Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Cytokinetic phenotype in Kif5b knockdown ATDC5 cells. a Western blot of protein extracts from single cell clones with stable expression of sh-ctl or sh-kif5b constructs. b Immunofluorescence of KIF5B (green) in sh-Kif5b clone #4. Scale bar: 10 μm. c sh-ctl and sh-Kif5b cells stained with α-tubulin (red) and DAPI (blue). Yellow asterisks denote typical binucleated cells. Scale bar: 20 μm. d Quantification of the bi- and multi-nucleation rate in control cell clones ( n = 3, sh-ctl clone #1–3) and Kif5b-knockdown cell clones ( n = 5, sh-Kif5b clone #4–8). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Re-introduced GFP tagged full length KIF5B reduced the bi- and multi-nucleation rate in Kif5b knockdown ATDC5 cells clone #4 ( n = 10 independent experiments) and #5 ( n = 5 independent experiments). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. f Time-lapse images of sh-ctl and sh-Kif5b cells in mitosis. Scale bar: 10 μm. g Quantification of duration of cytokinesis in sh-ctl cells ( n = 84 cells from sh-ctl clone #1–3) and sh-Kif5b cells ( n = 68 cells from sh-Kif5b clone #4, #5 and #8). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 5% to 95% percentile (whiskers). Duration of cytokinesis was calculated from the furrow ingression to the final separation of the two daughter cells. Cells fusing back were not included in this analysis

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Knockdown, Western Blot, Clone Assay, Expressing, Construct, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Whisker Assay

Journal: Cell & Bioscience

Article Title: KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes

doi: 10.1186/s13578-019-0344-5

Figure Lengend Snippet: Midbody microtubule organization is impaired in Kif5b deficient cells. a Immunofluorescence of α-tubulin in primary chondrocytes in late cytokinesis. Scale bar: 10 μm. b Quantification of tubulin intensity in midbody in primary chondrocytes ( Kif5b fl/ + cells: n = 24; Col2cre ; Kif5b fl/ − cells: n = 27). *** P < 0.0001; two-tailed Mann–Whitney U -test. The whisker plot shows median (lines), interquartile range (boxes) and 10% to 90% percentile (whiskers). c Electron micrographs of midbody regions from sh-ctl and sh-Kif5b ATDC5 cells. Red lines denote the region of Flemming body. Yellow arrows and boxes denote the broken regions in Flemming body. Scale bar: 0.5 μm. d Quantification of the length of Flemming body in sh-ctl and sh-Kif5b cells (sh-ctl cells: n = 12; sh-Kif5b cells: n = 9). *** P < 0.0001; unpaired two-tailed t-test. Data are mean ± S.D. e Live imaging of sh-ctl and sh-Kif5b cells expressing GFP-tubulin in cytokinesis. Scale bar: 10 μm. f Quantification of the bi- and multi-nucleation rate in sh-Kif5b cells transiently expressing GFP, GFP-Kif5b and GFP-Kif5bΔMT ( n = 5 independent experiments). *** P < 0.0001; NS, P = 0.4039; unpaired two-tailed t-test. Data are mean ± S.D. g A model showing the function of KIF5B in cytokinesis. In late cytokinesis, KIF5B alone or together with other unknown molecules cross-links microtubules in the midbody, and therefore the structure of midbody can be stably maintained

Article Snippet: Other antibodies were as follow: anti-actin (Sigma, WB 1:2000), anti-α-tubulin (Sigma, WB: 1:10,000; immunofluorescences: 1:2000), anti-Aurora B (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-clathrin heavy chain (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-GM130 (BD transduction, WB: 1:1000; immunofluorescence: 1:100), Anti-PRC1 (Abcam, WB: 1:1000; Santa cruz, immunofluorescence: 1:100), phalloidin conjugated with Alexa Fluor 488 (Molecular probes) for F-actin labeling (1:300), CellLightTM Tubulin-GFP BacMam 2.0 (Molecular probes) for labeling tubulin in live cells, transferrin from human serum conjugated with rhodamine (Molecular probes).

Techniques: Immunofluorescence, Two Tailed Test, MANN-WHITNEY, Whisker Assay, Imaging, Expressing, Stable Transfection

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 1. Representative Western blot analyses of lung CEACAM6 in adult CEABAC mice compared to human infants. (A) Whole lung homogenate. 10 lg protein was loaded for each sample. Bands are observed at approximately 90, 70, and 50 kDa for most CEABAC and term human infant samples but not in wt mice. CEACAM6 in CEABAC mice consistently ran at slightly higher molecular weight than human lung CEACAM6, probably reflecting different amount of glycosylation. By scanning densitometry, total CEACAM6 signal is similar for CEABAC mice and human lungs. (B) Large aggregate surfactant fraction of BAL. 2 lg protein was loaded for mouse samples and 0.1 lg for a sample from an intubated premature human infant. A single 90 kDa band is observed for CEABAC samples and human surfactant but not wt mouse samples. (C) Supernatant of samples from B. 5 lg protein was loaded for mouse samples and 1 lg for the human. CEACAM6 was detected in the human specimen as previously reported (Chapin et al. 2012) but not in mouse samples.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Western Blot, Molecular Weight, Glycoproteomics

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 2. Immunohistochemistry of CEACAM6 in cryosections of lung from adult wt and CEABAC mice. (A) Immunostaining of wt mouse lung. OTS-8 red staining shows alveolar type I cells, blue nuclei (DAPI) and absence of CEACAM6 (green). (b) Corresponding phase contrast image. (C) OTS-8 and CEACAM6 immunostaining of CEABAC lung. Relatively low intensity CEACAM6 staining (green) is observed in some alveoli and does not appear to co-localize with OTS-8 as a marker of type I cells. (D) Corresponding phase contrast image. (E) SP-B and CEACAM6 immunostaining (red) of CEABAC lung. Low intensity CEACAM6 staining (green) is observed in some alveoli and does not appear to co-localize with SP-B (red) as a marker of type II cells. (F) Corresponding phase contrast image. Insets in c and d show ~ twofold magnified views of CEACAM6+ alveolar regions (arrows). Images are representative of multiple sections from lungs examined. Bar = 20 lm.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Immunohistochemistry, Immunostaining, Staining, Marker

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 4. Effect of bleomycin and saline on CEACAM6 content in lung tissue and lavage surfactant at 10 days. (A) Representative Western blots of lung homogenate. Equal amounts of total protein were loaded. Compared to No Treatment and Saline, increased total CEACAM6 signal is observed for lungs of bleomycin-treated animals. (B) CEACAM6 content of homogenate by scanning densitometry (mean SD). (C) Representative immunodot blot for large aggregate surfactant (with duplicate rows containing serial dilutions of equal amounts of PL/well). Increased signal is observed for bleomycin versus saline that is most evident at higher dilutions. (D) CEACAM6 content of surfactant by scanning densitometry. Data are mean SE, and n = 5; *P < 0.05 versus saline treatment.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Saline, Western Blot

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 5. Immunohistochemistry for CEACAM6 expression in cryosections of lung from adult CEABAC mice treated with saline (A–C) or bleomycin (D–F) for 10 days. (A) OTS-8 staining in saline-instilled lung. (B) punctate CEACAM6 staining (green, arrow) of one cell that is immunopositive for OTS-8. (C) phase contrast image corresponding to A,B. (D) OTS-8 staining in bleomycin-instilled lung. (E) CEACAM6 staining of multiple cells that are either positive (e.g., arrows) or negative (e.g., arrowheads) for OTS-8. (F) phase contrast image corresponding to d and e; note thickened interstitium compared to saline (C), reflecting bleomycin injury. Bar = 20 lm. All fluorescence images were obtained at the same exposure settings.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Immunohistochemistry, Expressing, Saline, Staining

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 6. CEACAM6 staining score for lungs after bleomycin instillation. A, score for cryosections of uninjured lungs (day 0) and at days 5–19 after bleomycin instillation (mean SE for 10 fields on each of 2 sections for lungs of 8 mice). (B) representative images for scores of 2, 3 and 4. The score is a composite of staining intensity and number of positive cells per field by a blinded observer. Maximal signal occurs by day 11; *P < 0.05 versus day 0. Results from a second experiment were similar.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Staining

