Journal: The Journal of biological chemistry

Article Title: Cryopyrin-induced interleukin 1beta secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC.

doi: 10.1074/jbc.M401178200

Figure Lengend Snippet: FIG. 4. Cryopyrin requires oligomerization for ASC binding and IL-1 secretion. A, schematic representation of the cryopyrinPD-Fpk3 or Fpk3 vector. B, 1 108 THP-1 cryopyrinPD-Fpk3 and Fpk3 stable cells were immunoprecipitated (IP) with polyclonal myc Ab (top panel), and total lysates (bottom panel) from stable cells were immunoblotted with monoclonal ASC Ab. C, 0.5 106 THP-1 cells stably expressing with cryopyrinPD-Fpk3 or Fpk3 vector in the presence () or absence () of Fpk3 ligand AP1510 for 0, 4, and 8 h followed by detection of IL-1 secretion. The results are given as the mean S.D. of triplicate cultures.

Article Snippet: The antibodies used for immunoprecipitation and immunodetection were anti-FLAG monoclonal (Santa Cruz), anti-Myc polyclonal (Santa Cruz), anti-ASC polyclonal ( ProSci), anti-ASC monoclonal (21), and anti-tubulin monoclonal (Sigma).

Techniques: Binding Assay, Plasmid Preparation, Immunoprecipitation, Stable Transfection, Expressing

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. ASC/TMS1 was frequently silenced or reduced in RCC cell lines by promoter hypermethylation. HEK293, normal human embryonic kidney cell line. M, methylated. U, unmethylated. B. Pharmalogic demethylation with 5-Aza alone or combined with trichostatin A (A + T) restored ASC/TMS1 mRNA expression and induced its demethyation in RCC cell lines. C. Methylation status of individual CpG sites in the ASC/TMS1 promoter was confirmed by bisulfite genomic sequencing. Each row represents one bacterial clone with one circle symbolizing one CpG site. Filled ovals indicate methylated. Open ovals indicate unmethylated. D. Immunofluorescence staining of ASC/TMS1 protein in 786-O cells. Pharmalogic demethylation with 5-Aza alone or combined with trichostatin A (A + T) restored ASC/TMS1 protein expression in 786-O cells. Green pellet in the cytoplasm and nucleus represents positive staining (indicated by arrows).

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Methylation, Expressing, Genomic Sequencing, Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. The mRNA expression levels of ASC/TMS1 in paired primary RCC tissues as determined by quantitative real-time PCR. ASC/TMS1 mRNA was significantly downregulated in RCC samples compared with their adjacent normal tissues ( p = 0.0001). B. Representative immunohistochemical staining of a pair of RCC specimens and corresponding nontumor tissues. In adjacent nontumor tissues, intense immunostaining for ASC/TMS1 was detected in a cytoplasmic and nuclear distribution, whereas absent/weak immunostaining was observed in the cytoplasm and nucleus of tumor tissues. C. Evaluation and statistical analysis of ASC/TMS1 protein expression in 67 paired primary RCC tissues. ASC/TMS1 protein expression was significantly downregulated in RCC samples compared with adjacent normal tissues ( P < 0.0001).

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Immunostaining

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. ASC/TMS1 methylation in primary RCC. M, methylated; U, unmethylated. B. ASC/TMS1 methylation in paired RCC (T) and matched normal renal tissue (N) samples. C. Methylation status of ASC/TMS1 was confirmed by bisulfite genomic sequencing (BGS). Each row represents one bacterial clone with one circle symbolizing one CpG site. Filled ovals indicate methylated. Open ovals indicate unmethylated.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Methylation, Genomic Sequencing

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: Association between ASC/TMS1 methylation and clinicopathological features of patients with RCC

