Journal: Human genetics

Article Title: MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract

doi: 10.1007/s00439-019-02095-5

Figure Lengend Snippet: Literature analysis of top 150 lens-enriched proteins with regards to eye/lens defects

Article Snippet: The following primary antibodies were purchased from Abcam and Proteintech and used in the given dilutions in the blocking buffers: Eml2 (13529-1-AP, 1:25 diln.), Igfbp7(13529-1-AP, 1:25 diln.), Nol3(13529-1-AP, 1:25 diln.) and Slc7a5(13752-1-AP, 1:25 diln.).

Techniques: Expressing, Variant Assay

Journal: Human genetics

Article Title: MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract

doi: 10.1007/s00439-019-02095-5

Figure Lengend Snippet: Immunofluorescence using rabbit primary antibodies demonstrates robust expression of select MS/MS predicted proteins in the mouse embryonic day E14.5 lens. The proteins confirmed to be expressed (green) in the lens were Eml2 (Echinoderm microtubule associated protein like 2), Igfbp7 (Insulin-like growth factor binding protein 7), Nol3 (Nucleolar protein 3) and Slc7a5 (Solute carrier family 7 member 5). DNA was stained by DAPI. Lens epithelium (e) and fiber cells (f) are indicated. Scale bar represents 50 µm.

Article Snippet: The following primary antibodies were purchased from Abcam and Proteintech and used in the given dilutions in the blocking buffers: Eml2 (13529-1-AP, 1:25 diln.), Igfbp7(13529-1-AP, 1:25 diln.), Nol3(13529-1-AP, 1:25 diln.) and Slc7a5(13752-1-AP, 1:25 diln.).

Techniques: Immunofluorescence, Expressing, Tandem Mass Spectroscopy, Binding Assay, Staining

Journal: Human genetics

Article Title: MS/MS in silico subtraction-based proteomic profiling as an approach to facilitate disease gene discovery: application to lens development and cataract

doi: 10.1007/s00439-019-02095-5

Figure Lengend Snippet: Identification of lens-enriched membrane proteins in mouse

Article Snippet: The following primary antibodies were purchased from Abcam and Proteintech and used in the given dilutions in the blocking buffers: Eml2 (13529-1-AP, 1:25 diln.), Igfbp7(13529-1-AP, 1:25 diln.), Nol3(13529-1-AP, 1:25 diln.) and Slc7a5(13752-1-AP, 1:25 diln.).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 1. RNASET2 negatively regulates HCC invasion and metastasis: A) Levels of RNASET2 in 10 pairs of HCC tumor tissues (T) and matched adjacent noncancerous tissues (A) were determined by WB (n = 10). B) Representative IHC staining images of RNASET2 in tumor tissues and matched adjacent noncancerous tissues of HCC patients (n = 12). Scale bars, 50 μm. C) Detection of RNASET2 levels in DEN-induced mouse HCC tumor tissues and normal mouse liver tissues by WB (n = 5). D) ELISA test of soluble RNASET2 levels in plasma samples of HCC patients (n = 30) and healthy control group (n = 7). E) PLC/PRF/5 cells were treated with the indicated concentrations of TGF-𝛽1 for 48 h. The expression of RNASET2 and EMT-related proteins (N-cadherin, E-cadherin, MMP9, MMP2, and Vimentin) were measured by WB (n = 3). F) Determination of EMT-related proteins (N-cadherin, E-cadherin, MMP9, MMP2, and Snail) in RNASET2KO HuH-7 (n = 4) and PLC/PRF/5 (n = 3) monoclonal cell lines utilizing WB. G-I) Migration and

