Journal: The Journal of Cell Biology

Article Title: Septins suppress the release of vaccinia virus from infected cells

doi: 10.1083/jcb.201708091

Figure Lengend Snippet: Loss of septin promotes virus release and spread. (A) Schematic depicting key events at the plasma membrane during vaccinia virus egress. A36, the actin tail nucleator phosphorylated by Src and Abl family kinases, and B5, exposed on the surface of CEV, are integral viral membrane proteins. (B) Immunoblot analysis confirms that SEPT7 siRNA treatment of A549 cells for 72 h leads to loss of SEPT7 as well as reduction in the levels of SEPT2, SEPT9, and SEPT11. (C) Images of plaques formed on A549 cell monolayers treated with control (Allstar) or SEPT7 siRNA for 72 h under liquid overlay after 3 d of infection with WR or the YdF strain of virus, which is deficient in actin tail formation. Plaque comets are seen as a diffuse spray emanating from WR plaques in liquid overlay conditions. The graphs show the quantification of plaque size ( n > 190) with error bars representing the SEM from three independent experiments. (D) Quantification of virus release and total intracellular virus in A549 cells in the presence or absence of SEPT7 at 18 h after infection with WR or the YdF virus. Error bars represent SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Bar, 1 cm.

Article Snippet: The following primary antibodies were used for immunofluorescence and immunoblotting: A36 , AP-2 (2730; Abcam), Clathrin Heavy Chain (21679; Abcam), B5 ( ; the B5 antibody was labeled with the Molecular Probes Alexa Fluor 488 Monoclonal Antibody Labeling kit [Invitrogen] to generate the Alexa Fluor 488-B5 antibody [ ]), dyn II for immunofluorescence ( ; provided by M. McNiven, Mayo Clinic, Rochester, NY) and Western (A303-513A; Bethyl), FHOD1 (A304-825A; Bethyl), GAPDH (ab8245; Abcam), SEPT2 (60075–1-Ig; ProteinTech), SEPT7 (18991; IBL), SEPT9 (10769–1-AP; ProteinTech) for immunofluorescence and SEPT9 (A302-353A; Bethyl) for Western, and SEPT11 (A304-176A; Bethyl).

Techniques: Virus, Clinical Proteomics, Membrane, Western Blot, Control, Infection

Journal: The Journal of Cell Biology

Article Title: Septins suppress the release of vaccinia virus from infected cells

doi: 10.1083/jcb.201708091

Figure Lengend Snippet: Septin suppresses actin tail formation. (A) Immunoblot analysis confirms that SEPT7 siRNA treatment of HeLa cells for 72 h reduces the levels of SEPT7, SEPT2, SEPT9, and SEPT11. (B) Representative immunofluorescence images showing that loss of SEPT7 in uninfected HeLa cells has no obvious impact on the actin cytoskeleton (green). Bar, 50 µm. (C) Immunoblot analysis confirming that infection of HeLa cells with WR vaccinia virus does not alter the level of SEPT2, SEPT7, SEPT9, or SEPT11. (D) Representative images of actin tails (green) induced by WR (red labeled with B5 antibody) in HeLa cells treated with Allstar (control) or SEPT7 siRNA. Bar, 2 µm. The graphs show the quantification of the number of CEVs inducing actin tails and their length. Error bars represent SEM from three independent experiments in which a total of 30 cells were analyzed for actin tail number and 370 tails were measured for length. (E) Quantification of the velocity, directionality, time to form, and duration of actin tails in the absence of SEPT7. Error bars represent SEM from three independent experiments in which a total of >150 actin tails were measured for duration, >50 events for tail initiation from four independent experiments, and >300 actin tails for speed and directionality. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence and immunoblotting: A36 , AP-2 (2730; Abcam), Clathrin Heavy Chain (21679; Abcam), B5 ( ; the B5 antibody was labeled with the Molecular Probes Alexa Fluor 488 Monoclonal Antibody Labeling kit [Invitrogen] to generate the Alexa Fluor 488-B5 antibody [ ]), dyn II for immunofluorescence ( ; provided by M. McNiven, Mayo Clinic, Rochester, NY) and Western (A303-513A; Bethyl), FHOD1 (A304-825A; Bethyl), GAPDH (ab8245; Abcam), SEPT2 (60075–1-Ig; ProteinTech), SEPT7 (18991; IBL), SEPT9 (10769–1-AP; ProteinTech) for immunofluorescence and SEPT9 (A302-353A; Bethyl) for Western, and SEPT11 (A304-176A; Bethyl).

Techniques: Western Blot, Immunofluorescence, Infection, Virus, Labeling, Control

Journal: BMC Cancer

Article Title: Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

doi: 10.1186/1471-2407-13-398

Figure Lengend Snippet: Genomic organization of SEPT9. The UCSC genome browser ( http://genome.ucsc.edu ) was used to display the location and genomic organization of SEPT9 on chromosome 17q25. (A) Genomic region covering approximately 300 kb: the eight amplicons (IDs given in red letters and within brackets) are shown after aligning their sequences using BLAST; CpG islands are shown in the track above the amplicons and gene transcripts are shown in the tracks below the amplicons. CGIs were numbered for easier reference within this manuscript. The UCSC display of transcripts is annotated with the combined IDs from Ensembl and NCBI/gene map for easier cross platform identification. (B) Detail of approximately 6 kb showing a higher resolution of the locations of the closely neighboring amplicons 4 to 7.