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 7. Increased CEACAM6 immunostaining in epithelial cells after bleomycin instillation. Panel A, A–C, co-localization of CEACAM6 and AQP5 in alveolar epithelium. By confocal imaging, some alveolar cells co-stain (yellow) for CEACAM6 (green) and AQP5 (red, e.g., arrows) and other CEACAM6+ cells are negative for AQP5 (e.g., arrow heads). Bar = 50 lm. Panel B, CEACAM6 and CC10 staining in airways of control (A,C) and bleomycin-treated (B,D) CEABAC mice. Most cells of control animals are CC10-positive with sparse CEACAM6 signal (A and C— higher power). After bleomycin (B,D) co-localization of CEACAM6 and CC10 is observed in airway epithelial cells (arrows) and CEACAM6 signal occurs basal to the epithelium. Bars = (A) 40 lm; (B) 100 lm; (C) and (D) 40 lm.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Immunostaining, Imaging, Staining, Control

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 8. CEACAM6 and EGFP fluorescence signal in control and bleomycin-treated CEABAC/CBG mice. In control animals (A–D), cuboidal type II cells (green) are evident along with 2 CEACAM6+ (red) cells, with one example of co-localization (arrow). In bleomycin-treated mice (E–H), there is increased CEACAM6 signal (red) in injured areas and altered cell shape: F versus B, shape of many EGFP+ cells is flattened after bleomycin (arrowheads) with one cell showing apical CEACAM6 and cytoplasmic EGFP signal (G—arrow and inset); Phase micrographs are shown in D and H. Bar = 40 lm. * alveolus with EGFP+ epithelium.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Control

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 9. Representative scatter plots for sorting of lung cells. (A) Cells isolated from a bleomycin-treated CEABAC mouse. In this 2-way sort of live/single cells, 0.16% were CEACAM6+ and negative for CD11b, a lymphoid marker (box). 100,000 cells were recorded. B, Cells isolated from bleomycin-treated CEABAC/CBG mice. A 2-way sort provided populations of EGFP+ cells that were negative (square) or positive (ellipse) for CEACAM6. 1,000,000 cells were recorded.

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Isolation, Marker

Journal: Physiological reports

Article Title: Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

doi: 10.14814/phy2.12657

Figure Lengend Snippet: Figure 10. Lung structure and CEACAM6 expression in mice exposed to hyperoxia. Representative images of newborn CEABAC mice exposed to room air (A,C) or 80% oxygen (B,D) for 7 days. Note the simplified septal morphology and increased airspace size in mice exposed to hyperoxia (B vs. A, bar = 10 lm). C versus D, CEACAM6 and AQP5 staining. In mice exposed to hyperoxia there was a 1.8-fold increase in CEACAM6+ cell (green, arrows and arrowheads) compared to normoxic mice. There were similar numbers of cells that expressed both CEACAM6 and AQP5 (arrows) in each group. Bar = 40 lm

Article Snippet: All blots were run with recombinant human CEACAM6 (R & D Systems Inc., Minneapolis, MN) and adult human lung homogenate as internal controls.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 μg of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean ± standard deviation of the amount of immunoblot signal normalized with the signal for β-actin, and also for the total amount of protein in each fraction (for more details see ). *** P < 0.001; ns , not statistically significant.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) Samples of a membrane fraction (70 μg of proteins) from MCF 10A (A), MCF7 (B), and MDA-MB-231 (C) cells were incubated on ice for 1 hour with either 10 mM Tris HCl pH 7.4 ( Control ), 1 M KCl in 10 mM Tris HCl pH 7.4 ( KCl ) or 0.2 M Na 2 CO 3 pH 11.3 ( Na 2 CO 3 ). After centrifugation, pelleted membranes ( P ) and extracted proteins in the supernatant ( S ) were processed by SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. Syn16 , Syntaxin 16. The position of molecular mass markers is indicated on the left. (D-F) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in pellets ( P ) and supernatants ( S ) as shown in A-C of membranes incubated in control conditions (D), in 1 M KCl (E), or in 0.2 M Na 2 CO 3 (F). Bar represents the mean ± standard deviation of the amount of immunoblot signal. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Incubation, Control, Centrifugation, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: NRK (A and B), MCF 10A (C and D), MDA-MB-231 (E and F), and MCF7 (G and H) cells were left untreated ( Control ) or treated with 5 μg/ml BFA for 60 min ( BFA ). Cells were fixed, permeabilized, and immunolabeled with rabbit polyclonal antibody to GOLPH3, mouse monoclonal antibody to GM130, and either sheep antibody to TGN38 (A and B) or sheep antibody to TGN46 (C to H). Secondary antibodies were Alexa-594-conjugated donkey anti-rabbit IgG (red channel), Alexa-488-conjugated donkey anti-mouse IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Stained cells were examined by fluorescence microscopy. Merging red, green, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Insets show 1.7x magnifications. Bar, 10 μm.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Control, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C and examined by fluorescence microscopy. The time after initiation of imaging is shown in the bottom right corner of each panel in minutes:seconds. Images are representative of 15–20 videos of up to 200 seconds of recording. In B, filled arrows indicate a vesicular structure moving from the Golgi to the periphery of the cell. In C, filled arrows indicate a vesicular structure moving from the periphery of the cell to the Golgi area, and filled arrowheads indicate a tubular structure elongating from the Golgi. Empty arrows and empty arrowheads indicate the initial position of mobile structures. Bars, 5 μm. (D-F) The number of tubule-vesicular structures moving centrifugally (D), the number of tubular structures elongating from the Golgi (E), or the number of tubule-vesicular structures moving centripetally (F), were quantified from videos corresponding to 180 seconds of imaging. Bar represents the mean + standard deviation of the observed profiles (n = 15). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Imaging, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C. The area indicated by a white dotted-line rectangle in each set of images was bleached with a 488-nm laser set to 100% power. The fluorescence recovery after photobleaching (FRAP) was tracked by laser confocal microscopy with the 488-nm laser set to 2% power. Images were acquired before bleaching ( Pre-bleaching ), immediately after bleaching ( Bleaching ), and during the recovery of the fluorescence ( Recovery ) at approximately every 0.4-sec. Images of a representative experiment performed on each cell line are shown in each set of panels. Two images of the recovery of fluorescence are depicted with the time indicated in parenthesis in seconds. Bar, 10 μm. (D) Plot of the FRAP analysis of GFP-GOLPH3 in MCF 10A (black circles; n = 10), MDA-MB-231 (white circles; n = 10), and MCF7 (white squares; n = 10) cells. P , pre-bleaching; B ; bleaching. For simplicity, error bars are not depicted. *** P < 0.001; ns , not statistically significant. The halftime ( t 1/2 ) of maximal fluorescence recovery is indicated on the right in seconds (s).

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: Samples (30 μg of proteins) of rat liver cytosol ( Cyt ), rat liver Golgi membranes, and of cytosolic ( Cyt ) and membrane ( Memb ) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase ( CIAP ) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point ( pI ) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 ( GOLPH3 ) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. LysoPtdA , lysophosphatidic acid; LysoPtdCho , lysophosphatidylcholine; PtdIns , phosphatidylinositol; PtdIns(3)P , phosphatidylinositol 3-phosphate; PtdIns(4)P , phosphatidylinositol 4-phosphate; PtdIns(5)P , phosphatidylinositol 5-phosphate; PtdEth , phosphatidylethanolamine; PtdCho , phosphatidylcholine; S1P , sphingosine 1-phosphate; PtdIns(3 , 4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3 , 5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4 , 5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3 , 4 , 5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdA , phosphatidic acid; PtdSer , phosphatidylserine; Blank , no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Incubation, Recombinant, Western Blot