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Expressing, Migration

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 2. RNASET2 deficiency leads to cholesterol accumulation in HCC cells: A) GO analysis of HuH-7 cells. Data were defined as log2 (fold enrichment; RNASET2KO versus scrambled control; n = 2). B,C) The Biological Process (B) and KEGG (C) analysis of all differentially expressed proteins in proteomic data. Data were defined as log2 (fold enrichment; RNASET2KO versus scrambled; n = 2). D) Heatmap of significantly changed proteins associated with cholesterol metabolism in proteomic data. The protein levels were normalized as log2 (fold change; RNASET2KO versus scrambled; n = 2). E,F) WB analysis of the levels of ABCA1, SREBP1, SREBP2, HMGCR, APOE, and APOA1 in RNASET2KO and scrambled control HuH-7 (E) and PLC/PRF/5 (F) cells (n = 3). G) WB analysis of the levels of ABCA1, SREBP2, and APOA1 in scrambled, RNASET2KO, and RNASET2KO/ OE-RNASET2 HuH-7 cells. H,I) The levels of free cholesterol in RNASET2KO HuH-7 (H) (n = 3) and PLC/PRF/5 (I) (n = 4) cells were stained with Filipin III and tested by flow cytometry.

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Control, Staining, Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 3. Cholesterol was required for RNASET2KO-mediated migration and invasion of HCC cells: A-C) Representative images of migration (A,B) and invasion (C) assays of RNASET2 ablation HuH-7 cells treated in the presence or absence of exogenous cholesterol (n = 3). D,E) MHCC97-H cells intravenously inoculated nude mice were fed with regular chow or 1.25% cholesterol-supplemented diet for 6 weeks. Macroscopic images of lung metastatic foci (D) (n = 5). Representative H&E staining of lung metastasis (E) (n = 5). The zoomed images are shown below and the scale bars represented 400 μm. F,G) WB analysis of EMT-related proteins (MMP2, and Snail) in scr- and RNASET2KO HuH-7 cells treated with 10 μM simvastatin (F) and 3 μM lovastatin (G) for 48 h (n = 3). H-K) Representative images of trans-well migration (H,J) and invasion (I,K) assays of RNASET2KO HuH-7 cells supplemented with or without simvastatin (H,I) and lovastatin (J,K) for 48 h (n = 3). The distance of the wound gaps was determined using ImageJ. The wound width was normalized against that at time 0 h. Quantification was shown right. Scale bars for wound healing assays: 200 μm, for trans-well assays: 400 μm. Data are presented as mean ± SD. *p < 0.05, **p <0.01, ***p <0.001,****p <0.0001, ns = not significant by two-tailed unpaired Student’s t-test (A-C and H-K).

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Migration, Staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 4. UGT1A1 downregulation is involved in RNASET2 deficiency-induced cholesterol accumulation. A,B) Heatmap (A) and volcano plot (B) of RNA-seq data for differentially expressed genes after RNASET2 elimination. Differentially expressed genes were defined as genes with p < 0.01 and log2 (fold change) more (less) than 1 (−1) (scrambled versus RNASET2KO; n = 3). C,D) GO analysis (C) and KEGG analysis (D) depicting the top enriched pathways based on the significantly altered genes derived from RNA-seq data (n = 3). E,F) LC-MS assays indicating the levels of pyrimidine salvage biosynthesis related metabolites uridine, UMP, UDP, and UTP (E), and glucuronate metabolism related metabolites G1P, UDPG, and UDPGA (F) of RNASET2KO HuH-7 cells (n = 6). G) qRT-PCR assessed mRNA levels of enzymes involved in glucuronate metabolism of RNASET2KO HuH-7 cells (n = 3). H) WB analysis of UGT1A1 level in RNASET2KO HuH-7 cells (n = 3). I) LC-MS measured the levels of bile acids CA, DCA, and glucuronidation-

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: RNA Sequencing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Quantitative RT-PCR

