Journal: Investigative ophthalmology & visual science

Article Title: Nicotinamide Riboside Mitigates Retinal Degeneration by Suppressing Damaged DNA-Stimulated Microglial Activation and STING-Mediated Pyroptosis.

doi: 10.1167/iovs.66.4.14

Figure Lengend Snippet: FIGURE 4. NR mitigated damaged DNA-stimulated cGAS-STING pathway. (A) Representative microscopic images of dsDNA immunopositivity (in red) and DAPI-counterstained nuclei (in blue). Scale bar: 50 μm. The boxed areas within the images were magnified and displayed in the right panels (arrowheads). Scale bar: 10 μm. (B) Colocalization of Tom20 (green), dsDNA (red), and DAPI (blue) expressions in retinas of each group (arrows). Scale bar: 10 μm. (C) Heatmap of cGAS and STING from RNA sequencing of naive and LIRD retinas with or without NR treatment (n = 4/group). (D) Colocalization of cGAS (red) and Iba1-positive microglia (green) expression in the retinas of each group (arrows). Scale bar: 30 μm. (E) Quantification of cGAS-positive microglia of each group in the ONL at three days after light exposure (n = 4–6/group). (F) Colocalization of STING (red) and Iba1-positive microglia (green) expression in the retinas of each group (arrows). Scale bar: 30 μm. (G) Quantification of STING-positive microglia of each group in the ONL at three days after light exposure (n = 5–6/group). NR treatment significantly decreased the expression of cGAS and STING compared with the PBS-treated group, ****P < 0.0001.

Article Snippet: Iba-1 Rabbit 1:500 Abcam* ab178846 Iba-1 Goat 1:800 Abcam* ab5076 CD68 (FA-11) Rat 1:200 Thermo† 14-0681-82 dsDNA Mouse 1:200 Santa Cruz‡ sc-58749 Tom20 (D8T4N) Rabbit 1:200 Cell Signaling Technology§ 42406 Cgas Mouse 1:200 Santa Cruz‡ sc-515777 STing Rabbit 1:200 Proteintech║ 19851-1-AP NLRP3 Rabbit 1:200 Abmart¶ P60622R3 Casepase-1 Mouse 1:100 Santa Cruz‡ sc-56036 IL-1β Mouse 1:100 Cell Signaling Technology§ 12242 GSDMD Mouse 1:200 Santa Cruz† sc-393581 Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) Goat 1:400 Jackson# 111-545-003 Goat Anti-Rat IgG H&L (Alexa Fluor 594) Goat 1:300 Jackson# 112-505-175 Donkey Anti-Rabbit IgG H&L (Alexa Fluor 488) Donkey 1:400 Thermo† A21206 Donkey Anti-Mouse IgG H&L (Alexa Fluor 555) Donkey 1:400 Thermo† A31570 Donkey Anti-goat IgG H&L (Alexa Fluor 568) Donkey 1:400 Thermo† A11057 * Abcam, Cambridge, MA, USA.

Techniques: RNA Sequencing, Expressing

Journal: Frontiers in Microbiology

Article Title: SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

doi: 10.3389/fmicb.2021.780768

Figure Lengend Snippet: SARS-CoV-2 dsRNA localizes in mitochondria. (A,B) Vero E6 and Huh-7 cells were fixed and visualized by fluorescence microscopy using antibodies against Hsp60 (red) and dsRNA (green) at 3, 6, and 12 h post infection (h.p.i.). (C,D) Vero E6 and Huh-7 cells were infected with SARS-CoV-2 for 3, 6, and 12 h and visualized by fluorescence microscopy with antibodies recognizing Tom20 (red) and dsRNA (green). (E) Vero E6 cells were visualized by immunoelectron microscopy using antibodies against dsRNA at 12 h post infection. (F) Vero E6 cells were visualized by fluorescence microscopy using antibodies against Calnexin (red), Lamp2b (red) and dsRNA (green) at 12 h post SARS-CoV-2 infection. White scale bars = 5 μm; black scale bars = 500 nm.

Article Snippet: The control siRNA (sc-37007), siRNA against Tom20 (sc-36691) and siRNA against Pink1 (sc-44598) were purchased from Santa Cruz Biotechnology (California, United States).

Techniques: Fluorescence, Microscopy, Infection, Immuno-Electron Microscopy

Journal: Frontiers in Microbiology

Article Title: SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

doi: 10.3389/fmicb.2021.780768

Figure Lengend Snippet: Tom20 facilitates SARS-CoV-2 dsRNA localization in mitochondria. (A) Western blotting analysis showing Tom20 expression at 3, 6, 12, 24 and 48 h post SARS-CoV-2 infection in Vero E6 cells. (B) Western blotting analysis showing knockdown efficiency of Tom20 siRNA in Vero E6 cells. (C) Vero E6 cells transfected with control or Tom20 siRNA were stained with TMRM at 6 and 12 h post infection showing MPTP opening. (D) Vero E6 cells transfected with control or Tom20 siRNA were visualized by fluorescence microscopy using antibodies against Hsp60 (red) and dsRNA (green) at 12 h post SARS-CoV-2 infection. The amounts of dots with overlapping red and green fluorescence per cell were calculated from at least 30 cells from each group. White scale bars = 5 μm. (E) Viral load at 48 h in Vero E6 cells transfected with control or Tom20 siRNA. (F) Western blotting analysis showing N protein expression at 3, 6, 12, and 24 h post SARS-CoV-2 infection in Vero E6 cells transfected with control or Tom20 siRNA. (G) Vero E6 cells transfected with control or Tom20 siRNA were infected with SARS-CoV-2 to detect cellular reactive oxygen species at 12 and 24 h. Data were expressed as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, # p < 0.05, ## p < 0.01.

