Journal: FEBS Open Bio

Article Title: HSF1 is required for induction of mitochondrial chaperones during the mitochondrial unfolded protein response

doi: 10.1002/2211-5463.12863

Figure Lengend Snippet: Different requirements of SSBP1 for the activation of mitochondrial chaperone genes. (A) Knockdown of HSF1 or SSBP1. MEF cells were infected with an adenovirus expressing scrambled RNA (SCR) or shRNA for SSBP1 (SSBP1‐KD) or HSF1 (HSF1‐KD) for 2 h, maintained with normal medium for 70 h. Cell extracts were prepared and subjected to western blotting. (B–D) Activation of the mitochondrial chaperone genes in SSBP1‐ or HSF1‐knockdown cells. SSBP1 or HSF1 was knocked down as described in (A). The cells were treated with 10 μ m GTPP (B), 5 μ m CDDO (C), and 20 μ m rotenone (D) for 6 h. mRNA levels of HSP60, HSP10, mtHSP70, and HSP70 were quantified by RT‐qPCR ( n = 3). Mean ± SD is shown. Asterisks indicate P < 0.01 or 0.05 by Student's t ‐test (ns, not significant).

Article Snippet: After proteins were transferred onto nitrocellulose or PVDF (SSBP1 blot) membranes, the membranes were blocked in PBS/5% milk at a room temperature for 1 h and then were immunoblotted using rabbit antibodies against HSF1 (anti‐mHSF1j, Millipore ABE1044; dilution, 1 : 1000) [ ], TRAP1 (anti‐mTRAP1a; dilution, 1 : 1000) (this study) HSP60 (anti‐HSP60‐1; 1 : 2000) [ ], HSP10 (Santa Cruz, CA, USA sc‐20958; 1 : 1000), mtHSP70 (or GRP75) (Santa Cruz sc‐13967; 1 : 1000), and SSBP1 (anti‐mSSBP1x; dilution, 1 : 1000) (this study), and mouse antibody for HSP70 (Santa Cruz W27; 1 : 1000) and β‐actin (AC‐15; Sigma, St. Louis, MO, USA) diluted in PBS/2% milk at a room temperature for 1 h or at 4 °C overnight.

Techniques: Activation Assay, Knockdown, Infection, Expressing, shRNA, Western Blot, Quantitative RT-PCR

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

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, Isolation, Infection, Injection

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 2. Ang II affects the function of cardiac mitochondria. A-C: The activities of citrate synthase, complex I, and complex III in mouse heart tissues were determined. D: NADPH Oxidase 1 (Nox1) and NADPH Oxidase 4 (Nox4) mRNA expression in mouse heart tissues were ana- lyzed by qRT-PCR. E, F: The activities of antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured in mouse heart tissues. G: Single-stranded DNA-binding protein 1 (SSBP1) mRNA expression was analyzed by qRT-PCR. H, I: SSBP1 protein expression was analyzed by Western blot. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Western Blot

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 3. Ang II influences proliferation and collagen expression in mouse cardiac fibroblasts. A: Cell proliferation of mouse adult cardiac fibroblasts (MCFs) and mouse embryo fibroblast-NIH 3T3 cells (3T3) treated with multiple doses (0.0, 0.5 and 1.0 μmol/L) of Ang II and valsartan. B, C: Nox1, Nox4, COL1A1, COL3A1, and SSBP1 protein expression in MCFs and 3T3 cells with Ang II and valsartan exposure. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Expressing

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 4. The effect of SSBP1 knockdown on fibroblast cell proliferation and collagen expression in the presence or absence of Ang II. A, B: Cell viability of MCFs and 3T3 cells with SSBP1 knockdown. C, D: Analysis of SSBP1 protein expression in MCFs and 3T3 cells after the transfection of SSBP1 shRNA. E, F: Evaluation of COL1A1, COL3A1, Nox1, and Nox4 protein expression in MCFs and 3T3 cells after the knockdown of SSBP1 expression. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Knockdown, Expressing, Transfection, shRNA

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 5. The effect of SSBP1 overexpression on fibroblast cell proliferation and collagen expression in the pres- ence or absence of Ang II. A, B: Determination of MCFs and 3T3 cell viability with SSBP1 overexpression. C, D: Analysis of SSBP1 protein expression in MCFs and 3T3 cells after SSBP1 vector transfection. E, F: Evaluation of COL1A1, COL3A1, Nox1, and Nox4 protein expression in MCFs and 3T3 cells after SSBP1 overexpression. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Over Expression, Expressing, Plasmid Preparation, Transfection

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 6. SSBP1 regulates p53 protein expression in cardiac fibroblasts in the presence of Ang II. A, B: p53 mRNA expression in MCFs and 3T3 cells with SSBP1 knockdown. C, D: p53 protein expression in MCFs and 3T3 cells with SSBP1 knockdown. E, F: p53 protein expression in MCFs and 3T3 cells with SSBP1 overexpression. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Expressing, Knockdown, Over Expression

Journal: International heart journal

Article Title: Single-Stranded DNA-Binding Protein 1 Abrogates Cardiac Fibroblast Proliferation and Collagen Expression Induced by Angiotensin II.

doi: 10.1536/ihj.17-650

Figure Lengend Snippet: Figure 7. p53 expression affects fibroblast proliferation and collagen expression in the presence or absence of Ang II. A, B: Cell proliferation of MCFs and 3T3 cells with p53 overexpression by Ad-p53-GFP. C, D: Analysis of SSBP1 protein expression in MCFs and 3T3 cells after Ad-p53-GFP infection. E, F: Evaluation of COL1A1 and COL3A1 expression in MCFs and 3T3 cells with p53 overexpression. *P < 0.05, **P < 0.01.

Article Snippet: The primary antibodies included COL1A1 (1:1200, #NB600-408, Novus Biologicals, Littleton, CO, USA), COL3A1 (1:1200, #NB600594, Novus Biologicals), Nox1 (1:1000, #NBP1-31546, Novus Biologicals), Nox4 (1:1000, #NB110-58849, Novus Biologicals), SSBP1 (1:1000, #NBP1-80720, Novus Biologicals), p53 (1:1000, #NB200-103, Novus Biologicals), and GAPDH (1:1200, #NB300-221, Novus Biologicals).

Techniques: Expressing, Over Expression, Infection

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: SiRNA oligonucleotides for SSBP1 and negative control siRNA were purchased from Santa Cruz Biotechnology (USA).

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

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: The primary antibodies were: anti-Ki-67 (1:200, Dako), anti-ATG12 (100 ng/ml; R&D Systems, MAB6807), anti-LC3 (1:100; Nanotools), anti-SSBP1 (1:100; Novus Biologicals), anti-LAMP1 (1:100; Abcam), and anti-ERAP1 (1:100; GeneTex).

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