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (a) HepG2 with or without various treatments for 24 hours and stable HepG2 cells that expressed a control shRNA (sh-Ctrl) or the Atg5 shRNA (sh-Atg5) were subjected to flow cytometry analysis for CD133+ cells. Results represent the mean ± SEM of three independent experiments. None, no treatment. (B) HepG2 cells with the treatments shown in (A) were lysed for immunoblot analysis. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The β-actin protein was also analyzed to serve as the loading control. (C) Sphere-formation assay of CD133+ and CD133− HepG2 cells. The panels shown to the left are representative results of spheres formed by CD133+ and CD133− HepG2 cells with and without stable ATG5 knockdown. Scale bar=200 μm. The histogram shown to the right indicated the number of spheres larger than 100 μm in diameter when 500 CD133+ cells were seeded. The results represent the mean ± SEM of three independent experiments. (D) HepG2 cells with various treatments for 24 hours were incubated with MicroBeads (Miltenyi Biotec) for the isolation of CD133+ cells, which were then analyzed for their sphere-forming ability. 500 cells were seeded for the assay. Also see Figure S1.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Control, shRNA, Flow Cytometry, Western Blot, Tube Formation Assay, Knockdown, Incubation, Isolation

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Hep3B and Huh7 cells with various treatments for 24 hours were subjected to flow cytometry analysis for CD133+ cells. (B) HepG2 and Huh7 cells were transfected with the p53-expressing plasmid for two days, treated with 3-MA or rapamycin for another 24 hours and then subjected to flow cytometry analysis for CD133+ cells. (C) HepG2 cells transfected with the control siRNA (si-Ctrl) or the p53 siRNA (si-p53) for two days or treated with PFTα or DMSO for one day were analyzed for their CD133+ cells by flow cytometry (top panel) or sphere-forming ability of their CD133+ cells (bottom panel). The results shown in (A), (B) and (C) represent the mean ± SEM of three independent experiments. (D) HepG2 cells treated with DMSO or PFTα for one day or with siRNA for two days were lysed for immunoblot analysis. (E) Stable HepG2 cells that expressed control shRNA (sh-Ctrl), sh-Atg5, or both sh-Atg5 and sh-p53 were lysed for immunoblot analysis. (F) Cells mentioned in (E) were used for the sphere-formation assay. Also see Figure S2.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Transfection, Expressing, Plasmid Preparation, Control, Western Blot, shRNA, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) HepG2 cells with various treatments for 24 hours or stably expressing the control shRNA or the Atg5 shRNA were lysed for immunoblot analysis. (B) Immunoblot analysis of HepG2 cells transfected with either the control vector or the expression vector for various p53 proteins. Cells were lysed two days after DNA transfection for immunoblot analysis. None, control cells with no DNA transfection. (C) The experiments were conducted the same way as in (B), with the exception that Hep3B cells were used for the expression studies. (D) Hep3B or HepG2 cells were transfected with various p53-expressing plasmids or the control vector as indicated for two days followed by flow cytometry analysis for CD133+ cells. (E) HepG2 cells were transfected with the p53-expressing plasmids for two days, and CD133+ cells were then isolated for the sphere-formation assay. (F) The experiments were conducted the same way as in (E), with the exception that Hep3B cells were used for the studies. The results in (D–F) represented the mean ± SEM of three independent experiments. Also see Figure S3.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Stable Transfection, Expressing, Control, shRNA, Western Blot, Transfection, Plasmid Preparation, Flow Cytometry, Isolation, Tube Formation Assay

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Top panel, HepG2 cells were treated with DMSO, Mdivi-1 or CCCP for one day and then subjected to flow cytometry analysis for CD133+ cells; bottom panel, CD133+ HepG2 cells were isolated and treated with Mdivi-1 or CCCP for two days and then analyzed for their sphere-forming ability. (B) HepG2 cells without treatment or with the treatment of DMSO, CCCP or Mdivi-1 for one day were lysed for immunoblot analysis. Cells were also subjected to subcellular fractionation for the isolation of mitochondria, cytosol, and nuclei for immunoblot analysis. Tom20, β-actin and lamin B1 were used as the loading controls for mitochondria, cytosol and nucleus, respectively, to ensure equal amount of proteins were loaded on the gel. (C) Confocal microscopy for the analysis of the subcellular localization of p53(pS392) in HepG2 cells treated with DMSO, Mdivi-1 or CCCP. TOM20 was used as the marker for mitochondria. The areas boxed are enlarged at the bottom. Scale bar, 10 μm. (D) The results shown in (C) were quantified with a Leica TCS SP8 fluorescent confocal microscope. The results indicated the percentages of p53(pS392) that colocalized with TOM20. The results represent the mean ± SEM of at least 30 cells that were analyzed. See also Figure S5.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Flow Cytometry, Isolation, Western Blot, Fractionation, Confocal Microscopy, Marker, Microscopy

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: (A) Effects of PINK1 knockdown on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter using the Nanog-luc1 reporter (bottom panel). HepG2 cells transfected with either the control siRNA or the PINK1 siRNA for two days were analyzed. In the bottom panel, HepG2 cells were also transfected with the Nanog-luc1 reporter (see Figure 3B) for the analysis of luciferase activity. The luciferase activity of cells without the transfection of siRNA was arbitrarily defined as 1. The results represented the mean ± SEM of three independent experiments. (B) Effects of PINK1 over-expression on CD133+ HepG2 cells (top panel), their sphere-forming ability (middle panel) and their effects on the Nanog promoter (bottom panel). The experiments were conducted the same way as in (A), except that instead of using siRNA, cells were transfected with either the control vector or the PINK1-expressing plasmid. (C) Immunoblot analysis of HepG2 cells with PINK1 knockdown (left panels) or PINK1 over-expression (right panels) were lysed for immunoblot analysis. Total cell lysates as well as the nuclear lysates (bottom two panels) were analyzed. (D) PINK1 in HepG2, Hep3B or Huh7 cells was immunoprecipitated with a control antibody (−) or the anti-PINK1 antibody (+) and then incubated with GST-p53 in the presence of ATP. The GST-p53 phosphorylated at S392 was analyzed using the anti-p53 antibody that recognized phosphoserine-392. GST-p53 added in the reaction and PINK1 immunoprecipitated were also analyzed by immunoblot (bottom two panels). Numbers to the left of the top panel indicate protein molecular weight markers. (E) GST-p53 was mixed with GST-PINK1 or GST and incubated in the presence of ATP. The phosphorylation of p53 at S392 was then analyzed with the antibody that recognized phosphoserine-392. GST-p53, GST-PINK1 and GST used for the reaction was also analyzed by anti-p53, anti-PINK1 and anti-GST antibodies, respectively (bottom three panels). Numbers to the left indicate protein molecular weight markers. (F) Co-immunoprecipitation of p53 and p53(pS392) with PINK1. HepG2 cells were lysed and immunoprecipitated using the anti-PINK1 antibody or the control antibody followed by immunoblot analysis for p53, p53(pS392) and PINK1. (G) Co-immunoprecipitation of p53 and p53(pS392) with PINK1 using the anti-PINK1 antibody in different subcellular fractions (top 3 panels). β-actin, lamin B1 and Tom20 were used as the markers for cytosolic (C), nuclear (N) and mitochondrial (M) fractions. Equal amounts of p53 were used for the co-immunoprecipitation experiment (bottom 2 panels). See also Figure S6.

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Knockdown, Transfection, Control, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Incubation, Molecular Weight, Phospho-proteomics

Journal: Molecular cell

Article Title: Mitophagy Controls the Activities of Tumor Suppressor p53 to Regulate Hepatic Cancer Stem Cells

doi: 10.1016/j.molcel.2017.09.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sphere-formation assay The Human CD133 MicroBead Kit (Miltenyi Biotec) was used to isolate CD133 + cells from HepG2, Hep3B and Huh7 cells.

