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

Journal: Scientific Reports

Article Title: Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout

doi: 10.1038/srep38622

Figure Lengend Snippet: ( A–C ) 293T cells were transiently transfected with the iGLuc luciferase reporter plasmid (100 ng) and expression plasmids. Luminescence derived from iGLuc activation in each sample was normalized by β-galactosidase activity transfected as an internal control in each sample. ( A ) *Significantly different from NLRP3 + ASC + caspase-1, 0.5: p = 0.0064, 1–10: p =< 0.0001. ( B ) *Significantly different from ASC + caspase-1, 0.5: p = 0.0036, 1: p = 0.0001, 5–10: p =< 0.0001. ( D ) In vitro assay for caspase-1 enzyme activity was performed using a fluorometric caspase-1 assay kit with recombinant human caspase-1 (rCaspase-1; Bio-vision) in the presence or absence of CAPE or Z-VAD-FMK according to the manufacture’s instruction. The fluorescence was recorded at 400 nm after excitation at 505 nm with SpectraMaxM5 (Molecular Devices, Sunnyvale, CA). *Significantly different from rCaspase-1 alone, p = 0.0007. The values represent the means ± SEM (n = 3).

Article Snippet: Recombinant human ASC protein(Abnova) was captured with anti-ASC antibodies via antibody-antigen binding.

Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Derivative Assay, Activation Assay, Activity Assay, Control, In Vitro, Recombinant, Fluorescence

Journal: Scientific Reports

Article Title: Targeting ASC in NLRP3 inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout

doi: 10.1038/srep38622

Figure Lengend Snippet: ( A ) The structure of biotin-tagged CA (BT-CA), biotin-tagged DMC (BT-DMC), and biotin-tagged DHC (BT-DHC). ( B ) LPS-primed BMDMs were treated with CAPE, BT-CA, BT-DMC, and BT-DHC (10 μM) for 1 hr and then stimulated with monosodium uric acid (MSU) crystals (500 μg/ml) for 6 hr. The cell culture supernatants were analyzed for secreted IL-1β using ELISA. The values represent the means ± SEM (n = 3). # Significantly different from vehicle alone, p < 0.0001. *Significantly different from MSU alone, p < 0.0001. ( C ) After BMDM cell lysates were treated with BT-CA, BT-DMC, and BT-DHC (1 μM) at room temperature for 4 hr, cell lysates were precipitated with NeutrAvidin beads and subjected to immunoblotting analysis. The amount of ASC expression in cell lysates were determined as “input”. CAPE (1 μM) was added to cell lysates treated with BT-CA. ( D ) After 293T cells were transfected with ASC-expression plasmids, the cell lysates were treated with BT-CA, BT-DMC, and BT-DHC (1 μM) at room temperature for 4 hr. The cell lysates were precipitated with NeutrAvidin beads and subjected to immunoblotting analysis. CAPE (1 μM) was added to cell lysates treated with BT-CA. ( E ) Sensograms of CAPE binding to recombinant ASC protein in the presence of detergent (0.005% Tween-20) were obtained from surface plasmon resonance (SPR) analysis. Different concentrations of CAPE are presented as an overlay plot aligned at the start of injection. ( F ) The line graph of dose-binding response unit curve and the table showing kinetic parameters of the binding between CAPE and ASC calculated using a simple 1:1 interaction model were from SPR analysis in ( E ). The maximal expected binding level (Rmax c ) was calculated by Biocore T200 evaluation software and Rmax e value was obtained from experimental maximum response unit. Veh, vehicle. P, precipitation. IB, immunoblotting. WB, western blotting.

Article Snippet: Recombinant human ASC protein(Abnova) was captured with anti-ASC antibodies via antibody-antigen binding.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Binding Assay, Recombinant, SPR Assay, Injection, Software

Journal: International Journal of Molecular Sciences

Article Title: Appraising the Role of Astrocytes as Suppliers of Neuronal Glutathione Precursors

doi: 10.3390/ijms24098059

Figure Lengend Snippet: Glutathione synthesis, export, and extracellular cleavage. The figure shows the enzymes involved in the synthesis of the glutathione reduced form (GSH) and the reduction in its oxidized form (GSSG) in the cytoplasm. Transporters carrying out the efflux to the extracellular space are placed at the plasma membrane together with enzymes involved in extracellular GSH cleavage into its constituent amino acids. The proteins involved are γ-glutamylcysteine ligase (GCL), glutathione synthetase (GSS), glutathione reductase (GSR), γ-glutamyltranspeptidase (GGT), dipeptidase (DPEP), and multidrug resistance proteins (MRPs). ATP-consuming steps are indicated with a green background on the protein’s name. Extracellular metabolites are indicated in blue. Both GSH and GSSG are exported by cells.

