Journal: Cell Death and Differentiation

Article Title: ATG12 deficiency leads to tumor cell oncosis owing to diminished mitochondrial biogenesis and reduced cellular bioenergetics

doi: 10.1038/s41418-019-0476-5

Figure Lengend Snippet: a ATG12 was co-stained with MTO in MDA-MB-231 and H1299 cells. Scale bars: 20 µm. Images (n ≥ 3) were analyzed with Imaris software and the Pearson’s correlation coefficients plotted. Values are means ± SEMs. Related data are also presented in Fig. S7. b Mitochondrial fractions (M) from MDA-MB-231 and H1299 cells (16 × 106 cells) were separated from the cytosolic fractions (C) and were applied to western blotting. Conjugated ATG12 in cytosol and mitochondria is indicated with white and black stars, respectively. c Subcellular fractionation was performed, followed by western blotting with vector control and shATG12 H1299 cells (20 × 106 cells). C cytosol, M mitochondria. The mitochondrial ATG12 level is reduced in shATG12 cells (black stars) and the level of ATG5-ATG12 conjugate diminished, together with the appearance of free ATG5 in the cytosolic fraction (white rectangle). d ATG12 was co-stained with SSBP1 in freshly isolated human dermal fibroblasts and MDA-MB-231 cells. Scale bar: 20 µm. e ATG12 was co-stained with SSBP1 in tissue sections from paired colon and non-small cell lung cancer patients. Scale bars: 20 µm. f ATG12 was stained in a multicancer TMA. ATG12-positive cells from the normal and the corresponding tumor tissues were counted. Values are means ± SEMs. g H1299 cells were infected with either vector control virus or virus containing shATG12 plasmid. Three days post infection, cells were injected subcutaneously into NSG mice (four mice per group/cell type) and the tumor formation was assessed three times per week. The tumor sizes (mm3) of the vector control and the shATG12 cells were plotted. Values are means ± SDs from four mice per group. Two-way ANOVA with Bonferroni posttests was used to evaluate the statistical significance between the vector control cells and the cells deficient in ATG12 (pgenotype). The p values at different time points are also presented in the graph.

Article Snippet: After antigen retrieval in sodium citrate buffer (10 mM, pH 6.0), immunofluorescence staining was performed by incubating the paraffin sections with anti-ATG12 antibody (100 ng/ml; R&D Systems) and anti-SSBP1 antibody (1:100; Novus Biologicals) or appropriate control antibodies in blocking buffer.

Techniques: Staining, Software, Western Blot, Fractionation, Plasmid Preparation, Control, Isolation, Infection, Virus, Injection

Journal: Acta Neuropathologica Communications

Article Title: Hippocampal mitophagy alterations in MAPT -associated frontotemporal dementia with parkinsonism

doi: 10.1186/s40478-025-01955-8

Figure Lengend Snippet: Increased pS65-Ub localization in lysosomes of the subiculum in FTDP-17 MAPT P301L mutant cases. ( A ) Comparison of pS65-Ub cell density between the subiculum and dentate gyrus in low count- and high count-level groups in MAPT P301L cases. ( B ) Comparison of pS65-Ub cell percentages between subiculum and dentate gyrus. ( C ) Comparison of the granular and vacuolar pS65-Ub cell densities between the subiculum and dentate gyrus in low- and high-count pS65-Ub cells within MAPT P301L cases. ( D ) Comparison of the percentage of granular and vacuolar pS65-Ub cells between the subiculum and dentate gyrus. Higher percentage of vacuolar pS65-Ub cells were found in the subiculum compared to the dentate gyrus. ( E-F ) Representative images of pS65-Ub positive cells co-localized with SSBP1 (E) and CTSD (F) in the subiculum and dentate gyrus. Scale bars: 6.5 μm. Dashed lines represent outline of cells. ( G ) Number, size, and signal intensity of pS65-Ub inclusions per cell in the subiculum compared to the dentate gyrus. ( H-I ) Percentage of pS65-Ub positive puncta that co-localized with SSBP1 (H) or CTSD (I) in the subiculum and dentate gyrus. Data are shown as mean ± SEM. Statistical analysis was performed using unpaired t-test. P301L: n = 3. Approximately 30 pS65-Ub positive cells from the subiculum and 60 cells from the dentate gyrus were randomly selected for the analysis. DG = dentate gyrus

Article Snippet: Following 1 h blocking with the serum-free Protein Block (Dako, X0909), sections were incubated overnight at 4˚C with primary antibodies against pS65-Ub (in-house [ ], 1:300), the mitochondrial marker SSBP1 (Santa Cruz Biotechnology, sc-34727, 1:50), the lysosomal marker CTSD (MilliporeSigma, MA1013, 1:500), the autophagy marker p62/SQSTM1 (BD Biosciences, 610832, 1:250) or pS202-tau (CP13, 1:250) diluted in Dako Antibody Diluent (Dako, S3022).