Techniques: Virus, Recombinant, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Mouse Assay, Mutagenesis, Software, Imaging, Extraction, Isolation, DNA Labeling

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

doi: 10.1038/labinvest.3700534

Figure Lengend Snippet: Figure 1 Dose effect of GTE on annexin-I expression and actin cytoskeleton. (a) Portion of a Sypro Ruby stained 2-D PAGE gel showing the region containing the annexin-I protein spots separated by isoelectric point (pI) in the first dimension and by molecular weight (MW) in the second dimension. 2-D PAGE and protein identification are described in the Materials and methods section. (b) Western blot analysis of annexin-I expression relative to b-actin in A549, H157, and H460 cells treated with 0, 10, 20, and 40 mg/ml of GTE for 24 h; Graph shows the densitometry measurement of annexin-I level relative to b-actin level. Data represent one of the two independent experiments. (c) RT-PCR analysis of annexin-I expression relative to b-actin in A549 cells treated with different concentrations of GTE (0, 10, 20, and 40 mg/ml) for 24 h. Graph represents the mean7s.d. of three independent experiments. (d) Immunofluorescence analysis of annexin-I in A549 cells treated with 0, 10, 20, and 40 mg/ml of GTE for 24 h were washed, fixed, and labeled sequentially for annexin-I (red fluorescence), DNA (blue fluorescence) and F-actin (green fluorescence, data not shown); (e) Immunofluorescence of F-actin (green fluorescence) in A549 cells tripled labeled with F-actin, annexin-I, and DNA as described in (d). Note that the GTE treated-A549 cells had an increased F-actin staining intensity. Images were taken using a Nikon Eclipse E400 microscope at 40 object.

Article Snippet: Human lung adenocarcinoma A549 cell line, large cell lung carcinoma NCI-H460 cell line (American Type Culture Collection, Rockville, MD, USA) and squamous cell carcinoma NCI-H157 cell line (National Cancer Institute, Bethesda, MD, USA) were grown in 90% RPMI 1640 (Mediatech Inc., Herndon, VA, USA) medium with 1% penicillin and streptomycin mix solution (Invitrogen Corporation, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS).

Techniques: Expressing, Staining, Molecular Weight, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Labeling, Fluorescence, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

doi: 10.1038/labinvest.3700534

Figure Lengend Snippet: Figure 2 Effect of annexin-I RNA interference on annexin-I and F-actin in GTE treated cells. The procedures for RNA interference and immunofluorescence labeling are described in the Materials and methods section. (a) Images of fluorescence labeling of annexin-I (red fluorescence) and F-actin (green fluorescence) under various conditions as specified. As expected, siRNA blocks the GTE (20 mg/ml) induced annexin-I expression (red), which resulted in a decreased F-actin (green), as observed by comparing images of annexin-I and F-actin in cells treated with GTE without siRNA to the images with siRNA. As a comparison, siRNA with negative control (siRNA()) complex had no such an effect (ie, increased F-actin and annexin-I with GTE). Images were taken using a Nikon Eclipse E400 microscope at 20 object. Confirmation of differential expression of annexin-I in A549 cell lines treated with GTE by (b) Western blot results of conditions as specified. Bar graph represents mean7s.d. of densitometry measurements of annexin-1 relative to b-actin in two independent experiments. (c) Quantitative real-time PCR analysis of annexin-I expression under various conditions as specified. Bar graph in (b) shows the densitometry result of annexin-I relative to b-actin, and in (c) shows mean7s.d. of three independent experiments.

Article Snippet: Human lung adenocarcinoma A549 cell line, large cell lung carcinoma NCI-H460 cell line (American Type Culture Collection, Rockville, MD, USA) and squamous cell carcinoma NCI-H157 cell line (National Cancer Institute, Bethesda, MD, USA) were grown in 90% RPMI 1640 (Mediatech Inc., Herndon, VA, USA) medium with 1% penicillin and streptomycin mix solution (Invitrogen Corporation, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS).

Techniques: Immunofluorescence, Labeling, Fluorescence, Expressing, Comparison, Negative Control, Microscopy, Quantitative Proteomics, Western Blot, Real-time Polymerase Chain Reaction

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

doi: 10.1038/labinvest.3700534

Figure Lengend Snippet: Figure 4 Effects of GTE on (a) adhesion and (b) adhesion complex formation in A549 cells. (a) Cells growing on fibronectin-coated culture plates for 2 h were fixed with glutaraldehyde, stained with crystal violet, and measured by a microplate reader, as detailed in the Materials and methods section. Cells treated without or with GTE (40 mg/ml), siRNA, or combination for 24 h under various conditions as specified. Note that GTE increased adhesion significantly. Values represent one of two independent experiments. **Po0.01, *Po0.05. (b) Immunofluorescence analysis of paxillin in A549 cells treated with 40 mg/ml of GTE for 24 h were washed, fixed, and labeled with paxillin (red fluorescence), as detailed in the Materials and methods section. Images were taken using a Nikon Eclipse E400 microscope at 40 object. The white arrowhead indicates the focal adhesion complex.

Article Snippet: Human lung adenocarcinoma A549 cell line, large cell lung carcinoma NCI-H460 cell line (American Type Culture Collection, Rockville, MD, USA) and squamous cell carcinoma NCI-H157 cell line (National Cancer Institute, Bethesda, MD, USA) were grown in 90% RPMI 1640 (Mediatech Inc., Herndon, VA, USA) medium with 1% penicillin and streptomycin mix solution (Invitrogen Corporation, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS).

Techniques: Staining, Immunofluorescence, Labeling, Fluorescence, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

doi: 10.1038/labinvest.3700534

Figure Lengend Snippet: Figure 5 Effects of GTE on the migration of A549 cells. (a) Confluent monolayers of cells were maintained in a serum-free media, and a lane was scraped through the monolayers of the cells with a plastic micropipette tip. The cells were allowed to migrate across the lane at 371C for 24 h in the presence (40 mg/ ml) or absence of GTE with or without siRNA annexin-I transfection for 24 h. The distance that cells migrated into the area of the wound at different points was photographed using a computer imaging system. Data represent one of three independent experiments. (b) A549 cells were incubated with 40 mg/ml of GTE in the top chamber for 24 h. The number of cells that migrated through the filter to the lower surface was counted, as determined by OD measurement using a microplate reader. The results are the mean7s.d. of two different fields (*Po0.05).

Article Snippet: Human lung adenocarcinoma A549 cell line, large cell lung carcinoma NCI-H460 cell line (American Type Culture Collection, Rockville, MD, USA) and squamous cell carcinoma NCI-H157 cell line (National Cancer Institute, Bethesda, MD, USA) were grown in 90% RPMI 1640 (Mediatech Inc., Herndon, VA, USA) medium with 1% penicillin and streptomycin mix solution (Invitrogen Corporation, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS).

Techniques: Migration, Transfection, Imaging, Incubation

Journal: Nature Communications

Article Title: Fibroblastic reticular cells in lymph node potentiate white adipose tissue beiging through neuro-immune crosstalk in male mice

doi: 10.1038/s41467-023-36737-0

Figure Lengend Snippet: a–d Sham-operated and iLNX male C57BL/6 N mice were exposed to 6 °C or 30 °C for 2 days. a , b ELISA analysis of protein level (a) and the mRNA expression of IL-33 ( b ) in scWAT of sham-operated and iLNX mice ( n = 5). c The mRNA level of IL-33 in scWAT and iLN ( n = 8). d IL-33 protein level in iLN determined by Western blot (top) and densitometric quantification of IL-33 (bottom) ( n = 3). e – n Lentivirus encoding FLAG-tagged Cre and luciferase (Lenti-Ccl19-Cre) or luciferase only (Lenti-Ccl19-Luci) driven by the Ccl19 promoter was directly injected into iLNs (7.5 × 10 6 Transduction Units [TU] per side) of eight-week-old male IL33 fl/fl mice. Seven days after lentiviral injection, mice were housed at 30 °C for 3 weeks followed by 2-day cold exposure (6 °C) or continued to be housed at 30 °C for another 2 days. e IVIS Lumina imaging analysis showing luminescence intensity in mice with (Lenti-Ccl19-Cre) or without lentivirus injection (negative control) after intraperitoneal injection of luciferin (150 mg/kg) for 10 min. f Immunofluorescent staining with primary antibodies against Cre recombinase or gp38 (a cell surface marker of FRCs) in iLN section; Scale bar, 50 μm g The mRNA level of IL-33 in iLN ( n = 5). h ELISA analysis for IL-33 protein level in scWAT ( n = 5). i , j Quantification of IL-5, IL-13 ( i ) and MetENK ( j ) in ILC2s using flow cytometric analysis ( n = 5). k , l Flow cytometric analysis of absolute numbers of eosinophils ( k ) and M2 macrophages ( l ) ( n = 5). m The mRNA expression of thermogenic genes in scWAT, determined by real-time PCR ( n = 5). n Western blot analysis for UCP1 protein expression in scWAT (top) and densitometric quantification of UCP1 (bottom) ( n = 3). All samples are biologically independent replicates. Data are presented as mean ± SEM. Statistical data were assessed using unpaired two-tailed Student’s test ( c , d, n ) or Mann–Whitney U test (a , g – m) . All the p values were two-sided. Source data are available as a Source Data file. kDa, relative molecular weight in kilodalton. See also Fig. , – .

