Journal: bioRxiv

Article Title: Mitophagosomes induced during EV-D68 infection promote viral nonlytic release

doi: 10.1101/2024.12.05.627125

Figure Lengend Snippet: (A) H1HeLa cells were transfected with the scramble and Parkin siRNAs for 48 h. The cells were then infected with EV-D68 for 5 h and viral titers were measured by a plaque assay. (B) H1HeLa cells were transfected with siRNAs against the mitophagy receptor, BCL2L13 for 48 h. The cells were then infected as in A for a plaque assay-based viral titer measurement. (C) A549 cells were transfected with scramble, Parkin, and PINK1 siRNAs for 48 h. The cells were infected with EV-D68 for 5h and viral titers were determined by a plaque assay. (D) A549 cells were transfected with the indicated siRNAs for 48 h. The cells were then infected and tittered as in A. (E) A549 cells were transfected with the indicated mitophagy-associated siRNAs for 48 h, followed by viral infection and titer determination as in A. An MOI of 0.1 was used for all infections. Error bars indicate mean ± SEM of at least 2 independent repeats. Unpaired student’s t-test was used for the statistical analyses (**= p< 0.01; *= p< 0.05; ns=not significant.).

Article Snippet: Rabbit polyclonal-anti-Pink1 (Novus Biologicals, BC100-494).

Techniques: Transfection, Infection, Plaque Assay

Journal: Clinical Science (London, England : 1979)

Article Title: Impaired cardiac non-neuronal acetylcholine synthesis triggers mitochondrial dysfunction with the loss of nicotinic receptor-mediated calcium handling, causing the failing heart

doi: 10.1042/CS20257026

Figure Lengend Snippet: A . LC3-II or LC3-I/II ratio was altered in hChAT KO mouse hearts, associated with the up-regulation of p62 and nitrotyrosine protein expression. B , Protein levels of both Parkin (PRK) and PINK1 were altered reciprocally between cytosolic and mitochondrial fractions of hChAT KO mouse hearts, compared with control mouse hearts. The hChAT KO reduced PRK and PINK1 protein levels in the cytosolic fraction ( P <0.05 and P <0.05, respectively), however alternatively, their levels both increased in the mitochondrial fractions ( P <0.01 and P <0.01, respectively).

Article Snippet: Furthermore, proteins from mitochondrial or cytosol fraction were subjected to western blot analysis using a rabbit anti-PARK2/Parkin polyclonal antibody (1:1000, #14060–1-AP; Proteintech), rabbit anti-PINK1 polyclonal antibody (1:1000, #23274–1-AP; Proteintech), rabbit anti-GAPDH monoclonal ab (Cell Signaling Technology), and rabbit anti-VDAC polyclonal ab (Abcam).

Techniques: Expressing, Control

Journal: The FASEB Journal

Article Title: Tmx2 Maintains Mitochondrial Function to Support Preimplantation Embryogenesis

doi: 10.1096/fj.202500640r

Figure Lengend Snippet: FIGURE 6 | Tmx2 knockdown inhibits mitophagy and autophagy in morula-stage embryos. (A-D) Immunostaining for PINK1 (A), PARKIN (B), MAP1LC3B (C), and LAMP1 (D) in control and Tmx2-knockdown embryos. Scale bars: 75 μm. (E-H) Quantification of immunofluorescence inten- sity for PINK1 (E), PARKIN (F), MAP1LC3B (G), and LAMP1 (H) in control and Tmx2-knockdown morula-stage embryos. Sample sizes: PINK1: Control = 16, siRNA2 = 14, siRNA3 = 19; PARKIN: Control = 21, siRNA2 = 20, siRNA3 = 23; MAP1LC3B: Control = 22, siRNA2 = 20, siRNA3 = 21; LAMP1: Control = 30, siRNA2 = 32, siRNA3 = 31. Statistical significance: **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Article Snippet: The primary antibodies used included: rabbit anti- TMX2 (Origene, TA341391, 1:50), goat anti- OCT4 (Abcam, ab27985, 1:200), mouse antiCDX2 (Biogenex, MU392A- UC, 1:100), goat anti- SOX17 (R&D Systems, AF1924, 1:100), rabbit anti- NANOG (Abcam, ab80892, 1:50), rabbit anti- PINK1 (Affinity, DF7742, 1:50), rabbit antiPARKIN (Abcepta, AP6402B, 1:50), rabbit anti- MAP1LC3B (CUSABIO, CSB- PA013403GA01HU, 1:50), and rabbit antiLAMP1 (Beyotime, AF7353, 1:100).