Techniques: Mutagenesis, Comparison

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Transcripts that showed high homology to known genes.

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Sequencing, Ubiquitin Proteomics, Immunopeptidomics

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Human cytomegalovirus (HCMV) down-regulated the mRNA and protein expression of single-stranded DNA-binding protein (SSBP1). A , Expression levels of SSBP1 assayed by real-time PCR. B , Protein expression levels of SSBP1 assayed by western blot. Data are reported as means±SE, (n=4). *P<0.05 and **P<0.01 compared to time 0 (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein (SSBP1)-expression vector or siRNA were transfected into human umbilical vein endothelial cells. A , mRNA expression levels of SSBP1 assayed by real-time PCR. B , Protein expression levels of SSBP1 assayed by western blot. Data are reported as means±SE, n=4. **P<0.01 vs control (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein SSBP1 affected the expression levels of lipid metabolism-associated genes in human umbilical vein endothelial cells. The results of real-time PCR showed that over-expression of SSBP1 inhibited the expression of LDLR and SCARB, while knockdown of SSBP1 significantly promoted the mRNA expression of LDLR, HMGR, CETP and SCARB. LDLR: low-density lipoprotein receptor; SCARB: scavenger receptor B; HMGCR: HMG-CoA reductase; CETP: cholesteryl ester transfer protein. Data are reported as means±SE, n=4. *P<0.05 and **P<0.01 vs control (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Control

Journal: Brazilian Journal of Medical and Biological Research

Article Title: Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

doi: 10.1590/1414-431X20176389

Figure Lengend Snippet: Single-stranded DNA-binding protein (SSBP1) affected the total cholesterol content. The results showed that cholesterol in the cells decreased significantly in SSBP1-expression cells ( A ) and increased significantly in SSBP1 knock-down cells in a time-dependent manner ( B ). Data are reported as means±SE, n=6. *P<0.05 and **P<0.01 (ANOVA).

Article Snippet: The membranes were blocked in 5% BSA, and then incubated with Polyclonal sheep anti-human SSBP1 antibodies (R&D System, USA) followed by the appropriate horseradish peroxidase-conjugated secondary antibodies.

Techniques: Binding Assay, Expressing, Knockdown

Journal: The Journal of Neuroscience

Article Title: Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson's Disease-Relevant Stress

doi: 10.1523/JNEUROSCI.0541-18.2018

Figure Lengend Snippet: Incorporation of BrdU and EdU into neuronal mitochondria in cell bodies and axons within 1–3 h of exposure. A–C, Primary neurons (DIV14) expressing mitochondrially targeted photoactivatible GFP (Mitochondria) were treated with 10 μm BrdU for 1 h (A) or 3 h (B). Control cells were incubated with 100 μm ddC, an inhibitor of mitochondrial DNA polymerase gamma, for 6 h before and then during 3 h of BrdU exposure (C). Cells were immunofluorescently stained for BrdU and confocal imaging revealed BrdU puncta associated with mitochondria in cell bodies (A, B), but minimal, if any, incorporation when ddC was present (C), confirming specificity for mtDNA replication. D, BrdU puncta were also found in mitochondria of distal axons after 1 h of BrdU exposure. E, Primary neurons (DIV14) expressing mitochondrially targeted DsRed2 (Mitochondria) were treated with EdU for 3 h and then stained using the EdU Click-iT system. EdU-positive puncta were again observed in mitochondria of the cell body and axons (E, arrow).

Article Snippet: Cells were then rinsed in PBS and incubated with either MAP2 (rabbit anti-MAP2, 1:1000; Millipore catalog #ab5622) or mitochondrial single-stranded DNA binding protein (rabbit anti-mtSSBP, 1:2000; Origene catalog #TA314569) primaries again overnight at 4°C, followed by 1 h incubation with secondary (donkey anti-rabbit Cy3, 1:500, Jackson ImmunoResearch; donkey anti-rabbit 647, 1:500, Thermo Fisher Scientific).

Techniques: Expressing, Incubation, Staining, Imaging