Article Snippet: Subsequently, cells were fixed and permeabilized before staining with rabbit mAb anti-Cre recombinase (1:400, Cell Signaling Technology, D3U7F) followed by a secondary goat anti-rabbit Alexa Fluor® 568 antibody (1:800, Thermo Fisher Scientific, #A11011).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Luciferase, Injection, Transduction, Imaging, Negative Control, Staining, Marker, Real-time Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Molecular Weight

Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics

Article Title: Fasudil inhibits α-synuclein aggregation through ROCK-inhibition-mediated mechanisms.

doi: 10.1016/j.neurot.2025.e00544

Figure Lengend Snippet: Fig. 1. Experimental design and schematic represen- tation of the in vitro experiments. (a): Schematic rep- resentation of the α-synuclein-T/Synphilin-1 method. Co-expression of SynT and V5-Sph-1 leads to the for- mation of inclusions that can be observed after immunofluorescence. (b): Schematic representation of the VN-α-synuclein/α-synuclein-VC BiFC technique. α-synuclein tagged to the VN fragment of venus in- teracts with α-synuclein tagged to the VC fragment of venus leading to fluorescence. (c): Schematic repre- sentation of the RT-QuiC amplification. Small α-syn- uclein aggregates will break after quaking, creating nucleation centers (seeds) that will incorporate monomeric α-synuclein, leading to the formation of bigger aggregates. ThT binds to aggregates of a certain size, changing its conformation and leading to fluo- rescence emission. (d): Schematic representation of the incorporation of EGFP-tagged α-synuclein into microglia. AngII or AngII plus fasudil-treated microglial cells are co-cultured with neuron cells that express EGFP-tagged α-synuclein. EGFP-tagged α-synuclein is then incorporated into microglia, which is then identified by immunofluorescence and flow cytometry.

Article Snippet: Immunofluorescence was visualized with a fluorescence detection system (Molecular Imager ChemiDoc MP imaging System, BioRad).

Techniques: In Vitro, Expressing, Cell Culture, Cytometry

Journal: Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics

Article Title: Fasudil inhibits α-synuclein aggregation through ROCK-inhibition-mediated mechanisms.

doi: 10.1016/j.neurot.2025.e00544

Figure Lengend Snippet: Fig. 4. RT-QuiC analyses. (a–b): Coomassie staining of acrylamide gels shows the presence of PFFs 8 days after starting the preparation process. (c): Quantifi- cation of Coomassie-stained gels showed a higher significant quantity of protein in supernatants before starting the PFFs preparation process and after 4 days. *P < 0.05 relative to the previous column. n ¼ 3. Mann-Whitney U test and t-test. Error bars represent SD. (d): ThT assay of PFFs shows a significant increase in ThT fluorescence in samples collected at 8 days after starting the preparation process. R: Recombinant monomeric protein; sup: supernatant; prec: precipi- tate; PFFs: Pre-formed fibrils (4): Sample after four days of shaking; PFFs (8): Sample after eight days of shaking. *P < 0.05 relative to R. n ¼ 3 independent measurements. One-way ANOVA and Tukey's multiple comparison test. Error bars represent SD. (e): RT-QuiC graph of the monomeric α-synuclein amplification in the presence and absence of 20 μM fasudil. (f–i): Treatment with fasudil does not lead to significant changes in AUC, lag time, aggregation constant, nor in normalized RFU values at the end of the assay for unseeded RT-QuiC reactions. Treatment with fasudil significantly increases the lag time of the aggregation process in seeded RT-QuiC reactions. (j): Treatment with fasudil does not affect already aggregated α-synuclein. (k): RT-QuiC graph of monomeric α-synuclein amplification in the presence and absence of 40 μM fasudil, added at the onset of reaction and after 20 h (dotted line). (l–o): A second fasudil treatment 20 h after the onset of the process does not lead to significant changes in AUC, lag times, aggre- gation constant, nor in normalized RFU values at the end of the assay for seeded RT-QuiC reactions. n ¼ 3–6 independent amplifications. *P < 0.05 relative to Monomer þ PFFs. Error bars represent SEM. Mann- Whitney U test (f, i) or two-tailed unpaired Student's t-test (g-h, l-o).

Article Snippet: Immunofluorescence was visualized with a fluorescence detection system (Molecular Imager ChemiDoc MP imaging System, BioRad).

Techniques: Staining, MANN-WHITNEY, ThT Assay, Recombinant, Comparison, Two Tailed Test

Journal: eLife

Article Title: Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

doi: 10.7554/eLife.69207

Figure Lengend Snippet: ( A ) Western blot analysis for Gja1 knock-down by siRNA. Tubulin was used as internal loading control. n = 3 independent experimental repeats. ( B ) The representative confocal live cell imaging of Gja1 knocked-down HEK293 cells with Cx43-M6L or GJA1-20k transfection. ( C ) The fold change in the average area of individual mitochondria. n = 52 (Control), 57 (siGja1), 60 (siGja1+ M6 L), or 64 (siGja1+ GJA1-20 k) cells from five independent experiments. The images and the values of Control and siGja1 are also shown in . ( D ) The representative live cell imaging of mitochondria in WT mouse neonatal CMs with adenovirus-mediated GFP induction. The right panel indicates magnified image surrounded by square. ( E ) The fold change in the average area of individual mitochondria between WT (no virus introduction, the image and the value shown in ) and GFP-V5 introduction. n = 46 (WT) or 35 (GFP) cells from four hearts. Graphs were expressed as mean ± SD ( C ) or SEM ( E ). p values were determined by two-tailed Mann-Whitney U-test or Kruskal-Wallis test with Dunn’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001; n.s., not significant. Scale bars, 10 μm or 5 μm in magnified image. Exact p values and statistical data are provided in the source data. Figure 1—figure supplement 1—source data 1. All data points of the mitochondrial size and the statistical data for .

Article Snippet: Recombinant DNA reagent , pDEST-GJA1-20k-GFP (plasmid) , (DOI: 10.3389/fphys.2017.00905 ) , , GFP version of Addgene_#49,861.

Techniques: Western Blot, Knockdown, Control, Live Cell Imaging, Transfection, Virus, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

doi: 10.7554/eLife.69207

Figure Lengend Snippet: ( A ) and ( B ) Western blot analysis for mitochondrial dynamics related proteins. Transfection was confirmed by GFP bands and the band size difference in GFP is due to the difference in molecular weight between GST and GJA1-20k ( A ). Tubulin was used as internal loading control. n = 5 independent experimental repeats. ( C ) Representative fixed cell images of mitochondria (visualized by Tom20) with DRP1 siRNA, K38A treatment, or Control. ( D ) The fold change in the average area of mitochondria in each treatment. n = 34 (GST, control siRNA), 32 (GJA1-20k, control siRNA), 36 (GST, DRP1 siRNA), 31 (GJA1-20k, DRP1 siRNA), 34 (GST, K38A), or 36 (GJa1-20k, K38A) cells from three independent experiments. Graphs were expressed as mean ± SD. p values were determined by two-tailed Mann-Whitney U-test or two-way ANOVA with Bonferroni’s post-hoc test. ***p < 0.001; n.s., not significant. Scale bars, 5 μm ( C ). Exact p values and statistical data are provided in the source data. Figure 2—source data 1. All data points of the protein expression and the mitochondrial size and the statistical data for .

Article Snippet: Recombinant DNA reagent , pDEST-GJA1-20k-GFP (plasmid) , (DOI: 10.3389/fphys.2017.00905 ) , , GFP version of Addgene_#49,861.

