Journal: Redox Biology
Article Title: A clickable CoQ imaging probe reveals that cellular uptake and lysosomal trafficking depend on CD36 and NPC1
doi: 10.1016/j.redox.2025.103936
Figure Lengend Snippet: NPC1, CD36, LDLR involvement in CoQ uptake a ) Relative expression (log10-transformed RPKM value) of genes in murine brown adipocytes treated with 4CBA (24 h). b ) Relative expression (RPKM) of selected transcription factors in murine brown adipocytes treated with 4CBA or vehicle (CTL), based on RNA-seq analysis (n = 3 independent RNA pools per treatment). c ) Volcano plot obtained from DESeq2 analysis of vehicle control– and 4CBA-treated murine brown adipocyte RNA pools (n = 3 independent experiments per treatment). The Wald test was used for statistical analysis. d ) Strategy to verify NPC1, CD36, LDLR involvement in CoQ internalization by HPLC approach with CoQ 10 supplementation and imaging using CoQ Azide-SiR-DBCO SPAAC reaction. e ) Gene expression of NPC1, f ) CD36 and g ) LDLR siRNA-treated cells, n = 6. h-j ) CoQ 10 levels of murine brown adipocytes treated with vehicle or CoQ 10 ND (5 μM) for 24 h after transfection with siRNA, n = 6. Cells were treated on day 7, and harvested on day 8 for HPLC analysis. Data are presented as mean ± SEM. Results were compared using an unpaired two-tailed Student's t-test. Statistical significance is indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001 compared to controls. Data represent biological replicates. For the colocalization analysis, BAT cells were treated with siRNAs as outlined in d . Cells were treated with 50 μM CoQ AzideND for 24 h at day 7 and analyzed the following day. SPAAC reaction and staining was performed as described in . To visualize the plasma membrane, the fluorescent probe MemGlow 488 (Cytoskeleton) was used at a final concentration of 1 μM after the SPAAC reaction. l ) First row, from left to right, representative confocal images of cells costained with CoQ Azide, Hoechst, and LysoTracker Green, along with m ) the corresponding colocalization analysis of CoQ Azide signal with lysosomes. n ) Costaining of CoQ Azide, Hoechst, and MitoTracker Green in the second row, and o ) respective colocalization of CoQ Azide signal with MitoTracker Green. p ) Third row, representative confocal images of cell costained with CoQ Azide, Hoechst and MEMGlow and q ) respective colocalization analysis of CoQ Azide signal with plasma membrane. Scale bar, 20 μm. Colocalization analysis was performed using JaCoP plugin on ImageJ. Data represent two independent experiments. A minimum of five images were analyzed for each condition. Results were compared using a one-way ANOVA test. Statistical significance is indicated as ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.
Article Snippet: Lentiviral particles were produced by co-transfecting HEK293T cells with the transfer plasmid, pMD2.G (Addgene #12259), and psPAX2 (Addgene #12260) at a 5:3.75:1.25 ratio using PEI transfection reagent (Polysciences, 23966).
Techniques: Expressing, Transformation Assay, RNA Sequencing, Control, Imaging, Gene Expression, Transfection, Two Tailed Test, Staining, Clinical Proteomics, Membrane, Concentration Assay
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