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 5. RNASET2 suppresses the activation of MET signaling: A) Human phospho-RTK antibody array analysis in scr- or RNASET2KO-HuH-7 cells (n = 1). B) Immunofluorescent staining of MET in scr- or RNASET2KO-HuH-7 cells. Bars represent 20 μm (n = 3). C) WB assays of MET level in scr- or RNASET2KO-HuH-7 cells (n = 5). D) MS/MS validation of MET. The fragmentation spectrum of the tryptic MET peptide -GPVLEHPDCFPCQDCSSK- was shown with annotated y-ions (blue) and b-ions (red) (n = 2). E) Scatter plot showing the rank of differentially expressed proteins in RNASET2 deletion versus scrambled HuH-7 cells (n = 2). The protein levels were defined as log2 (fold change) more (less) than 1 (-1). F) WB analysis of the levels of RNAST2, MET, ERK1/2 and pERK1/2 in multiple liver cancer cells (n = 3). G-I) Roles of RNASET2 loss- or gain-of-function in the levels of the indicated proteins in HuH-7 (G), PLC/PRF/5 (H) and MHCC97-H (I) cells were individually identified by WB (n = 2 to 5). J) RNASET2 expression was rescued in RNASET2KO HuH-7 cells, then the levels of the indicated proteins were evaluated (n = 3).

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Activation Assay, Ab Array, Staining, Tandem Mass Spectroscopy, Biomarker Discovery, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 7. RNASET2 deficiency caused reduction of UGT1A1 promoted HCC metastasis: A) Cholesterol levels of RNASET2KO HuH-7 cells incubated with uridine (200 μM) for 48 h were measured with filipin III staining. Quantification was shown right (n = 3). B) WB assays of the indicated proteins in Scr- and RNASET2KO HuH-7 cells cultured with or without uridine for 48 h. C) The levels of MET phosphorylation and EMT makers production in RNASET2KO HuH-7 cells transfected with OE-UGT1A1 (n = 3). D,E) The mRNA (D) and protein (E) levels of UGT1A1 in shRNA transfected HuH-7 cells (n = 3). F,G) Representative images of migration (F) and invasion (G) of the indicated subgroups. Quantification was shown right (n = 3). Scale bars for trans-well assays: 400 μm. H,I) Representative images of migration (H) and invasion (I) in the control and shUGT1A1 HuH-7 cells. Quantification was shown on the right panel (n = 3). Scale bars for trans-well assays: 400 μm. Data are presented as mean ± SD. *p < 0.05, **p <0.01, ***p <0.001, ****p <0.0001 by two-tailed unpaired Student’s t-test (A,D, and F-I).

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Incubation, Staining, Cell Culture, Phospho-proteomics, Transfection, shRNA, Migration, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: RNASET2 Deficiency Induces Hepatocellular Carcinoma Metastasis through Cholesterol-Triggered MET Activation.

doi: 10.1002/advs.202411888

Figure Lengend Snippet: Figure 8. Targeting MET attenuates RNASET2 deletion-mediated HCC metastasis: A) the level of RNASET2 in tumor tissues versus adjacent tissues of HCC patients from GEO data (GSE76427). B) The survival probability of patients with low and high RNASET2 expression in GSE76427 data. C) Representative IHC staining images of RNASET2 and MET in tumor tissues and matched adjacent noncancerous tissues of HCC patients (n = 5). Scale bars, 50 μm. D-F) Macroscopic views (D), lung weight (E), and metastatic foci number (F) of the mouse models i.v. injected with scrambled control, RNAST2KO, scrambled-shMET, and RNAST2KO-shMET HuH-7 cells (n = 5). G) H&E staining of lung metastasis of the indicated groups. The images shown in the lower panel were with higher magnification (n = 5). Scale bars: 400 μm. H) Survival of lung metastatic mouse model triggered by the labeled groups (n = 8). I–K) Macroscopic views (I), lung weight (J), and metastatic foci number (K) of the lung metastasis mouse model groups: scrambled,

Article Snippet: Afterwards, the slices were stained with RNASET2 antibody (13753-1-AP, Proteintech).

