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