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Methylation

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. Ectopic expression of ASC/TMS1 protein was confirmed by western blot. B. Cell growth curve was inhibited by ASC/TMS1 in 786–0 and A498 cells. C. ASC/TMS1 suppressed colony formation in 786–0 and A498 cells. D. ASC/TMS1 causes cell cycle arrest in G0/G1 phase. E. Re-expression of ASC/TMS1 suppresed the protein expression of CyclinD1 and PCNA in 786–0 and A498 cells. F. ASC/TMS1 knockdown by si-ASC/TMS1 increased cell growth ability in a normal HEK293. The data are means ± s.d. of three separate experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. The migration of 786–0 cells in wound healing experiment. Ectopically expressed ASC/TMS1 suppressed RCC cell migration in 786-O cells. Photographs were taken at 0, 24 and 36 h to determine the different mobility between 786–0/control and 786–0/ASC/TMS1. B. Cell invasion of 786–0 and A498 cells in matrigel assay. ASC/TMS1 suppressed RCC cell invasion in 786–0 and A498 cells. The data are means ± s.d. of three separate experiments. * P < 0.05; and ** P < 0.01.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Migration, Matrigel Assay

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. Flow cytometry assay with Annexin V:PE and 7AAD double staining. B1. Overexpression of ASC/TMS1 induced the protein expression of cleaved caspase-8, cleaved caspase-9, cleaved PARP in 786–0 and A498 cells by western blot. B2. Ectopic expression of ASC/TMS1 enhanced the protein expression of p-p53 and p21 using western blot. GAPDH was used as an internal control. C. Knockdown of ASC/TMS1 reduced the protein expression of cleaved caspase-8, cleaved caspase-9, cleaved PARP in 786–0 and A498 cells by western blot. * P < 0.05; and *** P < 0.001.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Flow Cytometry, Double Staining, Over Expression, Expressing, Western Blot

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A1. Subcutaneous tumor growth curve of ASC/TMS1-expressing 786–0 cells in SCID mice was compared with control empty vector transfected cells. A2. A representative picture of tumor growth in SCID mice subcutaneously inoculated with ASC/TMS1 or control vector. A3. Pictures of the isolated tumors from each mouse ( n = 5/group). A4. Histogram represents mean of the tumor weight from the ASC/TMS1 and control vector groups. Data are mean ±SD. B1. Immunohistochemical staining of ASC/TMS1 in xenograft tumors in SCID mice. Brown cytoplasmic and nuclear signals indicate the ASC/TMS1 protein expression. B2. Representative ki-67 staining of xenografted tumor derived from 786–0 cells transfected with ASC/TMS1 or control vector. An decrease in the number of Ki-67-positive cells (brown-stained nuclei) is evident in ASC/TMS1-transfected tumors. ** P < 0.01.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Plasmid Preparation, Transfection, Isolation, Immunohistochemical staining, Staining, Derivative Assay

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: A. 786–0 cells with or without 5-Aza-priming (10 μm, 72 h) were treated with etoposide or doxorubicin at the indicated concentration for additional 36 h. 5-Aza-primed 786–0 cells showed a significant decrease in cell survival compared with unprimed 786–0 cells, as revealed by the CCK8 assay. B. 786–0-control or 786–0-ASC/TMS1 cells were treated with etoposide or doxorubicin at the indicated concentration for 36 h. Cell death was significantly higher in the 786–0-ASC/TMS1 cells than that in the control 786–0 cells, as revealed by the CCK8 assay. C. Caki-2 cells were transfected with si-control or si-ASC/TMS1 RNA (50 nM), and after 48 h of transfection, cells were treated with etoposide (50 uM) or doxorubicin (1 uM) and analyzed by Western blot for the indicated antibodies.

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Concentration Assay, CCK-8 Assay, Transfection, Western Blot

Journal: Oncotarget

Article Title: Epigenetic inactivation of the candidate tumor suppressor gene ASC/TMS1 in human renal cell carcinoma and its role as a potential therapeutic target

doi:

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: The siRNA specific for ASC/TMS1 and its control siRNA were purchased from OriGene (Rockville, MD, USA).

Techniques: Sequencing