31-gauge insulin syringes 24 hr post-SNC.

Techniques: Injection, Electron Microscopy, Recombinant, Software, Isolation, Light Microscopy, Imaging