Journal: The Journal of Biological Chemistry

Article Title: NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway *

doi: 10.1074/jbc.M116.752261

Figure Lengend Snippet: Nox4 silencing increases mitochondrial mass via TFAM. A, bar graph representing the effect of silencing Nox4, TFAM, or Nox4 plus TFAM on the mitochondrial-to-nuclear DNA ratio in IMR-90 cells. NT siRNA was transfected in with Nox4 siRNA alone or TFAM siRNA alone to keep equal the amount of siRNA between each experimental group (final concentration of siRNA 100 nm). B–D, bar graphs representing the silencing efficiency of Nox4 or TFAM siRNA in IMR-90 cells. Values represent the mean ± S.E.; error bars represent S.E.; *, p < 0.005 compared with control; A–D, n = 9 (3 biological replicates total, 3 technical replicates per group per experiment). E, total cell lysates were isolated from IMR-90 cells transfected with NT, Nox4, TFAM, or Nox4 plus TFAM siRNA (cells were harvested 5 days post transfection). NT siRNA was transfected in with Nox4 siRNA alone or TFAM siRNA alone to keep the amount of siRNA between each experimental group equal. Then the levels of the mitochondrial proteins TFAM, citrate synthase, VDAC, COX IV, and α-tubulin were determined by Western blotting; molecular weight markers are indicated on the left side of the corresponding panel. F, densitometry analysis of the signal associated with the detection of TFAM, citrate synthase, VDAC, and COX IV in total cell lysates of IMR-90 cells deficient in Nox4, TFAM, or Nox4 and TFAM. Values represent the mean ± S.E.; error bars represent S.E.; * p < 0.05 compared with control (NT siRNA transfected cells); #, p < 0.05 compares Nox4 siRNA to Nox4 + TFAM siRNA; n = 4 (4 biological replicates total, 1 technical replicate per group per experiment).

Article Snippet: We purchased antibodies (Ab) to β-actin (clone AC-15, mouse monoclonal Ab against N-terminal peptide, catalogue #A1978, lot #043M4840V) and α-tubulin (clone B-5–1-2, mouse mAb against C-terminal peptide, catalogue #T5168, lot #103M4773V) from Sigma; TFAM (clone D5C8 rabbit mAb against peptide containing Val-225, catalogue #8076S, lot #1), total PGC-1α (rabbit mAb against PGC-1α fusion protein, catalogue #2178S, lot #2), c-Myc (clone D3N8F, rabbit mAb against central region of human c-Myc, catalogue #12987S, lot #1), NRF-1 (rabbit Ab against peptide near Leu167, catalogue #12381, lot #1), VDAC (clone D73D12, rabbit mAb against N-terminal peptide, catalogue #4661S, lot #5), citrate synthase (clone D7V8B, rabbit mAb against residues near the C terminus of the protein, catalogue #14309S, lot #1) and COX IV (clone 3E11, rabbit mAb against synthetic peptide corresponding to residues surrounding Lys29 of human COX IV, catalogue #4850S, lot #6) from Cell Signaling Technology (Boston, MA); lamin A/C (clone 636, mouse mAb against lamin preparation of porcine origin, catalogue #sc-7292, lot #F3011) was purchased from Santa Cruz Biotechnology (Dallas, TX); Nrf2 (rabbit Ab against fusion protein from amino acids 256–605, catalogue #16396-1-AP, lot #00043173) was from Proteintech Group Inc. (Rosemont, IL); phospho-PGC-1α rabbit Ab against Ser-571, catalogue #AF6650, lot #CEBA0114011) was from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Concentration Assay, Isolation, Western Blot, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway *

doi: 10.1074/jbc.M116.752261

Figure Lengend Snippet: Nox4 silencing increases TFAM transcript level and nuclear Nrf2 expression. A, bar graph representing the effect of silencing Nox4 on TFAM mRNA levels in IMR-90 cells. Values represent the mean ± S.E.; error bars represent S.E.; *, p < 0.01 compared with control, n = 15 (5 biological replicates total, 3 technical replicates per group per experiment). B, nuclear or cytosolic fractions were isolated from IMR-90 cells transfected with NT or Nox4 siRNA (cells were harvested for experimentation 3 days post transfection). Then the protein levels of NRF-1, Nrf2, c-Myc, lamin A/C, and α-tubulin were determined by Western blot; molecular weight markers are indicated on the left side of the corresponding panel. C–E, densitometry analysis of NRF-1, Nrf2, and c-Myc expression in the nuclear fractions of control or Nox4-silenced lung fibroblasts. Values represent the mean ± S.E.; error bars represent S.E.; * p < 0.005 compared with control; n.s., non-significant difference between means. B–E, n = 4 (4 biological replicates total, 1 technical replicate per group per experiment).