Article Snippet: Import of Gly into cells depends on several transporters of the ASC family, such as GLYT, B 0 AT, and VIAAT systems [ ], which, according to the Human Protein Atlas, show differential transcript levels between cell types.

Techniques: Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Appraising the Role of Astrocytes as Suppliers of Neuronal Glutathione Precursors

doi: 10.3390/ijms24098059

Figure Lengend Snippet: Schematic representation of the main sources of amino acids for glutathione synthesis. The figure shows the import of amino acids through transporters at the plasma membrane and the main pathways used for the cellular production of Cys, Glu, and Gly (green forest background) required for glutathione synthesis. For simplification, the contribution of protein degradation as an amino acid source is not shown and discontinuous lines indicate conversion through several steps in the route that are not detailed. Amino acids and peptides are depicted in a salmon background, whereas green was the color used for other metabolites. Cytoplasmic (dark blue), mitochondrial (orange), and peroxisomal (red) enzymes involved in these pathways are: AGT, alanine glyoxylate aminotransferase (EC 2.6.1.44); AHCY, adenosylhomocysteinase (EC 3.13.2.1); BHMT, betaine homocysteine methyltransferase (EC2.1.1.5); CBS, cystathionine β-synthase (EC 4.2.1.22); CTH, cystathionine γ-lyase (EC 4.4.1.1); DMGDH, dimethylglycine dehydrogenase (EC 1.5.8.4); GCL, γ-glutamylcysteine ligase (EC 6.3.2.2); GDH, glutamate dehydrogenase (EC 1.4.1.3); GLS, glutaminase (EC 3.5.1.2); GSS, glutathione synthetase (EC 6.3.2.3); MAT, methionine adenosyltransferase (EC 2.5.1.6); MTR; methionine synthase (EC 2.1.1.13); MTs, S-adenosylmethionine-dependent methyltransferases; SARDH, sarcosine dehydrogenase (EC 1.5.8.3); SHMT, serine hydroxymethyltransferase (EC 2.1.2.1); SHMT2, mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1).

Article Snippet: Import of Gly into cells depends on several transporters of the ASC family, such as GLYT, B 0 AT, and VIAAT systems [ ], which, according to the Human Protein Atlas, show differential transcript levels between cell types.

Techniques: Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Appraising the Role of Astrocytes as Suppliers of Neuronal Glutathione Precursors

doi: 10.3390/ijms24098059

Figure Lengend Snippet: Expression levels of human amino acid transporters as shown in the Human Protein Atlas. Data collected from the Human Protein Atlas refer to expression levels of amino acid transporters in astrocytes, excitatory neurons, and inhibitory neurons, although not all transcripts reach significant levels in these cell types. The figure shows a comparison of data obtained from single-cell RNA sequencing ( https://www.proteinatlas.org/ (accessed on 14 November 2022)). Both Cis and Cis transporters are expressed in astrocytes and neurons (upper panels), although higher levels for Cis transporters are found in astrocytes, and differences for Cys transporters are less pronounced but still larger for astrocytes. Similar levels of expression for different types of carriers allowing Gly or Ser transport are also reported in this database (lower panel). Expression of Gln transporters is detected both in neurons and astrocytes (left center panel), although levels in neurons are higher. Levels of Glu transporters are extraordinarily high in astrocytes compared to neurons and well above those detected among the amino acid transporters considered in the present work (right center panel). Again, transcript levels found in this database may not reflect the corresponding protein levels. The RNA nomenclature shown in the figure is that found in the database and the encoded proteins/carriers (in parenthesis) are as follows: SLC7A11 and SCL3A2 (xCT and 4F2hc subunits of the X c − antiporter, respectively); SLC1A3 (EAAT1); SLC1A2 (EAAT2); SLC1A1 (EAAT3); SLC1A6 (EAAT4); SLC1A7 (EAAT5); SCL1A4 (ASCT1); SCL1A5 (ASCT2); SLC7A9 (CSNU3); SLC7A10 (ASC-1); SLC7A13 (AGT-1); SLC6A9 (GLYT1); SLC6A5 (GLYT2); SLC6A17 (B o AT); SLC25A38 (FLJ20551); SLC32A1 (Gly export system VIAAT); SLC38A1 (ATA1) and SLC38A2 (ATA2); SLC38A3 (SN1); SLC38A4 (ATA3); SLC38A5 (SN2); SLC38A7 (FLJ10815); SLC25A12 (ARALAR); SLC25A22 (GC1); and SLC66A4 (lysosomal CTNS Cis carrier).