Techniques: Knockdown, Immunostaining, Control, Immunofluorescence

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Primary antibodies.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Affinity Purification

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Decreased MIC60 immunosignals in the OPL of MOG/CFA-injected mice in comparison to CFA-injected mice at day 9 post-injection. The 0.5 µm- thin retina sections from MOG/CFA-injected EAE mice ( B1 – B3 , D1 – D3 , F1 – F3 ) and CFA-injected control mice ( A1 – A3 , C1 – C3 , E1 – E3 ) double-immunolabelled with rabbit polyclonal MIC60 antibody (green channel) and mouse monoclonal RIBEYE antibody (red channel). Signals from the green ( A1 , B1 , C1 , D1 , E1 , F1 ) channels and red channels ( A2 , B2 , C2 , D2 , E2 , F2 ) were merged in ( A3 , B3 , C3 , D3 , E3 , F3 ). ( A1 – B3 ) Representative low-magnification images. Low-magnification micrographs show MIC60-labelled mitochondria in the OPL and in the photoreceptor inner segments in the same section. ( C1 – D3 ) MIC60-positive mitochondria in the OPL close to the synaptic ribbon at intermediate magnification. ( E1 – F3 ) High-magnification confocal images of single immunolabelled synaptic mitochondria in the OPL. Ring-like MIC60 immunosignals of individual presynaptic mitochondria are visible close to the synaptic ribbons (arrows). Ribbons appear as single horseshoe-labelled structures (arrowheads). ( G1 , G2 , H1 , H2 ) MIC60 immunosignals from the OPL and IS were quantified as integrated density. ( G1 , H1 ) Bar graphs depict normalized arithmetic means ± S.E.Ms. MOG/CFA integrated density immunofluorescence values were normalized to the mean of the CFA control values (set to 100%). In ( G2 , H2 ), the same data as in ( G1 , H1 ) are depicted as box-and-whisker plots to show the individual datapoints and their distribution. ( G2 , H2 ) Boxes represent 25th–75th percentiles of the data points, mean and median values are indicated by thin and thick horizontal lines, respectively, and whiskers are equal to 1.5 times the interquartile range (IQR). Mann–Whitney U-test was conducted to determine if there were significant differences in MIC60 immunosignals in the OPL between CFA and MOG/CFA groups. The distributions significantly differed between both groups (effect size d = 1.0751; power = 0.9999). MIC60 immunosignals in the inner segments did not differ between both groups (effect size d = 0.1497; power: 0.1517). p -values < 0.05 were considered statistically significant. Abbreviations: CFA, complete Freund’s adjuvant; MOG, myelin oligodendrocyte glycoprotein; EAE, experimental autoimmune encephalomyelitis; IF, immunofluorescence; OS, outer segment; IS, inner segment; OPL, outer plexiform layer; ONL, outer nuclear layer; S.E.M., standard error of the mean; N = number of independent experiments; n = number of images analyzed from retinal sections; n.s., non-significant; ****, p < 0.0001. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Injection, Comparison, Control, Immunofluorescence, Whisker Assay, MANN-WHITNEY, Adjuvant

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Decreased expression of MIC60 in individual mitochondria of rod photoreceptor synapses in MOG/CFA-injected EAE mice at day 9 after immunization. The 0.5 µm thin retina sections immunolabelled with rabbit polyclonal MIC60 antibody ( A1 , B1 ) and with mouse monoclonal RIBEYE antibody ( A2 , B2 ). Overlay images are shown in ( A3 , B3 ). Highly magnified confocal images of the immunolabelled OPL (A1-B3) were used for the quantification of RIBEYE puncta ( C1 , C2 ) and MIC60 puncta ( C3 , C4 ). ( D1 , D2 ) Quantification of MIC60 fluorescence intensities of each single MIC60-positive synaptic mitochondrion close to rod synaptic ribbons, measured as integrated density. ( C1 , C3 , D1 ) Bar graphs depict normalized arithmetic means ± S.E.M. In ( C2 , C4 , D2 ), the individual data from ( C1 , C3 , D1 ) are depicted as box-and-whisker plots. Boxes represent 25th–75th percentiles; the mean and median values are indicated by thick and thin horizontal lines, respectively. Whiskers are equal to 1.5 times the interquartile range (IQR). Unpaired Student’s t -test was conducted to determine if there were significant differences in RIBEYE puncta in the OPL between CFA and MOG/CFA groups. The distributions did not differ significantly between both groups (effect size d = 0.2738, power = 0.4827). To determine differences in MIC60 puncta between both groups the Mann–Whitney U-test was conducted and showed significant difference between both groups (effect size d = 1.8338; power =1.0). Differences in MIC60 expression in individual mitochondria were evaluated with Mann–Whitney U-test. Analysis revealed significant differences between the CFA and MOG/CFA groups (effect size d = 0.7443; power = 1.0). Data were considered statistically different with p < 0.05. Abbreviations: EAE, experimental autoimmune encephalomyelitis; CFA, complete Freund’s adjuvant; MOG, myelin oligodendrocyte glycoprotein; OPL, outer plexiform layer; S.E.M., standard error of the mean; N = number of independent experiments; n = number of images analyzed from retinal sections; ****, p < 0.0001; n.s., non-significant. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Expressing, Injection, Fluorescence, Whisker Assay, MANN-WHITNEY, Adjuvant

