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: 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.

Techniques: Expressing, Transformation Assay, RNA Sequencing, Control, Imaging, Gene Expression, Transfection, Two Tailed Test, Staining, Clinical Proteomics, Membrane, Concentration Assay

Journal: Journal of Advanced Research

Article Title: Palmitoylated COX-2 Cys555 reprogrammed mitochondrial metabolism in pyroptotic inflammatory injury in patients with post-acute COVID-19 syndrome

doi: 10.1016/j.jare.2025.05.005

Figure Lengend Snippet: Regulation of SARS-CoV-2 S protein expression on mitochondrial quality control and metabolic reprogramming in lung epithelial cells. (A-F, H-L) BEAS-2B cells were transfected with pCMV3-SARS-CoV-2 S (pCMV-S, 1 μg for 24 h) to induce the S protein expression. ( A) TEM observation of mitochondrial morphology in cells. N, nucleus. M, mitochondria. Scale bar, 5 μm for the gobal view and 2 μm for the zoom view. ( B) IF assay of MitoTracker (green) and LysoTracker (red) was used to assess the intensity and co-localization in cells. Scale bar, 10 μm. ( C) WB analysis of mitochondrial dynamics-associated proteins (Drp1, p-Drp1 Ser616 , and Mfn2). ( D) Flow cytometry (FCM) assay of mitochondrial ROS levels using MitoSOX (red) in cells. ( E) WB analysis of mitochondrial damage-related proteins (Bax, Bcl2, and Cyto C). ( F) JC-1 assay of mitochondrial membrane potential. ( G) The GEO dataset ( GSE147507 ) from SARS-CoV-2-infected A549 cell samples (n = 6) was screened, while mock samples (n = 5) used as controls. The heatmap depicts the DEGs of glycolysis-related genes. ( H) Relative mRNA levels of mitochondrial energy metabolism-related genes in cells. ( I) FCM assay of glucose flux using fluorescent glucose analog 2-NBDG in cells. ( J) Detection of ATP content in cells. ( K) WB analysis of glycolysis-related proteins (HIF1α, HK2, and PKM2). ( L) Levels of glycolysis rate-limiting enzymes (hexokinase and pyruvate kinase) activities and glycolysis product lactic acid. (M−O) BEAS-2B cells were pre-treated with the hexokinase inhibitor 2-deoxy-D-glucose (2-DG, 20 μM for 3 h), followed by transfection with pCMV-S (1 μg for 24 h) or mock transfection. (M) WB analysis of NLRP3 inflammasome activation (NLRP3, ASC, and p10) and pyroptosis-related proteins (GSDMD and IL-1β). ( N) LDH release assay and ELISA detection of IL-1β release levels in the supernatant. ( O) Cell viability was measured using the MTT assay. *, P < 0.05, compare to the control group or corresponding group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The impact of SARS-CoV-2 S protein expression on glucose uptake in lung epithelial cells was evaluated using the 2-NBDG fluorescent glucose probe (MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Expressing, Control, Transfection, Flow Cytometry, Membrane, Infection, Activation Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, MTT Assay