Techniques: Western Blot, Transfection, Molecular Weight, Control, Two Tailed Test, MANN-WHITNEY, Expressing

Journal: eLife

Article Title: Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

doi: 10.7554/eLife.69207

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pDEST-GJA1-20k-GFP (plasmid) , (DOI: 10.3389/fphys.2017.00905 ) , , GFP version of Addgene_#49,861.

Techniques: Plasmid Preparation, Expressing, Transfection, Construct, Recombinant, Negative Control, DC Protein Assay, Isolation, Luminescence Assay, Purification, Software, Imaging, Staining

Journal: Neuron

Article Title: Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

doi: 10.1016/j.neuron.2018.09.010

Figure Lengend Snippet: Figure 1. Endothelial ASM Is Increased in Aging and Causes Brain Endothelial Cell Death (A) ASM activity in young and aging human plasma (n = 24 for human samples per group). (B) ASM activity in plasma derived from 3-, 12-, and 20-month-old WT and Smpd1+/ mice (n = 8 mice per group). (C) ASM activity in liver, kidney, spleen, heart, lung, stomach, genitals, muscle, fat, and brain from 3- and 20-month-old WT mice (n = 3–8 mice per group). (D) Left: ASM activity in total brain from 3-, 12-, and 20-month-old WT and Smpd1+/ mice (n = 6–8 mice per group), Middle: representative immunoblotting of CD31, Tuj-1, and GFAP in brain microvessels and microvessel-depleted brains. Right: ASM activity in isolated microvessels and microvessel-depleted brains from 3- and 20-month-old WT and Smpd1+/ mice (n = 6–8 mice per group). (E) Percentage of ASM in endothelial cells (CD31+), pericytes (PDGFR-b+), or smooth muscle cells (a-SMA+) derived from brain microvessels of 3- and 20-month- old WT and Smpd1+/ mice (n = 5 mice per group). (F) Representative images (top) and quantification (bottom) of lectin-positive microvascular profiles in cortex of 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). (G) Representative images (top) and quantification (bottom) of active caspase-3 (arrow) in cortical endothelial cell of 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). (H) Representative images (top) and quantification (bottom) of string vessels (arrow) in cortex of 3- and 20-month-old WT and Smpd1+/ mice (n = 4 mice per group). Scale bars, 50 mm. (A and C) Student’s t test. (B, D, and E–H) One-way ANOVA and Tukey’s post hoc test. *p < 0.05. All error bars indicate SEM. See also Figures S1–S3.

Article Snippet: We also used human SMPD1 antibody (3 mg/mL, R&D Systems, MAB5348) or IgG isotype antibody (3 mg/mL, R&D Systems, MAB003) to examine the effects of ASM inhibition.

Techniques: Activity Assay, Clinical Proteomics, Derivative Assay, Western Blot, Isolation

Journal: Neuron

Article Title: Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

doi: 10.1016/j.neuron.2018.09.010

Figure Lengend Snippet: Figure 2. Genetic Inhibition of ASM Reduces BBB Disruption in Old Mice (A) Water content in brains of 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). (B) Left: immunofluorescence images of extravascular fibrin deposits in cortex of mice brain. Scale bars, 50 mm. Right: quantification of extravascular fibrin deposits (n = 6 mice per group). (C) Left: representative immunoblotting of fibrin and thrombin levels in microvessel-depleted brain tissue. Right: quantification of fibrin and thrombin (n = 6 mice per group). (D) Representative in vivo time-lapse multiphoton imaging and quantification data of tetramethylrhodamine (TMR) dextran (molecular weight [MW] = 40 kDa; red) leakage from cortical vessels in 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). Arrows in WT old mice point to leakages from cortical vessels. Scale bar, 50 mm. (E) Concentrations of fluorescently labeled dextrans of various molecular sizes in 3- and 20-month-old WT and Smpd1+/ mice brain lysates (n = 3–4 mice per group). (F) Representative image of fluorescent 150 kDa dextran in 20-month-old WT and Smpd1+/ mice after staining with anti-CD31 and anti-active caspase-3. Arrow, apoptotic vessels. Scale bar, 20 mm. (G) Left: immunoblotting and quantification of Cav-1 phosphorylation in total brain of 3- and 20-month-old WT and Smpd1+/ mice (n = 4 mice per group). Right: immunoblotting and quantification of Cav-1 phosphorylation, Cavin1, and EHD2 in isolated microvessels of 3- and 20-month-old WT and Smpd1+/ mice (n = 4–6 mice per group). (H) EM analysis and quantification of cortical endothelial cells for vesicles in 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group and 8 capillary cross-sections per mouse). Arrow, cytoplasmic vesicles; arrowhead, luminal vesicles. Scale bar, 500 nm. (I) EM image and quantification of cortical endothelial cells for HRP transcytosis in 3- and 20-month-old WT and Smpd1+/ mice (n = 4 mice per group and 8 capillary cross-sections per mouse). Scale bar, 500 nm. (J) EM analysis of tight junctions in endothelial cells from 3- and 20-month-old WT and Smpd1+/ mice. Scale bar, 100 nm. Black arrows point to endothelial junctions. Red arrows indicate microvesicles filled with HRP in endothelium in 20-month-old WT and Smpd1+/ mice.

Article Snippet: We also used human SMPD1 antibody (3 mg/mL, R&D Systems, MAB5348) or IgG isotype antibody (3 mg/mL, R&D Systems, MAB003) to examine the effects of ASM inhibition.

Techniques: Inhibition, Disruption, Western Blot, In Vivo, Imaging, Molecular Weight, Labeling, Staining, Phospho-proteomics, Isolation

Journal: Neuron

Article Title: Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

doi: 10.1016/j.neuron.2018.09.010

Figure Lengend Snippet: Figure 3. Genetic Inhibition of ASM Delays Neurodegenerative Changes in Old Mice (A) Immunofluorescence images and quantification of NeuN+ neurons in the brain of 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). Scale bars, 50 mm. (B) Representation of brain morphology and quantification of maximal brain diameter in 3- and 20-month-old WT and Smpd1+/ mice (n = 5 mice per group). (C) Representative high-magnification, bright-field microscopy analysis and quantification of Golgi-Cox staining showing dendritic spine density in the CA1 region of 3- and 20-month-old WT and Smpd1+/ mice (n = 6 mice per group). Scale bars, 10 mm. (D) Representative immunofluorescence images and quantification of synaptophysin in the brain of 3- and 20-month-old WT and Smpd1+/ mice (n = 5 mice per group). Scale bars, 50 mm. (E) Morris water maze test in the 3- and 20-month-old WT and Smpd1+/ mice (n = 10–13 mice per group). (F) Results of contextual and tone tasks during fear conditioning test (n = 7–9 mice per group). (G) Light and dark transition test. Moving distance, time spent, transition number, and first transition time were measured in the 3- and 20-month-old WT and Smpd1+/ mice (n = 8–12 mice per group). (H) LTP induced by high-frequency stimulation (WT young mice, n = 15 slices from 8 mice; Smpd1+/ young mice, n = 13 slices from 7 mice; WT old mice, n = 13 slices from 7 mice; Smpd1+/ old mice, n = 14 slices from 8 mice). One-way ANOVA, Tukey’s post hoc test. *p < 0.05, **p < 0.01. All error bars indicate SEM.

Article Snippet: We also used human SMPD1 antibody (3 mg/mL, R&D Systems, MAB5348) or IgG isotype antibody (3 mg/mL, R&D Systems, MAB003) to examine the effects of ASM inhibition.