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Decreased expression of the mitochondrial ATP5B synthase subunit of the F 1 F 0 ATP synthase complex in synaptic mitochondria of photoreceptor synapses in MOG/CFA-injected EAE retinas at day 9 post-immunization. The 0.5 µm thin retina sections from CFA-injected mice and MOG/CFA-injected mice were incubated with mouse monoclonal ATP5B antibody ( A1 , B1 , C1 , D1 ) and with rabbit polyclonal monoclonal antibodies against RIBEYE (U2656) ( A2 , B2 , C2 , D2 ), as indicated. Overlay images are shown in ( A3 , B3 , C3 , D3 ). ( A1 – A3 , B1 – B3 ) show representative low-magnification micrographs of the photoreceptor compartments from MOG/CFA- and CFA-injected mice. The immunolabelled OPL is magnified in ( C1 – C3 , D1 – D3 ). ( E1 , E2 ) ATP5B immunosignals from the OPL were quantified as integrated density from 5 different littermate mice at day 9 post-injection. ( E1 ) shows column plots in which the arithmetic mean values ± S.E.M.s are depicted. MOG/CFA integrated density immunofluorescence values were normalized to the mean of the CFA control values. In ( E2 ), the same data as shown in ( E1 ) are depicted as box-and-whisker plot to show the individual datapoints. In ( E2 ), boxes represent 25th–75th percentile of the data points, with the mean and median values indicated by thick and thin horizontal lines, respectively, and whiskers are equal to 1.5 times the interquartile range (IQR). Mitochondrial ATP synthase 5B subunit expression is significantly reduced in the experimental group in comparison to the CFA control group as revealed by the Mann–Whitney U-test (effect size d = 1.3938; power = 1.0). p -values < 0.05 were considered statistically significant. Abbreviations: ATP5B, mitochondrial ATP synthase subunit 5B of the mitochondrial F 1 F 0 ATP synthase complex; CFA, complete Freund’s Adjuvant; EAE, experimental autoimmune encephalomyelitis IS, inner segment; MOG, myelin oligodendrocyte glycoprotein; ONL, outer nuclear layer; OPL, outer plexiform layer; S.E.M., standard error of the mean; N = number of independent experiments; n = number of images analyzed from retinal sections; ****, p < 0.0001. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Expressing, Injection, Incubation, Bioprocessing, Immunofluorescence, Control, Whisker Assay, Comparison, MANN-WHITNEY, Adjuvant

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Decreased expression of the mitochondrial COX1 protein in synaptic mitochondria of photoreceptor synapses in MOG/CFA-injected EAE retinas at day 9 post-immunization. The 0.5 µm thin retina sections from CFA-injected mice and MOG/CFA-injected mice were incubated with rabbit polyclonal antibody against COX1 ( A1 , B1 , C1 , D1 ) and with mouse monoclonal antibody against RIBEYE (2D9) ( A2 , B2 , C2 , D2 ), as indicated. Overlay images are shown in ( A3 , B3 , C3 , D3 ). ( A1 – A3 , B1 – B3 ) show low-magnification micrographs of the photoreceptor compartments. The immunolabelled OPL is magnified in ( C1 – C3 , D1 – D3 ). ( E1 , E2 ) COX1 immunosignals from the OPL were quantified as integrated density from 5 different littermate mice at day 9 post-injection. ( E1 ) shows column plots of the arithmetic mean values ± S.E.M.s. MOG/CFA integrated density immunofluorescence values were normalized to the mean of the CFA control values that was set to 100%. ( E2 ) Same data as in ( E1 ) depicted as box-and-whisker plot to show the individual datapoints and their distribution. ( E2 ) Boxes represent 25th–75th percentile of the data points, and mean and median values are indicated by thick and thin horizontal lines, respectively. Whiskers are equal to 1.5 times the interquartile range (IQR). Comparison of COX1 expression between control and experimental groups using a Mann–Whitney U-test revealed significant reduction in COX1 expression in the OPL of MOG/CFA-injected animals (effect size d = 1.2242; power = 1.0). p -values < 0.05 were considered statistically significant. Abbreviations: CFA, complete Freund’s Adjuvant; COX1, cytochrome c oxidase subunit 1; EAE, experimental autoimmune encephalomyelitis IS, inner segment; MOG, myelin oligodendrocyte glycoprotein; ONL, outer nuclear layer; OPL, outer plexiform layer; S.E.M., standard error of the mean; N = number of independent experiments; n = number of images analyzed from retinal sections; ****, p < 0.0001. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Expressing, Injection, Incubation, Immunofluorescence, Control, Whisker Assay, Comparison, MANN-WHITNEY, Adjuvant