Article Snippet: Sections were then incubated with the primary antibody against Septin-9 (polyclonal AB cat.# PAB4799, Abnova, Germany) in 1:50 dilution for 60 minutes at 37°C, washed again in PBS, and detected with Alexa Fluor 546 dye in 1:100 dilution after 30 minutes of incubating at 37°C.

Techniques:

Journal: BMC Cancer

Article Title: Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

doi: 10.1186/1471-2407-13-398

Figure Lengend Snippet: Bisulfite DNA sequencing results for SEPT9 amplicons. Each column displays results from one amplicon and each row displays results for one cell type. Within each of the rectangular boxes, the columns correspond to single CpGs and the three rows correspond to three patients. As all amplicons are drawn to the same scale the location of the amplicons is illustrated at the bottom with arrows pointing to the genomic map (compare to Figure ). Color coding: yellow = unmethylated (0%), green = partially methylated (50%), blue = fully methylated (100%), and lighter or darker green/blue colors provide methylation levels below or above 50%. White areas = no sequencing data available. Abbreviations: str = stromal cells, epi = epithelial cells, Norm = normal colon tissue, NAT1 = normal adjacent tissue (1 cm away from tumor), NAT2 = normal adjacent tissue (> 10 cm from tumor), Adeno = adenoma tissue, pat = patient no.: only the last number of the patient ID in Additional file : Table S1 is shown here.

Article Snippet: Sections were then incubated with the primary antibody against Septin-9 (polyclonal AB cat.# PAB4799, Abnova, Germany) in 1:50 dilution for 60 minutes at 37°C, washed again in PBS, and detected with Alexa Fluor 546 dye in 1:100 dilution after 30 minutes of incubating at 37°C.

Techniques: DNA Sequencing, Amplification, Methylation, Sequencing

Journal: BMC Cancer

Article Title: Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

doi: 10.1186/1471-2407-13-398

Figure Lengend Snippet: Box plot diagrams of SEPT9 methylation. Results from amplicons 4 and 5 in different LCM tissue specimens. Data are shown separately for stromal (str) and epithelial (epi) cells and for the three patients in each group. Red = epithelial cells, blue = stromal cells. The horizontal bar within boxes shows the median, dots within the boxes the means, the lower and upper boundaries of boxes show the 25th and 75th percentiles, the whiskers are determined by the 5th and 95th percentiles.

Article Snippet: Sections were then incubated with the primary antibody against Septin-9 (polyclonal AB cat.# PAB4799, Abnova, Germany) in 1:50 dilution for 60 minutes at 37°C, washed again in PBS, and detected with Alexa Fluor 546 dye in 1:100 dilution after 30 minutes of incubating at 37°C.

Techniques: Methylation

Journal: BMC Cancer

Article Title: Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

doi: 10.1186/1471-2407-13-398

Figure Lengend Snippet: Histopathology and Septin-9 immunohistochemistry. The images for H&E staining and IHC were obtained from adjacent sections and are from the same blocks used for LCM sampling. H&E staining in the left panel shows overall tissue morphology. The three IHC images per patient are from one slide: Hoechst staining (blue) marks nuclei, AlexaFluor 546 (red) shows Septin-9 protein; the right panel shows the combination of Hoechst and AlexaFluor 546. For better visualization of the Hoechst staining, these images were obtained with intensified brightness for the blue channel. Normal sections are from BSM0451; NAT1, NAT2, and tumor are from BSM0456, and adenoma is from BSM0457. All images are at 20x magnifications; the scale bars correspond to 100 μm.

Article Snippet: Sections were then incubated with the primary antibody against Septin-9 (polyclonal AB cat.# PAB4799, Abnova, Germany) in 1:50 dilution for 60 minutes at 37°C, washed again in PBS, and detected with Alexa Fluor 546 dye in 1:100 dilution after 30 minutes of incubating at 37°C.

Techniques: Histopathology, Immunohistochemistry, Staining, Sampling

Journal: BMC Cancer

Article Title: Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

doi: 10.1186/1471-2407-13-398

Figure Lengend Snippet: Quantification of immunohistochemistry results. Expression of Septin-9 protein in stromal (blue boxes) and epithelial (red boxes) cells. The Y axis displays arbitrary units for the normalized fluorescence signals (ratio AlexaFluor 546 to Hoechst) in IHC slides. Each box plot contains multiple scanned slide sections from three patients.

Article Snippet: Sections were then incubated with the primary antibody against Septin-9 (polyclonal AB cat.# PAB4799, Abnova, Germany) in 1:50 dilution for 60 minutes at 37°C, washed again in PBS, and detected with Alexa Fluor 546 dye in 1:100 dilution after 30 minutes of incubating at 37°C.

Techniques: Immunohistochemistry, Expressing, Fluorescence