Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Caspase‐1 activation and increased ubiquitinated protein apoptosis‐associated speck‐like proteins ( ASC ) in Gaucher macrophages ( GM s). (A) ELISA or (B) Total supernatants or total protein lysates from treated macrophages from patients (P) and controls (C) were probed for IL ‐1β. (C) Control (C) and GM s (P) were immuno‐stained for LC 3 (red) and NLRP 3 (green) after treatment with lipopolysaccharide ( LPS ) (100 ng) and ATP (5 m m ) for 1 h. Cells were imaged using a confocal microscope (Z‐stack with 0.5 μm thickness). Co‐localization was evaluated using Imaris software. Merged channels are shown as yellow. Pictures represent seven independent experiments performed on cells from seven different patients. Single channels are presented in the supplements. (D) Caspase‐1 was analyzed in total lysate and supernatant by immunoblotting, after treatment with LPS alone or LPS (100 ng)+ ATP (5 m m ) or rapamycin (25 n m ). Data represent five independent experiments. (E) Total protein from control and Gaucher macrophages was immunoblotted for ASC after treatment with LPS (100 ng) and ATP (5 m m ), and then rapamycin (25 n m ), 3 MA (5 m m ) or Baf.1A (10 μ m ) for 40 min. ASC expression was normalized to β‐actin. The blot is representative of seven independent experiments performed on samples from seven different patients and controls. P < 0.05(*) and P < 0.01(**), P < 0.001(***). (F) Control and GM s were immunostained for p62 (red) and ASC (green) in the absence and presence of LPS (100 ng) and ATP (5 m m ) and then imaged by confocal microscopy. Merged channel volumes are shown in yellow, and insets showing surface renderings of three‐dimensional reconstruction using Imaris software are shown in the far right panels.

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Software, Western Blot, Expressing, Confocal Microscopy

Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Inflammasome activation due to impaired autophagy in Gaucher macrophages ( GM s) (1) In both control and GM s, lipopolysaccharide ( LPS ) priming induces activation of p65‐ NF ‐ kB , which is translocated to the nucleus, leading to production of pro‐ IL ‐1β in the cytosol. In control macrophages (1a), LPS stimulates accumulation of ubiquitinated p65‐ NF ‐ kB , which is further recognized by p62, delivered to autophagosomes, and degraded in the lysosome (1a, solid line). In GM s (1b, dashed line), impaired autophagy prevents degradation of p65‐ NF ‐ kB through autophagy and results in its activation (1b). (2) In both control and GM s, stimulation by both LPS and extracellular ATP leads to inflammasome complex formation ( NLRP 3, apoptosis‐associated speck‐like proteins ( ASC ) and caspase1) and activation. (3) Activated inflammasomes undergo ubiquitination of ASC , leading to p62‐mediated engulfment of inflammasomes by autophagosomes. Pro‐ IL ‐1β conversion to active IL ‐1β is limited due to the destruction of activated inflammasomes by autophagolysosomes. In GM s, defective autophagy and lysosomal dysfunction inhibit the elimination of active inflammasomes through autophagy, resulting in the upregulation and secretion of IL ‐1β (3).

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay

Journal: Frontiers in Oncology

Article Title: Inter and intra-tumor heterogeneity of paediatric type diffuse high-grade gliomas revealed by single-cell mass cytometry

doi: 10.3389/fonc.2022.1016343

Figure Lengend Snippet: Summary of the 15 antibodies used for the mass cytometry analysis.

Article Snippet: Anti-Human CD49c , 161Dy , Fluidigm , 3161016B , .

Techniques: Mass Cytometry

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