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: Strong decrease in Pink1 immunosignals in the OPL of MOG/CFA-injected mice in comparison to CFA-injected control mice on day 9 post-injection. The 0.5 µm-thin retina sections from both groups (i.e., CFA ( A1 – A3 ) and MOG/CFA ( B1 – B3 )) were double-immunolabeled with rabbit polyclonal antibody against Pink1 ( A1 , B1 ) and mouse monoclonal antibody against RIBEYE (2D9) ( A2 , B2 ). Signals from the green ( A1 , B1 ) and red ( A2 , B2 ) channels were merged in ( A3 , B3 ). The double-immunolabelling data demonstrate that Pink1 puncta are located close to the RIBEYE puncta ( A3 , B3 ). ( C1 , C2 ) Quantification of the Pink1 immunosignals in the OPL. Column plots in ( C1 ) show normalized arithmetic mean fluorescence values (integrated densities) ± S.E.M. Pink1 immunosignals in the OPL of MOG/CFA-injected mice were normalized to the arithmetic mean of the values from CFA-injected control mice (that was set to 100%). The Mann–Whitney U-test was used to determine whether Pink1 is significantly altered in MOG/CFA-injected animals in comparison to CFA-injected control animals because data were non-normally distributed (effect size d = 1.1445; power = 1.0). p -values < 0.05 were considered statistically significant. The same data as shown in ( C1 ) are also depicted as box-and-whisker plots in ( C2 ) to show the individual datapoints. Boxes represent the 25th–75th percentile of the data points. The mean and median values in the box-and-whisker plot are indicated by thick and thin horizontal bars, respectively. Whiskers are equal to 1.5 times the interquartile range (IQR). Abbreviations: CFA, complete Freund’s adjuvant; Pink1, PTEN-induced kinase 1; MOG, myelin oligodendrocyte glycoprotein; OPL, outer plexiform layer; S.E.M., standard error of the mean; N = number of independent experiments; n = number of images analyzed from retinal sections; ****, p < 0.0001. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Injection, Comparison, Control, Immunolabeling, Fluorescence, MANN-WHITNEY, Whisker Assay, Adjuvant

Journal: Cells

Article Title: Early Synapse-Specific Alterations of Photoreceptor Mitochondria in the EAE Mouse Model of Multiple Sclerosis

doi: 10.3390/cells14030206

Figure Lengend Snippet: DRP-1 immunosignals in synaptic mitochondria of photoreceptor synapses are strongly increased in MOG/CFA-injected EAE mice compared to CFA-injected control mice on day 9 after immunization. The 0.5 µm-thin retina sections from CFA mice ( A1 – A3 ) and MOG/CFA mice ( B1 – B3 ) were double-immunolabeled with rabbit polyclonal antibody against DRP1 ( A1 , B1 ) and mouse monoclonal antibody against RIBEYE (2D9) ( A2 , B2 ). Signals from the green ( A1 , B1 ) and red ( A2 , B2 ) channels were merged in ( A3 , B3 ). Quantification of DRP1 immunosignals in the OPL is depicted in ( C1 , C2 ). Column plots in ( C1 ) show normalized arithmetic mean fluorescence values (integrated densities) ± S.E.M. DRP1 immunosignals in the OPL of MOG/CFA-injected mice were normalized to the arithmetic mean of the values from CFA-injected control mice (that was set to 100%). Statistical analysis using the Mann–Whitney U-test of the non-normally distributed data revealed a significant increase in DRP1 immunosignals in the OPL of MOG/CFA-injected animals in comparison to in the CFA control group (effect size d = 1.2141; power = 0.9999). p -values < 0.05 were considered statistically significant. The same data as shown in ( C1 ) are depicted in ( C2 ) as box-and-whisker plots to show the individual datapoints. Boxes represent the 25th–75th percentile of the data points. The mean and median values in the box-and-whisker plot are indicated by thick and thin horizontal bars, respectively. Whiskers are equal to 1.5 times the interquartile range (IQR). Abbreviations: CFA, complete Freund’s adjuvant; DRP1, dynamin-related protein 1; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; OPL, outer plexiform layer; S.E.M., standard error of the mean; ****, p < 0.0001; N = number of independent experiments; n = number of images analyzed from retinal sections. Scale bars: 5 μm.

Article Snippet: PTEN-induced kinase-1 (PINK1), affinity-purified rabbit polyclonal antibody, 23274-1-AP , ProteinTech, 23274-1-AP , [ , , ] , 1:100 (IF).

Techniques: Injection, Control, Immunolabeling, Fluorescence, MANN-WHITNEY, Comparison, Whisker Assay, Adjuvant