Techniques: Inhibition, Microscopy, Staining

Journal: Neuron

Article Title: Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

doi: 10.1016/j.neuron.2018.09.010

Figure Lengend Snippet: Figure 5. ASM Regulates Endothelial Cell Permeability via Caveolae-Mediated Transcytosis (A and B) Representative images (A) and fluorescence-activated cell sorting (FACS) analysis (B) of phalloidin in primary mouse brain endothelial cells 10 min after ASM treatment (n = 8 independent experiments). Scale bars, 20 mm. (C) Immunoblotting and immunocytochemistry of phospho-ERM in primary mouse brain endothelial cells 10 min after ASM stimulation with immunoglobulin G (IgG) isotype antibody or ASM antibody (n = 5 independent experiments). Scale bar, 20 mm. (D) Time- and concentration-dependent effects of ASM on ERM dephosphorylation (n = 4 independent experiments). (E) Western blot analysis of ERM dephosphorylation in ASM-treated cells after calyculin A or okadaic acid pre-incubation for 3 hr (n = 8 independent experiments). (F) Representative immunocytochemistry images of phospho-ERM in ASM-treated cells after calyculin A or okadaic acid pre-incubation for 3 hr. Scale bars, 20 mm. (G and H) Representative images (G) and FACS analysis (H) of phalloidin in ASM-treated cells after calyculin A pre-incubation for 3 hr (n = 8 independent experiments). Scale bars, 20 mm. (I) Western blot analysis of phospho-Cav-1 in ASM-treated cells after calyculin A pre-incubation for 3 hr (n = 6 independent experiments). (J) BSA transcytosis assay in ASM-treated cells after calyculin A pre-incubation (n = 8 independent experiments). Scale bars, 20 mm. (K) Western blot analysis and quantification of phospho-Erk1/2 and phospho-Akt in ASM-treated primary mouse brain endothelial cells after PP1 inhibition (n = 4–5 independent experiments). (L) Western blot analysis and quantification of phospho-Cav-1 in primary mouse brain endothelial cells after inhibition of the Erk1/2 pathway (n = 5 independent experiments). (M) Western blot analysis of ERM dephosphorylation in the isolated microvessels from 3- and 20-month-old WT and Smpd1+/ mice (n = 5 mice per group). (B, E, and H–L) Student’s t test. (C, D, and M) One-way ANOVA and Tukey’s post hoc test. *p < 0.05, **p < 0.01. All error bars indicate SEM.

Article Snippet: We also used human SMPD1 antibody (3 mg/mL, R&D Systems, MAB5348) or IgG isotype antibody (3 mg/mL, R&D Systems, MAB003) to examine the effects of ASM inhibition.

Techniques: Permeability, FACS, Western Blot, Immunocytochemistry, Concentration Assay, De-Phosphorylation Assay, Incubation, Inhibition, Isolation

Journal: Neuron

Article Title: Vascular and Neurogenic Rejuvenation in Aging Mice by Modulation of ASM.

doi: 10.1016/j.neuron.2018.09.010

Figure Lengend Snippet: Figure 7. ASM Knockdown in Endothelial Cells Reduces Aging-like Brain Pathology (A) Smpd1 mRNA level in brain microvessels and microvessel-depleted brain derived from Smpd1 miR RNAi and control miR RNAi-treated 13-month-old Slco1c1-CreERT2;Smpd1ox/ox mice or 18-month-old WT mice (n = 4 mice per group). (B) ASM activity in plasma, brain microvessels, and microvessel-depleted brain derived from each group (n = 6–7 mice per group). (C) Representative images and quantification of lectin-positive microvascular profiles in cortex of each group (n = 6 mice per group). (D) Left: immunofluorescence images of extravascular fibrin deposits in cortex of each group. Right: quantification of extravascular fibrin deposits (n = 6 mice per group). (E) Representative in vivo time-lapse multiphoton imaging and quantification of TMR dextran (MW = 40 kDa; red) leakage from cortical vessels in each group (n = 3 mice per group). (F) Left: representative immunoblotting of fibrin and thrombin levels in microvessel-depleted brain tissue from each group. Right: quantification of fibrin and thrombin (n = 4 mice per group). (G and H) Western blot analysis of Cav-1 phosphorylation, Cavin-1, and EHD2 (G) and ERM dephosphorylation (H) in the isolated microvessels from each group (n = 4 mice per group). (I) Quantification of NeuN+ neurons in the brain of each group (n = 6 mice per group). (J) Morris water maze test in the Smpd1 miR RNAi and control miR RNAi-treated 13-month-old Slco1c1-CreERT2;Smpd1ox/ox mice or 18-month-old WT mice (n = 8–10 mice per group). Scale bars, 50 mm. Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. All error bars indicate SEM. See also Figures S5–S7.

Article Snippet: We also used human SMPD1 antibody (3 mg/mL, R&D Systems, MAB5348) or IgG isotype antibody (3 mg/mL, R&D Systems, MAB003) to examine the effects of ASM inhibition.

Techniques: Knockdown, Derivative Assay, Control, Activity Assay, Clinical Proteomics, In Vivo, Imaging, Western Blot, Phospho-proteomics, De-Phosphorylation Assay, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Mass spectroscopy analysis of SCN5A-ATF2 protein-protein interaction

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Human macrophage SCN5A splice variant associates with the transcription factor ATF2. To confirm the results from LC/MS (Table 1), immunoprecipitation (IP) of human SCN5A was performed in BMDM from transgenic and wild type conditions. BMDM from the transgenic mice express the human macrophage SCN5A variant under the control of a cfms promoter (24). Co-immunoprecipitation of ATF2 (red) was observed in the transgenic (TG) but not wild type (WT) condition. Difference gel electrophoresis labeling for total protein content in the lysate is shown in blue.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Variant Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Transgenic Assay, Nucleic Acid Electrophoresis, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Pharmacological activation increases phospho-ATF2 levels. A, human SCN5A+ (hSCN5A) and wild type BMDM were treated with the sodium channel agonist, veratridine (100 μm), for 15 min and then stained for total ATF2 (red), phospho-ATF2 (green), and DNA (DAPI, blue). Scale bar, 10 μm. Quantitative analysis (AxioVision auto-measurement module) revealed a statistically significant difference in nuclear phospho-ATF2 staining between the two conditions (top micrographs). Fluorescent densitometric sum/nucleus was 5.58 × 106 ± 0.71 × 106 in hSCN5A cells versus 0.32 × 106 ± 0.14 × 106 in the wild type condition (p < 0.01, n = 4 cell samples; 46 cells analyzed for the hSCN5A condition and 54 cells for the wild type condition; ± S.E.). Baseline staining in the SCN5A BMDM without treatment is shown on the bottom. Scale bar, 10 μm. B, Western blot analysis was performed to confirm phospho-ATF2 cell staining. Veratridine treatment of mouse BMDM for 15 min led to markedly increased phospho-ATF2 levels in transgenic human SCN5A+ (hSCN5A) macrophages (Tg) but not in the wild type conditions (WT) or following vehicle (DMSO) treatment. Densitometric analysis (ImageJ) revealed relative fluorescent units of 0.24 ± 0.13 for vehicle-treated transgenic BMDM, 0.31 ± 0.08 for vehicle-treated WT, 0.87 ± 0.05 for veratridine-treated transgenic cells (p < 0.01 ANOVA, n = 4 cell preparations, + S.E.), and 0.28 ± 0.15 for veratridine-treated WT. TG, transgenic.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Activation Assay, Staining, Western Blot, Transgenic Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Increased expression of an Sp100 variant, Gm7609, is associated with human SCN5A expression and pharmacological activation. A, Affymetrix mouse 2.0ST whole transcriptome arrays were utilized to examine global gene expression in mouse BMDM that express the human macrophage SCN5A transgene and in control wild type cells. Data were analyzed using NetAffx software (Affymetrix). Fold change (transgenic versus wild type) and p values are based on three separate RNA preparations for each condition (error bars, ± S.E.). B, QPCR analysis confirmed increased expression of Gm7609 in transgenic cells but not the 3′ prime segment of Sp100 (Table 2, statistical analysis). C, pharmacological activation with the sodium channel agonist veratridine (100 μm for 2 h) of transgenic BMDM led to a further increase in Gm7609 mRNA that was not observed in wild type cells (Table 3). D, siRNA knockdown of ATF2 and Adcy8, a calcium-dependent isoform of adenylate cyclase, but not Adcy10, inhibited the veratridine-induced response (Table 4). The transgenic condition in the graphs is shown in red and the wild type condition in blue.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing, Variant Assay, Activation Assay, Software, Transgenic Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: SCN5A mediates a veratridine-induced calcium response in human monocyte-derived macrophages. Human MDM were labeled with the calcium indicator Fluo4 and imaged by time-lapse live cell microscopy. Stimulation of human MDM with veratridine (100 μm) elicited a biphasic peak response and a more prolonged elevation in cytosolic calcium. The initial peak response (Peak 1) and the sustained response, but not Peak 2, were dependent on expression of SCN5A and RYR1. Images were acquired at 10-s intervals for 10 min and subsequently analyzed in ImageJ and MATLAB. The top graph shows representative tracings for each condition. The bar graphs show quantitative analysis of peak responses and the cumulative response (AUC, area under the curve). For peak 1, the mean responses were as follows: 80.2 ± 5.8 RFUs for control siRNA, 15.1 ± 4.9 for SCN5A knockdown, and 13.4 ±3.2 for RYR1 knockdown (p < 0.001 for control condition, ANOVA, n = 10 regions of interest, error bars, ±S.E.). For peak 2, 32.3 ± 2.7 for control siRNA, 48.9 ±5.8 for SCN5A knockdown, and 31.7 ± 5.1 for RYR1 knockdown (not significant, ANOVA, n = 10). The AUC was 1909.0 ± 163.1 for the siRNA control condition, 803.3 ± 192.5 for SCN5A knockdown, and 164.6 ± 40.3 for RYR1 knockdown (p < 0.001 for control condition, ANOVA, n = 10).