Article Snippet: We purchased antibodies (Ab) to β-actin (clone AC-15, mouse monoclonal Ab against N-terminal peptide, catalogue #A1978, lot #043M4840V) and α-tubulin (clone B-5–1-2, mouse mAb against C-terminal peptide, catalogue #T5168, lot #103M4773V) from Sigma; TFAM (clone D5C8 rabbit mAb against peptide containing Val-225, catalogue #8076S, lot #1), total PGC-1α (rabbit mAb against PGC-1α fusion protein, catalogue #2178S, lot #2), c-Myc (clone D3N8F, rabbit mAb against central region of human c-Myc, catalogue #12987S, lot #1), NRF-1 (rabbit Ab against peptide near Leu167, catalogue #12381, lot #1), VDAC (clone D73D12, rabbit mAb against N-terminal peptide, catalogue #4661S, lot #5), citrate synthase (clone D7V8B, rabbit mAb against residues near the C terminus of the protein, catalogue #14309S, lot #1) and COX IV (clone 3E11, rabbit mAb against synthetic peptide corresponding to residues surrounding Lys29 of human COX IV, catalogue #4850S, lot #6) from Cell Signaling Technology (Boston, MA); lamin A/C (clone 636, mouse mAb against lamin preparation of porcine origin, catalogue #sc-7292, lot #F3011) was purchased from Santa Cruz Biotechnology (Dallas, TX); Nrf2 (rabbit Ab against fusion protein from amino acids 256–605, catalogue #16396-1-AP, lot #00043173) was from Proteintech Group Inc. (Rosemont, IL); phospho-PGC-1α rabbit Ab against Ser-571, catalogue #AF6650, lot #CEBA0114011) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Isolation, Transfection, Western Blot, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway *

doi: 10.1074/jbc.M116.752261

Figure Lengend Snippet: Nrf2 silencing reduces mitochondrial TFAM expression. A, total cell lysates or mitochondrial fractions were isolated from NT, Nox4, Nrf2, or Nox4 plus Nrf2 siRNA-transfected IMR-90 cells (cells were harvested for experimentation 5 days post transfection). NT siRNA was transfected in with Nox4 siRNA alone or Nrf2 siRNA alone to keep the amount of siRNA between each experimental group equal (final concentration of siRNA, 100 nm). Mitochondria were isolated from the same number of cells (2.4 × 106) for each experimental group. Then the entire mitochondrial fraction was submitted to SDS-PAGE analysis for each experimental group, and the expression levels of the proteins TFAM, citrate synthase, CoxIV, and α-tubulin were analyzed by Western blot; for each signal detected, molecular weight markers are indicated on the left side of the corresponding panel. B, densitometry analysis of TFAM, citrate synthase, and CoxIV expression in total cell lysates isolated from lung fibroblasts transfected with NT, Nox4, Nrf2, or Nox4 plus Nrf2 siRNA. C, densitometry analysis of TFAM, citrate synthase, and COX IV expression in mitochondrial fractions isolated from lung fibroblasts transfected with NT, Nox4, Nrf2, or Nox4 plus Nrf2 siRNA. B and C, values represent the mean ± S.E.; error bars represent S.E.; *, p < 0.05 compared with control (NT siRNA-transfected cells). #, p < 0.05 compares Nox4 siRNA to Nox4 + Nrf2 siRNA; n = 4 (4 biological replicates total, 1 technical replicate per group per experiment). D, bar graph representing the effect of silencing Nox4, Nrf2, or Nox4 plus Nrf2 on Nox4 mRNA expression in IMR-90 cells. E, bar graph representing the effect of silencing Nox4, Nrf2, or Nox4 plus Nrf2 on Nrf2 mRNA expression in IMR-90 cells. Values represent the mean ± S.E.; error bars represent S.E.; *, p < 0.005 compared with control, n = 9 (3 biological replicates total, 3 technical replicates per group per experiment). F, nuclear and cytosolic fractions were isolated from NT, Nox4, Nrf2, or Nox4 plus Nrf2 siRNA-transfected IMR-90 cells (cells were harvested for experimentation 3 days post transfection). Then the protein levels of NRF-1, Nrf2, c-Myc, lamin A/C, and α-tubulin were determined by Western blot.

