Journal: The Journal of Biological Chemistry

Article Title: NLRC3 protein inhibits inflammation by disrupting NALP3 inflammasome assembly via competition with the adaptor protein ASC for pro-caspase-1 binding

doi: 10.1074/jbc.M116.769695

Figure Lengend Snippet: Overexpression of NLRC3 inhibits NALP3-induced IL-1β secretion. A and B, IL-1β secretion in ectopically assembled NALP3 inflammasomes in HEK293FT cells transfected with 500 ng of pro-IL-1β, 250 ng of ASC, and 250 ng of pro-caspase-1 plasmids and the indicated amount of NALP3 (A) or 1 μg of NALP3 and the indicated amount of NLRC3 (B). Each condition was duplicated. Secreted IL-1β was quantified 24 h after transfection in cell-free supernatants by ELISA. C, IL-1β cleavage in HEK293FT cells with ectopically assembled NALP3 inflammasomes. NT, non-transfected. D, NLRC3 mRNA levels in the THP-1 OvCont and OvNLRC3 cell lines. Quantitative PCR results of relative mRNA of NLRC3/β-actin are given. The NLRC3/β-actin ratio of OvCont cells was assigned as 1 arbitrary unit. E, NLRC3 protein levels in THP-1 OvControl and OvNLRC3 stable lines. F and G, IL-1β secretion in THP-1 OvNLRC3 and THP-1 OvControl stable lines in response to nigericin (F) and MSU (G) treatment.

Article Snippet: Prof. Ömer Yilmaz, Broad Institute, Massachusetts Institute of Technology, Boston, MA) or newly generated pLenti-CMV-Blast-NLRC3 (OvNLRC3) vectors and their control pLenti-CMV-Blast (named OvCont) together with the helper plasmids pMD2G and psPAX2.

Techniques: Over Expression, Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Identification of candidate driver genes on chromosome 7p in colorectal cancer (CRC). A, Left, GET4 mRNA expression in 623 CRC tissues and 51 normal colon tissues obtained from The Cancer Genome Atlas (TCGA) dataset. Middle, GET4 mRNA expression in 17 CRC tissues and paired normal colon tissues in the GSE 32323 dataset. Right, GET4 mRNA expression assessed by RT‐quantitative PCR in 122 CRC tissues and paired normal colon tissues in our dataset. * P < .05. B, Immunohistochemical staining for GET4. Scale bar, 200 μm (left) and 50 μm (right). N, normal tissue; T, tumor tissue. C, Left, The frequency of mutations was 1.5% in GET4 among CRC cases in the COSMIC dataset. Middle, Status of GET4 in 613 CRC tissues in TCGA dataset. Right, Status of GET4 in 60 CRC tissues in the CCLE dataset. Gained, log 2 copy number ratios >0.1; Lost, log 2 copy number ratios <−0.1; Not called, −0.1 < log 2 copy number ratios < 0.1. D, Left, Correlation between GET4 copy number and GET4 mRNA expression in the TCGA dataset. Right, Correlation between GET4 copy number and GET4 mRNA expression in the CCLE dataset. r , Pearson’s correlation coefficient. E, Left, Overall survival rate in CRC patients according to GET4 mRNA expression in tumor tissues in our dataset. Right, Overall survival rate in CRC patients according to GET4 mRNA expression in tumor tissues in the TCGA cohort using the LinkedOmics database. F, BAG6 mRNA expression between 623 CRC tissues (T) and 51 normal colon tissues (N) in the TCGA dataset. n.s., not significant. G, Immunohistochemical staining for BAG6 in the normal colon (N) or tumor tissues (T). Scale bar, 200 μm (left) and 50 μm (middle and right). The bar graph shows the nuclear / cytoplasmic ratio of BAG6 intensity in the normal colon or tumor tissues. More than 200 cells were counted in each. * P < .05. H, Overall survival rate in CRC patients according to BAG6 mRNA expression in tumor tissues in the TCGA cohort using the LinkedOmics database. n.s., not significant. I, Left, The frequency of mutations was 3.7% in BAG6 among CRC cases in the COSMIC dataset. [Correction added on 18 December 2021, after first online publication: In Figure 1I caption, the dataset ‘TCGA’ in Left was corrected to ‘COSMIC’ in this version.] Middle, Status of BAG6 in 615 CRC tissues in the TCGA dataset. Right, Status of BAG6 in 60 CRC tissues in the CCLE dataset. Gained, log 2 copy number ratios >0.1; Lost, log 2 copy number ratios <−0.1; Not called, −0.1 < log 2 copy number ratios < 0.1