Article Snippet: Import of Gly into cells depends on several transporters of the ASC family, such as GLYT, B 0 AT, and VIAAT systems [ ], which, according to the Human Protein Atlas, show differential transcript levels between cell types.

Techniques: Expressing, Comparison, RNA Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Appraising the Role of Astrocytes as Suppliers of Neuronal Glutathione Precursors

doi: 10.3390/ijms24098059

Figure Lengend Snippet: Schematic representation of the differences in astrocytic and neuronal expression and uptake through amino acid transporters. Data collected from the Human Protein Atlas and the literature show differences in the expression and activity of transporters involved in the uptake of amino acids required for glutathione synthesis between neurons and astrocytes. These results are summarized in the figure, assuming the existence of a correlation between transcript and protein levels. Abbreviations shown correspond to protein nomenclature and specific carriers found in inhibitory neurons are labeled in red font. The font size attempts to reflect the comparative expression levels of the carriers and the glutathione concentrations, while the width of the arrows simulates the diverse uptake rates estimated in the literature. Discontinuous lines are used to depict the existence of several steps in a specific pathway. The lack of transsulfuration in neurons reported in the literature is indicated by a red cross.

Article Snippet: Import of Gly into cells depends on several transporters of the ASC family, such as GLYT, B 0 AT, and VIAAT systems [ ], which, according to the Human Protein Atlas, show differential transcript levels between cell types.

Techniques: Expressing, Activity Assay, Labeling

Journal: Frontiers in Immunology

Article Title: Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

doi: 10.3389/fimmu.2023.1214289

Figure Lengend Snippet: Some experimental studies reporting expression and function of the NLRP3 inflammasome in non-myeloid cells.

Article Snippet: The Human Protein Atlas reports transcript expression under basal conditions in endothelial cells from different organs that do not appear to express relevant levels of NLRP3 mRNA , although they do express ASC and CASP1 .

Techniques: Expressing, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis

doi: 10.3389/fimmu.2023.1214289

Figure Lengend Snippet: Inflammasome-dependent and –independent roles of NLRP3 in myeloid cells. Left: Canonical activation of NLRP3 starts with TLR activation, mediate by Myd88 and NF-κB, which triggers the expression of NLRP3, pro-IL-1β and -IL-18. Several stimuli can trigger the inflammasome assembly, such as K + efflux, Ca +2 influx, crystals, phagosome rupture, mtROS and mtDNA. The conjunction of NLRP3, ASC and Caspase 1 lead to Caspase 1 activation and proteolytic cleavage of pro-IL-1β and -IL-18, as well GSDMD resulting in IL-1β and -IL-18 secretion and/or pyroptosis. Right: Non-canonical NLRP3 activation is triggered by TLR activation, via Myd88 and TRIF, or type I interferon receptor (IFRA), which lead to NF-κB activation and induction of NLRP3, pro-IL-1β and -IL-18 expression. The type I interferon triggers caspase-11, and caspase 1 expression. LPS and LPG can bind and directly activate caspase 11, which cleaves GSDMD in GSDMD-N driving pyroptosis. Nlrp3 can act as a transcription factor in T cells by binding directly to the Il4 , Il13 , Icos , Maf , Il1r1 promoter region. Nlrp3 can induce phosphorylation of Smad2/4 in the TGFβR pathway, thereby regulating the transcription of target genes. Created with BioRender.com .

Article Snippet: The Human Protein Atlas reports transcript expression under basal conditions in endothelial cells from different organs that do not appear to express relevant levels of NLRP3 mRNA , although they do express ASC and CASP1 .

Techniques: Activation Assay, Expressing, Binding Assay, Phospho-proteomics