Article Snippet: The control siRNA (sc-37007), siRNA against Tom20 (sc-36691) and siRNA against Pink1 (sc-44598) were purchased from Santa Cruz Biotechnology (California, United States).

Techniques: Western Blot, Expressing, Infection, Knockdown, Transfection, Control, Staining, Fluorescence, Microscopy

Journal: Frontiers in Microbiology

Article Title: SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

doi: 10.3389/fmicb.2021.780768

Figure Lengend Snippet: Mitophagy is activated via Pink1/Parkin pathway in SARS-CoV-2-infected cells. (A) Western blotting analysis showing Pink1 and Parkin expression at 3, 6, 12, 24, 48, and 72 h post SARS-CoV-2 infection in Vero E6 cells. (B) Vero E6 cells were mock- or SARS-CoV-2-infected for 12 h and visualized by fluorescence microscopy with antibodies recognizing Hsp60 (red) and Pink1 (green). (C) Vero E6 cells were visualized by fluorescence microscopy using antibodies against Hsp60 (red) and Parkin (green) at 12 h post mock or SARS-CoV-2 infection. (D) Vero E6 cells were transfected with control or Pink1 siRNA and visualized by fluorescence microscopy using antibodies against Hsp60 (red) and Parkin (green) at 12 h post mock or SARS-CoV-2 infection. (E) Western blotting analysis showing knockdown efficiency of Tom20 siRNA in Vero E6 cells. The amounts of dots with overlapping red and green fluorescence per cell were calculated from at least 30 cells from each group. White scale bars = 5 μm. Data were expressed as mean ± SEM from three independent experiments. * p < 0.05, *** p < 0.001.

Article Snippet: The control siRNA (sc-37007), siRNA against Tom20 (sc-36691) and siRNA against Pink1 (sc-44598) were purchased from Santa Cruz Biotechnology (California, United States).

Techniques: Infection, Western Blot, Expressing, Fluorescence, Microscopy, Transfection, Control, Knockdown

Journal: Frontiers in Microbiology

Article Title: SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment

doi: 10.3389/fmicb.2021.780768

Figure Lengend Snippet: Schematic representation showing SARS-CoV-2-induced disruption of mitochondrial homeostasis. Upon infection, SARS-CoV-2 releases single strand RNA known to replicate in DMV structures. Tom20 facilitates the entry process of viral RNA into mitochondria, resulting in mitochondrial dysfunction, including the loss of ΔΨm, MPTP opening and increased ROS release. Concomitantly, mitophagy is initiated through Pink1/Parkin pathway by host cell for mitochondrial quality control and virus clearance. However, SARS-CoV-2 hinders the binding of p62 to LC3 protein, thus inhibiting the p62-labeled mitochondria to be encapsulated by autophagosomes. Mitophagy stays in the early stage. This figure is created with BioRender.com .

Article Snippet: The control siRNA (sc-37007), siRNA against Tom20 (sc-36691) and siRNA against Pink1 (sc-44598) were purchased from Santa Cruz Biotechnology (California, United States).

Techniques: Disruption, Infection, Control, Virus, Binding Assay, Labeling

Journal: bioRxiv

Article Title: TBCK-deficiency leads to compartment-specific mRNA and lysosomal trafficking defects in patient-derived neurons

doi: 10.1101/2025.03.02.641041

Figure Lengend Snippet: a, Immunoblot of mitochondrial marker, TOM20, in control (Ctrl) and TBCK-deficient neurons (p.R126X) at 14D. b, Quantification of TOM20 relative to GAPDH (n=4). c, Immunoblot of proteins from the oxidative phosphorylation complexes (I-V) in iNeurons at 14D in control (Ctrl) and TBCK-deficient neurons (p.R126X) (n=4). Significance was calculated using unpaired t-test analysis. d-i, Quantification of immunoblot in a relative to VDAC (n=4). Significance was calculated using unpaired t-test analysis. All graphs show error bars with mean ± SD from independent experiments (n).

Article Snippet: The following antibodies were used for western blot analysis: rabbit anti-LC3B (1:1000, Cell signaling, 27755), rabbit anti-p62 (1:1000, Cell signaling, 51145), mouse anti-mTOR (1:1000, Cell signaling, 45175), rabbit anti-phospho mTOR (1:1000, Cell signaling, 55365), rabbit anti-RAB5 (1:1000, Cell signaling, 35475), rabbit anti-Rab7 (1:1000, Cell signaling, 9367T), rabbit anti-TUJ1 (1:1000, Cell signaling, 5568), rabbit anti-Tom20 (1:1000, Cell signaling, 72610), mouse anti-Flag (1:1000, Sigma, F1804), goat anti-GAPDH (1:2000, RD Systems, AF5718), rabbit anti-TBCK (1:1000, Novus, NBP1-83166), rabbit anti-VDAC (1:1000, Abcam, ab15895), mouse anti-LAMP1 (1:500, DSHB, H4A3), rabbit anti-Cyt B (1:1000, Proteintech, 55090-1-AP), rabbit anti-RAB7 (1:1000, Cell signaling, 9367T), rabbit anti-PPP1R21 (1:1000, Atlas Ab, HPA036791), rabbit anti-C12ORF4 (1:1000, Sigma, HPA037871), rabbit anti-JIP4 (1:1000, Cell signaling, 5519), rabbit anti-TRIM27 (1:1000, IBL, 18791), rabbit anti-CRYZL1 (1:500, Novus, NBP1-89367), OXPHOS cocktail (1:500, Abcam, ab110413), goat anti-rabbit IgG (1:3000, LI-COR, 926-32211), goat anti-mouse IgG (1:3000, LI-COR, 926-68070), donkey anti-goat IgG (1:3000, LI-COR, 926-68074)

Techniques: Western Blot, Marker, Control