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

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

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Univariate and multivariate analyses of clinicopathologic factors affecting overall survival in study patients with colorectal cancer

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Expressing

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Association between GET4 mRNA expression of tumor tissues and clinicopathologic factors in colorectal cancer

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Expressing

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Effects of GET4 KO on cell proliferation in colorectal cancer (CRC) cells. A, Left, Direct sequencing analysis confirmed successful genome editing of GET4 exon 1. Middle, RT‐PCR of gene‐targeted GET4 . WT, 2100 bp; KO, approximately 1300 bp. Right, Immunoblotting for GET4 in WT cells and GET4 KO cells. B, Colony formation assays using GET4 KO SW620 and SW480 cells. C, Left, GET4 protein levels in the indicated cells. Immunoblotting for GET4 in these rescued cells. Right, Colony formation assays using indicated cells. D, In vivo analysis using xenograft mouse models. Tumor size using WT cells or GET4 KO CRC cells. E, Immunohistochemical staining for GET4 or Ki‐67 in tumor tissues from WT cells and GET4 KO CRC cells. These pairs were compared with the most intensely stained areas in each. Scale bar, 50 μm. * P < .05

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, In Vivo, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Effects of GET4 KO on the cell cycle in colorectal cancer (CRC) cells. Knockout of GET4 suspended cell cycle progression of CRC cells. A, Cell cycle assay of WT cells and GET4 KO cells after serum starvation for 96 h. Propidium iodide (PI) staining was carried out after refeeding of FBS for the indicated time periods. Bar graphs represent the fold change in the proportion of S‐phase distribution. B, Representative flow cytometry plots of EdU incorporation in GET4 KO cells and WT cells. Bar graphs represent the proportion of S‐phase distribution. * P < .05

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Knock-Out, Cell Cycle Assay, Staining, Flow Cytometry

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: GET4 regulates the intracellular localization of BAG6 and the acetylation of p53 in colorectal cancer (CRC) cells. A, Knockout of GET4 increased nuclear localization of BAG6. More than 200 cells were counted in each, and bar graphs show the nuclear / cytoplasmic ratio of BAG6 intensity. * P < .05. B, Immunoblotting for p53 and p53 (ace‐lys373) in WT cells and GET4 KO CRC cells

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Knock-Out, Western Blot

Journal: Cancer Science

Article Title: GET4 is a novel driver gene in colorectal cancer that regulates the localization of BAG6, a nucleocytoplasmic shuttling protein

doi: 10.1111/cas.15174

Figure Lengend Snippet: Knockout of GET4 upregulates p21 expression in colorectal cancer (CRC) cells. A, Left, RT‐quantitative PCR for p21 mRNA expression normalized to 18s in WT cells and GET4 KO cells. * P < .05. Right, Immunoblotting for p21 in WT cells and GET4 KO cells that were treated with doxorubicin (DOX, 0.5 μg/mL) and extracted at different time points. B, Schema indicating how GET4 promotes cell cycle progression and tumor proliferation

Article Snippet: Rescue studies were carried out using pLenti‐GIII‐CMV‐ GET4 vector (Applied Biological Materials).

Techniques: Knock-Out, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biomedicines

Article Title: Identification of DDX60 as a Regulator of MHC-I Class Molecules in Colorectal Cancer

doi: 10.3390/biomedicines10123092

Figure Lengend Snippet: The different fractions between normal tissues and cancerous tissues (from the Proteogenomic Analysis of Human Colon Cancer Study [ 38 ] for the 35 potential candidates correlated with MHC-I and IFN-γ expression. The genes highlighted in yellow are the final candidates.

Article Snippet: To knockdown the gene, 3 × 10 5 cancer cells were seeded in 6-well plate format, allowed to equilibrate overnight, and then treated with siRNA of target genes (10 µM) or nontarget control for 72 h. DDX60 overexpression was established using pLenti-GIII-CMV vector carrying DDX60 cDNA obtained from Applied Biological Materials Inc. (Richmond, BC, Canada, Cat# 178610610196).

Techniques: Expressing

Journal: Biomedicines

Article Title: Identification of DDX60 as a Regulator of MHC-I Class Molecules in Colorectal Cancer

doi: 10.3390/biomedicines10123092

Figure Lengend Snippet: The expression of DDX60 is correlated with MHC-I expression in CRC. ( A ) Linear regression analysis showing the correlation between DDX60 expression levels (log2) and MHC-I-correlated molecules expression levels (log2) based on mRNA sequencing data of 459 TCGA CRC samples. ( B ) Western blotting images showed the protein levels of DDX60 and Actin in six different CRC cell lines.