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Derivative Assay, Labeling, Microscopy, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Pharmacological activation of human monocyte-derived macrophages regulates signal transduction and transcription that is dependent on SCN5A expression. A, veratridine treatment (100 μm for 15 min) increased cAMP levels in human MDM in control siRNA conditions but not following knockdown of SCN5A, ADCY8, or RYR1 (Table 5). B, veratridine treatment (100 μm for 2 h) increased Sp100 mRNA levels in control siRNA-treated MDM but not following SCN5A knockdown (Table 6). C, veratridine treatment (100 μm for 2 h) markedly increased IFNB transcription in MDM treated with control siRNA. Knockdown of SCN5A, ADCY8, or ATF2 inhibited this response (Table 7). Error bars, ± S.E., from pooled replicates from a representative donor. Similar results were obtained from a second donor.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Activation Assay, Derivative Assay, Transduction, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Veratridine-induced increases in cAMP in human monocyte-derived macrophages is dependent on expression of SCN5A , ADCY8 , and RYR1

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Veratridine treatment increases transcription of SP100 in human monocyte-derived macrophages and is dependent on expression of SCN5A HMBS is hydroxymethylbilane synthase.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Veratridine-induced increases in IFNB transcripts in human monocyte-derived macrophages is dependent on expression of SCN5A , ATF2, and ADCY8 HMBS is hydroxymethylbilane synthase.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: I-V analysis Current measurements at −20 mV in the poly(I:C) condition. pF is picofarads.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Transfection of poly(I:C) complexes is similar in the presence or absence of macrophage SCN5A. The indicated cell types were challenged with rhodamine-labeled poly(I:C) (LMW, 200 nm for 20 min) and subsequently washed and fixed for microscopic analysis (scale bars, 20 μm).

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Transfection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Poly(I:C) treatment initiates SCN5A-dependent transcription. A, in mouse BMDM that express the human macrophage SCN5A variant, transfection of poly(I:C) (low molecular weight, 200 ng/ml for 2 h), but not treatment with naked poly(I:C) (200 ng/ml for 2 h), increased expression of Gm7609 transcripts. This increase was not observed in wild type, SCN5A-negative cells, or in transgenic cells following siRNA knockdown of Adcy8 or ATF2 (Table 8) (error bars, ± S.E. from pooled replicates of multiple RNA preparations). B, in human MDM treated with control siRNA, increased transcription of IFNB was observed following treatment with transfected poly(I:C) (200 ng/ml for 2 h). This increase was inhibited by siRNA knockdown of SCN5A or ATF2; knockdown of DDX58 (RIG-1) also reduced the response but to a lesser degree (Table 9). Error bars, ± S.E., from pooled replicates from a representative donor. Similar results were obtained from a second donor. C, knockdown of MyD88 or TRIF (TICAM-1) increased expression of IFNB in response to cytosolic poly(I:C) in human MDM (Table 10). D, human MDM were infected with HSV-1 for 24 h, and transcription of viral transcripts glycoprotein B (GB) and thymidine kinase (TK) was analyzed. Knockdown of SCN5A, but not treatment with control siRNA, increased transcription of glycoprotein B and thymidine kinase (Table 11).

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Variant Assay, Transfection, Molecular Weight, Expressing, Transgenic Assay, Infection

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Cytosolic poly(I:C)-induced increases in mouse Gm7609 transcripts in hSCN5A + bone marrow-derived macrophages is dependent on expression of SCN5A , ATF2, and ADCY8

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Cytosolic poly(I:C)-induced increases in IFNB transcripts in human monocyte-derived macrophages is dependent on expression of SCN5A and ATF2 but less so on DDX58 HMBS is hydroxymethylbilane synthase.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: SCN5A knockdown in human monocyte-derived macrophages increases viral transcripts in HSV-1 infected cells HMBS is hydroxymethylbilane synthase.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Infection

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: Poly(I:C) increases cAMP levels and currents in HEK-293 cells that heterologously express human macrophage SCN5A. A, cAMP levels were monitored by live cell imaging using the cyclic nucleotide-gated channel assay (Fig. 4). Poly(I:C) treatment (low molecular weight, 200 ng/ml) increased cAMP levels 10 and 15 min post-challenge in SCN5A-transfected cells but not mock-transfected HEK-293 cells. Normalized fluorescent ratios were 1.39 ± 0.13 (5 min), 2.42 ± 0.32 (10 min), and 2.44 ± 0.32 (15 min) for the SCN5A condition and 1.15 ± 0.10 (5 min), 1.02 ± 0.17 (10 min), and 1.24 ± 0.11 (15 min) for the control condition (error bars, ± S.E., n = 10 regions of interest for each condition, p < 0.05 for 10 and 15 min conditions). B, whole cell patch clamp analysis revealed outward and inward currents in SCN5A-expressing cells with cesium (Cs) as the predominant intracellular cation and sodium in the extracellular solution (second recording from top). Outward currents were reduced in control transfected cells (top current recording), and an inward current was not seen. Poly(I:C) in the intracellular solution (200 ng/ml; third from top) markedly increased a voltage-dependent inward current in SCN5A-positive cells and reduced the outward current. Replacement of sodium (Na) with NMDG in the extracellular solution (bottom current recording) prevented the poly(I:C) induced inward current. C, I-V curve analysis of the experiments shown in B. Ambroxol (50 μm), an inhibitor of tetrodotoxin-resistant sodium channels, reduced inward but not outward currents in SCN5A cells. Data in Tables 12​12​–15 show statistical analysis of currents at +40 mV and −20 mV in the absence and presence of intracellular poly(I:C). Data are pooled, and normalized (pA/picofarad (pF)) current recordings and are expressed as ± S.D.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: Live Cell Imaging, Molecular Weight, Transfection, Patch Clamp, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: I-V analysis Current measurements at +40 mV in the untreated condition. pF is picofarads.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: I-V analysis Current measurements at −20 mV in the untreated condition. pF is picofarads.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human Macrophage SCN5A Activates an Innate Immune Signaling Pathway for Antiviral Host Defense *

doi: 10.1074/jbc.M114.611962

Figure Lengend Snippet: I-V analysis Current measurements at +40 mV in the poly(I:C) condition. pF is picofarads.

Article Snippet: The following TaqMan primers were obtained from Applied Biosystems/Invitrogen: Hs00165693_m1 (human SCN5A ), Mm99999915_g1 (mouse Gapdh ), Mm00449735_m1 (mouse Sp100 ), Mm04204797_m1 (mouse Gm7609 ), Hs00162109_m1 (human SP100 ), Hs01077958_s1 (human IFNB1 ), AIGJQ71 (HSV thymidine kinase), and AIHSPDQ (HSV glycoprotein B ( 8 )).

Techniques: