Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of Mcu in RBL-2H3 cells. A , exon structure of rat Mcu indicating the target sites of guide RNA1 and guide RNA2. B , quantitative real-time PCR analysis of Mcu mRNA expression in wild-type (WT) RBL-2H3 cells and Mcu knockout (KO) clones A4 and D10 (n = 30 wells; 3 biological replicates). Medians ( bars ) with 95% confidence intervals (error bars ) are displayed on the scatter dot plots. C , immunoblot analysis of WT and Mcu KO clones A4 and D10 using MCU antibodies raised against either the C terminus or N terminus. Vinculin served as a loading control. Molecular weights are indicated in kilodaltons (kDa). MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: CRISPR, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: Mcu knockout by CRISPR/Cas9 causes widespread and divergent transcriptomic changes across clones generated using distinct guide RNAs. A and B , volcano plots showing differentially expressed genes (DEGs) in Mcu −/− clones A4 ( A ) and D10 ( B ) relative to WT cells. Red and blue dots represent significantly upregulated and downregulated genes, respectively. Mcu is shown as a green dot . Gray dots indicate unchanged genes, either with below absolute fold change 2.0 cutoff ( light gray ) or having false discovery rate(FDR) higher than 0.05 ( dark gray ). C , volcano plot comparing A4 and D10 clones directly. D and E , Venn diagrams of downregulated genes ( D ) and upregulated genes ( E ) in A4 versus WT and D10 versus WT, highlighting both overlapping and clone-specific expression changes. F , ingenuity pathway analysis (IPA) of the 213 DEGs common to both clones (64 downregulated and 149 upregulated) with a z-score cutoff of 1.0. G , bubble plot displays enriched canonical pathways predicted by IPA. The y -axis on the right shows enriched pathway names ordered according to hierarchical clustering of their z-scores across two conditions (A4 versus WT and D10 versus WT), with the corresponding dendrogram displayed on the left . The x -axis labels the two conditions. Each pathway is represented by two circles , one for each condition. Circle size corresponds to the gene ratio for that pathway in the given condition. The fill color of each circle reflects the z-score: red for positive z-scores (activation), blue for negative z-scores (inhibition), transparent for values near zero, and gray where the z-score is NA. Threshold of z-score was set to absolute z-score ≥ 1.5. Below cutoff p values ( i.e. , p value > 0.05) are indicated by small lines on the right of the bubble. MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: Knock-Out, CRISPR, Clone Assay, Generated, Expressing, Activation Assay, Inhibition