Article Snippet: We purchased antibodies (Ab) to β-actin (clone AC-15, mouse monoclonal Ab against N-terminal peptide, catalogue #A1978, lot #043M4840V) and α-tubulin (clone B-5–1-2, mouse mAb against C-terminal peptide, catalogue #T5168, lot #103M4773V) from Sigma; TFAM (clone D5C8 rabbit mAb against peptide containing Val-225, catalogue #8076S, lot #1), total PGC-1α (rabbit mAb against PGC-1α fusion protein, catalogue #2178S, lot #2), c-Myc (clone D3N8F, rabbit mAb against central region of human c-Myc, catalogue #12987S, lot #1), NRF-1 (rabbit Ab against peptide near Leu167, catalogue #12381, lot #1), VDAC (clone D73D12, rabbit mAb against N-terminal peptide, catalogue #4661S, lot #5), citrate synthase (clone D7V8B, rabbit mAb against residues near the C terminus of the protein, catalogue #14309S, lot #1) and COX IV (clone 3E11, rabbit mAb against synthetic peptide corresponding to residues surrounding Lys29 of human COX IV, catalogue #4850S, lot #6) from Cell Signaling Technology (Boston, MA); lamin A/C (clone 636, mouse mAb against lamin preparation of porcine origin, catalogue #sc-7292, lot #F3011) was purchased from Santa Cruz Biotechnology (Dallas, TX); Nrf2 (rabbit Ab against fusion protein from amino acids 256–605, catalogue #16396-1-AP, lot #00043173) was from Proteintech Group Inc. (Rosemont, IL); phospho-PGC-1α rabbit Ab against Ser-571, catalogue #AF6650, lot #CEBA0114011) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Isolation, Transfection, Concentration Assay, SDS Page, Western Blot, Molecular Weight

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.V5 epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.V5 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: L1 protein associated with condensed chromosomes retains the reactivity of the H16.56E epitope. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without Click-iT reaction buffer without dye. Next, the cells were incubated with L1 conformationally dependent mouse MAb H16.56E antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: The linear epitope of the 33L1-7 antibody is hidden in the L1 protein associated with condensed chromosomes. (A and B) At 24 hpi, HaCaT cells infected with EdU-labeled pseudovirus were fixed, permeabilized, and treated with or without with Click-iT reaction buffer without dye. Next, the cells were incubated with mouse MAb 33L1-7 antibody (green) and AF488-conjugated anti-α-tubulin (white). Lastly, the cells were treated with Click-iT reaction buffer with AF555 dye (red) to stain the EdU-labeled pseudogenome and mounted in DAPI (blue). Note that colocalization of the EdU puncta and L1 protein is denoted as a yellow color.

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Labeling, Incubation, Staining

Journal: Journal of Virology

Article Title: Human Papillomavirus Major Capsid Protein L1 Remains Associated with the Incoming Viral Genome throughout the Entry Process

doi: 10.1128/JVI.00537-17

Figure Lengend Snippet: Some full-length L1 protein accompanies the viral genome to the nucleus. (A) HeLa cells were infected with HPV16 pseudovirus with or without 1.5 μM Eg5 inhibitor III (Eg5i) and tracked via live-cell imaging using the IncuCyte Zoom for 48 h. Note that the images are depicted at 18 hpi. (B) HaCaT cells were infected with EdU-labeled HPV16 pseudovirus in the presence of 1.5 μM Eg5i. The cells were fixed at 24 hpi and permeabilized with either digitonin at 0.625 μg/ml or 0.5% TX-100 and then treated with AF555 (green) in Click-iT reaction buffer. The cells were permeabilized again with 0.5% TX-100 and treated with AF647 (red) in Click-iT reaction buffer. Lastly, the cells were mounted in DAPI (blue). (C) HaCaT cells were infected with EdU-labeled pseudovirus and treated with 1.5 μM Eg5i. At 24 hpi, the cells were fixed and permeabilized in 0.5% TX-100. Next, the cells were treated with AF555 (red) in Click-iT reaction buffer, followed by incubation with AF488-conjugated anti-α-tubulin (white) and MAb 33L1-7 (green). Lastly, the cells were mounted in DAPI (blue). Note the colocalization between EdU and L1 signal denoted by white arrows. (D) HeLa cells were infected with HPV16 pseudovirus in the presence of 1.5 μM Eg5i. Cells were trypsinized for 2 min, and monoastral cells were collected. Next, the cells were treated with 15 μl of 0.25% trypsin for 1 h at 37°C. The cells were lysed by passage through a 1-ml syringe with a 25-gauge needle 40 times. Cell lysates were incubated for 1 h at 37°C once more, the trypsin was inactivated, and the samples were analyzed by Western blot analysis with a cocktail of HPV16 L1-specific mouse MAbs (IID5, 33L1-7, and 312F).

Article Snippet: The microtubule network was detected using mouse MAb anti-α-tubulin conjugated to Alexa Fluor 488 (AF488; catalog no. 8058S; Cell Signaling).

Techniques: Infection, Live Cell Imaging, Labeling, Incubation, Western Blot