Article Snippet: To knockdown the gene, 3 × 10 5 cancer cells were seeded in 6-well plate format, allowed to equilibrate overnight, and then treated with siRNA of target genes (10 µM) or nontarget control for 72 h. DDX60 overexpression was established using pLenti-GIII-CMV vector carrying DDX60 cDNA obtained from Applied Biological Materials Inc. (Richmond, BC, Canada, Cat# 178610610196).

Techniques: Expressing, Sequencing, Western Blot

Journal: Biomedicines

Article Title: Identification of DDX60 as a Regulator of MHC-I Class Molecules in Colorectal Cancer

doi: 10.3390/biomedicines10123092

Figure Lengend Snippet: DDX60 regulates the expression of MHC-I molecules in CRC cells. ( A , B ). Western blotting images showed the expression of DDX60, B2M, and Actin in vector control or DDX60-overexpressed DLD-1 ( A ) and HCT-15 ( B ). ( C – F ) FACS analysis of MHC-I expression in vector control or DDX60-overexpressed DLD-1 ( A ) and HCT-15 ( B ) with/without IFNγ treatment. ( G ) Western blotting images showed the expression of DDX60, B2M, and Actin in NCI-H747 cells treated with siRNA of nontarget control (siCTL) or DDX60 for 48 h. ( H , I ) FACS analysis of MHC-I expression in control (siCTL) and DDX60-Knockdown NCI-H747 cells with/without IFNγ stimulation. p values were calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. ns: p ≥ 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: To knockdown the gene, 3 × 10 5 cancer cells were seeded in 6-well plate format, allowed to equilibrate overnight, and then treated with siRNA of target genes (10 µM) or nontarget control for 72 h. DDX60 overexpression was established using pLenti-GIII-CMV vector carrying DDX60 cDNA obtained from Applied Biological Materials Inc. (Richmond, BC, Canada, Cat# 178610610196).

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: Biomedicines

Article Title: Identification of DDX60 as a Regulator of MHC-I Class Molecules in Colorectal Cancer

doi: 10.3390/biomedicines10123092

Figure Lengend Snippet: DDX60 is downregulated in CRC and its expression is associated with patient prognosis. ( A , B ). Immunohistochemical staining images showing the expression level of DDX60 in normal and cancerous tissue. Data were obtained from Protein Atlas ( https://www.proteinatlas.org/ , accessed on 14 January 2022) ENSG00000137628-DDX60/pathology/colorectal+cancer. ( B ) Kaplan–Meier survival curves showing the survival probability of CRC patients with high or low DDX60 levels. Data were obtained from TGCA.

Article Snippet: To knockdown the gene, 3 × 10 5 cancer cells were seeded in 6-well plate format, allowed to equilibrate overnight, and then treated with siRNA of target genes (10 µM) or nontarget control for 72 h. DDX60 overexpression was established using pLenti-GIII-CMV vector carrying DDX60 cDNA obtained from Applied Biological Materials Inc. (Richmond, BC, Canada, Cat# 178610610196).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Biomedicines

Article Title: Identification of DDX60 as a Regulator of MHC-I Class Molecules in Colorectal Cancer

doi: 10.3390/biomedicines10123092

Figure Lengend Snippet: DDX60 expression is correlated with immune cell infiltration and immunotherapy response. ( A – C ) The correlation analysis of DDX60 expression and immune cell infiltration of dendritic cells (DCs), CD8+ T cells, and CD4+ T cells based on TCGA CRC RNA-seq data. ( D – F ) Immune cell infiltration of dendritic cells (DCs), CD8+ T cells, and CD4+ T cells in CRC tissues with high or low DDX60 expression. ( G ) The mRNA expression levels (fold change of Log2) of DDX60 in the tumors from the immunotherapy responders and non-responders in different solid cancer. The gene expression data were obtained from Tumor Immunotherapy Gene Expression Resource (TIGER) at http://tiger.canceromics.org/#/home accessed on 10 May 2022.

Article Snippet: To knockdown the gene, 3 × 10 5 cancer cells were seeded in 6-well plate format, allowed to equilibrate overnight, and then treated with siRNA of target genes (10 µM) or nontarget control for 72 h. DDX60 overexpression was established using pLenti-GIII-CMV vector carrying DDX60 cDNA obtained from Applied Biological Materials Inc. (Richmond, BC, Canada, Cat# 178610610196).

Techniques: Expressing, RNA Sequencing Assay