Techniques: Expressing, Immunohistochemistry, Injection, Control, Staining, Labeling

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: The expression of NPAS1 in COAD tissues. ( A) In 24 different cancer types in UALCAN database, the upregulation and downregulation level of NPAS1. **p < 0.01, ***p < 0.001, NA p>0.05. ( B ) The expression level of NPAS1 in COAD based on sample types. ( C ) The expression level of NPAS1 in COAD based on histological subtypes. ( D ) The expression level of NPAS1 in COAD based on TP53 mutant status. ( E ) The expression level of NPAS1 in COAD in GSE44076 gene expression profile. ( F ) The expression level of NPAS1 in COAD in GSE74602 gene expression profile. ( G ) The ROC curve was generated to assess the value of NPAS1 in identifying COAD tissues. ( H ) The expression level of NPAS1 in 10 pairs of COAD tissues and adjacent normal tissues was detected by WB. ( I ) The expression level of NPAS1 in a pair of COAD tissue and adjacent normal tissue was detected by IHC staining.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Expressing, Mutagenesis, Gene Expression, Generated, Immunohistochemistry

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: Kaplan-Meier survival plots were used to compare the low and high expression of NPAS1 in COAD. ( A ) OS survival curve of patients with COAD between high and low expression of NPAS1. ( B and C ) OS survival curves of stage I and stage II between NPAS1-high and -low patients with COAD. ( D ) OS survival curve of stage N0 between NPAS1-high and -low patients with COAD. ( E ) OS survival curve of female between NPAS1-high and -low patients with COAD. ( F ) OS survival curve of MSI stable between NPAS1-high and -low patients with COAD.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: Enrichment of biofunction and associated genes analysis of NPAS1 in COAD. ( A and B ) The GOKEGG analysis of NPAS1. ( C – H ) The GSEA analysis of NPAS1.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques:

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: Correlation between NPAS1 expression and immune characteristics. ( A ) Correlation between the expression level of NPAS1 and the proportions of 24 immune cell types. ( B–K ) Scatter plot of NPAS1 expression and individual immune cell infiltration levels, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: DNA methylation of NPAS1. ( A ) DNA methylation level of NPAS1 in COAD and normal tissues. ( B ) DNA methylation level of NPAS1 in COAD patients with different cancer stages. ( C ) DNA methylation level of NPAS1 in COAD patients with different nodal metastasis status. ( D ) DNA methylation level of NPAS1 in COAD patients with different ages. ( E ) DNA methylation level of NPAS1 in COAD patients with TP53 mutation status. (F ) Individual CPG in NPAS1 methylation levels with available patient characteristics and gene subregions.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: DNA Methylation Assay, Mutagenesis, Methylation

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: Alterations of NPAS1 in COAD. ( A ) Waterfall diagram illustrating the distribution and categorization of genetic mutations in NPAS1 gene in patients with COAD. ( B ) Specific mutation site of NPAS1. ( C–G ) Mutation site of NPAS1 and the relationship between mutations and neoplasm disease stage, cancer type, tumor grade, race category and MSI status. ( H–J ) NPAS1 variants in different COAD datasets.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Mutagenesis

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: Single-cell RNAseq analysis of NPAS1. ( A ) NPAS1 mRNA expression in various cell types. ( B and C ) The expression level of NPAS1 mRNA varies in different cell populations in the colon. ( D and E ) Expression of the NPAS1 and type markers in the various single-cell clusters of the colon, with Mas-norm method and Z-score method.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Single Cell, RNA sequencing, Expressing

Journal: International Journal of General Medicine

Article Title: Comprehensive Analysis of the Expression and Prognostic Significance of NPAS1 in Patients with Colorectal Adenocarcinoma Based on Bioinformatics

doi: 10.2147/IJGM.S556875

Figure Lengend Snippet: PPI Network Analysis. ( A ) PPI network diagram of NPAS1-associated genes. ( B ) Circular network diagram of NPAS1-associated genes. ( C ) Top ten Hub genes in the PPI network. ( D ) TF-Gene interaction network diagram constructed by the transcription factors of the top ten Hub genes. ( E ) Circular network diagram constructed by the transcription factors of the top ten Hub genes.

Article Snippet: The proteins that were the focus of this study were specifically recognized by the primary antibodies employed: GAPDH (Proteintech; 60004-1-Ig) (Wuhan, China) (1:5000), NPAS1 (proteintech; 13701-1-AP) (Wuhan, China) (1:500).

Techniques: Construct