plasmids encoding suv3-ha  (Addgene inc)

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Schematic of mitochondrial transcript unwinding and degradation by SUV3 and PNPase complexes. ( B ) Representative images of cells immunolabeled for TOM20 (grayscale) and dsRNA (J2; blue/rainbow lookup table) at 4 days versus 7 days of SUV3 depletion. Scale bars, 5 μm. ( C ) Three representative images of RNA bodies in cells fixed and immunolabeled with antibodies against dsRNA (J2; blue), TOM20 (green), and RNA-FISH targeting RNR2 (magenta). Scale bars, 1 μm.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Immunolabeling

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Representative image of control (top) and SUV3-depleted (bottom) cells pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect TOM20 (blue) and RNA-FISH against RNR2 (magenta). Scale bars, 1 μm. ( B ) Average HPG fluorescence intensity in segmented mitochondria in the presence or absence of CAP (50 μg/ml) [(no CAP) control: n = 9 mitochondrial networks, sgSUV3: n = 6 mitochondrial networks; (CAP) control: n = 4 mitochondrial networks, sgSUV3: n = 4 mitochondrial networks]. [** P < 0.01, Kruskal-Wallis test (** P < 0.01), followed by Dunn’s multiple comparisons]. ( C ) Diagram of proposed impact of SUV3 depletion on dsRNA accumulation and single-stranded RNA reorganization into mitochondrial stress bodies.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Control, Labeling, Immunolabeling, Fluorescence

Journal: Science Advances

Article Title: Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress

doi: 10.1126/sciadv.ads6830

Figure Lengend Snippet: ( A ) Average ROI area and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of imaged IMR90 cells when SUV3 depletion is induced in cells after incubation in CAP (50 μg/ml) (DMSO: n = 16, CAP: n = 12). (** P < 0.01, Mann-Whitney test). ( B ) Average size and fluorescence intensity of segmented RNR2-FISH signals in maximum intensity projections of IMR90 cells incubated in CAP (50 μg/ml), imaged 7 days after transfection with sgSUV3 RNP complexes (DMSO: n = 17, CAP: n = 18). (not significant, Mann-Whitney test). ( C ) Representative images of control (top) and 50 μM CAP-treated (bottom) cells 7 days after transfection with sgSUV3 RNP complexes, fixed, and immunolabeled to detect dsRNA (J2; blue), TOM20 (green), and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( D ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, fixed, and immunolabeled to detect dsRNA (J2; green), GRSF1 (blue), and RNR2-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( E ) Representative image of control (top) and day 4 sgSUV3 (bottom) cells, pulse-labeled with 50 μM HPG for 15 min (green), fixed, and immunolabeled to detect dsRNA (J2; blue) and RNA-FISH against RNR2 (magenta). Scale bars, 5 μm; 1 μm in zoom. ( F ) Proposed model for organization of mitochondrial gene expression into translational hubs and their remodeling during stress.

Article Snippet: Plasmids encoding SUV3-HA, SUV3 G207V -HA, and mCherry-DRP1 K38A have been deposited to Addgene.

Techniques: Fluorescence, Incubation, MANN-WHITNEY, Transfection, Control, Immunolabeling, Labeling, Gene Expression