Journal: Nutrients

Article Title: Long-Term Consumption of a Sugar-Sweetened Soft Drink in Combination with a Western-Type Diet Is Associated with Morphological and Molecular Changes of Taste Markers Independent of Body Weight Development in Mice

doi: 10.3390/nu14030594

Figure Lengend Snippet: Transcriptome analysis of 59 selected genes associated with oral chemosensation, displayed as a heatmap showing mean fold changes in gene expression of the SSB-fed diet groups in relation to the respective water-fed group (=1) in the form of a color code. The gene expression was analyzed using one customized cDNA microarray per group from pooled RNA samples of the CV from mice that received either a standard diet (chow, n = 10–11) or Western-type diet (WD, n = 7–8) with water (Water) or a sugar-sweetened beverage (SSB) as a drink for 24 weeks.

Article Snippet: The isolated RNA samples per mouse were reverse transcribed to cDNA using the LunaScript RT Supermix Kit (New England Biolabs GmbH, Frankfurt am Main, Germany).

Techniques: Expressing, Microarray, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Frontiers in Oncology

Article Title: SNHG9, a Papillary Thyroid Cancer Cell Exosome-Enriched lncRNA, Inhibits Cell Autophagy and Promotes Cell Apoptosis of Normal Thyroid Epithelial Cell Nthy-ori-3 Through YBOX3/P21 Pathway

doi: 10.3389/fonc.2021.647034

Figure Lengend Snippet: Identification and expression of PTC cell exosome-enriched lncRNA SNHG9. (A) High-throughput screening identification of PTC associated exosome lncRNAs. SNHG9 is PTC cell exosome-enriched lncRNA in TPC-1 and K-1 cells compared with Nthy-ori-3 cell. (B, C) Validation of SNHG9 overexpression in both TPC-1 and K-1 cells and their respective exosomes compared with Nthy-ori-3 cell and its exosome by qPCR. (D) Coregulation network of SNHG9 with mRNA/miRNA. SNHG9 had an interaction with autophagy related molecules. (E) Gene ontology enrichment analysis showed the highest regulation scores in autophagy and apoptosis. (F) KEGG-pathway-weighted analysis showed SNHG9 mainly targeted apoptosis and autophagy pathways. (G) SNHG9 in the PTC cell supernatant mainly derived from cell exosomes. QPCR showed significantly lower SNHG9 expression level in supernatant treated with Rnase and Triton compared with supernatant treated with only Rnase and control group. (H) QPCR confirmed no SNHG9 expression in cell supernatants after exosome extraction. (I) SNHG9 expression level between tumor and normal tissues in 70 PTC patients from FUSCC. The results were normalized to β-actin mRNA level. (J) Waterfall plot showed the distribution of SNHG9 expression level in each PTC patients from FUSCC. ***P < 0.001, data were pooled from three independent experiments. FUSCC, Fudan University Shanghai Cancer Center; PTC, papillary thyroid cancer.

Article Snippet: Next, we used the Arraystar Human LncRNA Array v2.0 gene chip to compare expression profile data of lncRNAs in Nthy-ori-3, TPC-1 and K-1 cells and their respective exosomes.

Techniques: Expressing, High Throughput Screening Assay, Biomarker Discovery, Over Expression, Derivative Assay, Control, Extraction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM expression is increased in HCC and fetal liver tissues and is associated with prognosis. A , Normalized (Norm) HNRNPM expression levels during mouse liver development from GSE57824 data. B , HNRNPM expression levels during mouse liver development from GSE13149 data. C , Western blot analysis of HNRNPM protein levels in human fetal liver and adult liver tissues. D , Real-time qPCR analysis of HNRNPM mRNA levels in human fetal liver and adult liver tissues. Data are mean ± standard deviation of n = 3 independent samples. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001 by the Student t test. E , Norm HNRNPM expression in HCC and normal liver tissues. ∗∗ P < .01 by the Student t test. F , Real-time qPCR analysis of HNRNPM mRNA levels in 60 paired HCC and normal liver tissues. G , Representative images of HNRNPM by IHC in HCC and normal tissues. H , Kaplan-Meier analysis of HNRNPM in HCC cohort. I , Kaplan-Meier analysis of HNRNPM in TCGA cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM was associated with clinopathological characteristics and poor prognosis in patients with HCC. A , Oncomine analysis showed the prognostic splicing factors from TCGA datasets. B , The selected prognostic splicing factors validated by real-time PCR in portal vein tumor thrombosis (PVTT) HCC, non-PVTT HCC, and normal liver tissues. C , The HNRNPM protein expression in metastasis and metastasis-free HCC tissues. D , The HNRNPM protein expression in tumor grade I/II and III/IV. E , The HNRNPM protein expression in no-microvascular invasion and microvascular invasion HCC tissues. F , The relative HNRNPM expression in tumor stage I/II/III/IV. G , The correlation analysis between Ki-67 and HNRNPM in TCGA database. H , Kaplan-Meier analyses of the correlations between HNRNPM level and overall survival in HCC tumor stage I/II and III/IV from our HCC cohort. I , Kaplan-Meier analyses of the correlations between HNRNPM level and OS in HCC tumor grade I/II and III/IV from our HCC cohort.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 240 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Association of HNRNPM Expression With Clinical Characteristics in 371 Patients With HCC

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Virus

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall Survival for HNRNPM (n = 240)

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Univariate and Multivariate Cox Regression Analysis of Overall and Disease-free Survival for HNRNPM (n = 370) From TCGA Database

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Cell stem cell transcriptional factors SOX2 and OCT4 bind with promoter and upregulate the expression of HNRNPM. A , The basic expression of HNRNPM in different HCC cell lines. B-C , Western blot analysis of HNRNPM expression when overexpressing ( B ) or depletion of ( C ) OCT4 and SOX2. D , The predicted binding site for OCT4 and SOX2 with HNRNPM promoter. E , OCT4 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. F , SOX2 directly binds with HNRNPM promoter by ChIP assays and luciferase assays. G-H , Correlation analysis between OCT4 ( G ), SOX2 ( H ), and HNRNPM from TCGA database.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Western Blot, Binding Assay, Luciferase

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The tumorigenesis effects of HNRNPM overexpression in MHCC97L and HepG2 cells. A-B , The mRNA and protein levels of HNRNPM in MHCC97L ( A ) and HepG2 cells ( B ) stably overexpressing HNRNPM. C-D , The cell proliferation by CCK-8 assays stably MHCC97L ( C ) and HepG2 cells ( D ) stably overexpressing HNRNPM. ∗∗∗∗ P < .0001 as compared with control. E-F , The cell apotosis by flow cytometry stably MHCC97L ( E ) and HepG2 cells ( F ) stably overexpressing HNRNPM. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test. G-H , The in vivo effects in BALB/c nude mice in MHCC97L ( G ; n = 6) and HepG2 cells ( H ; n = 6) stably overexpressing HNRNPM. ∗∗ P < .01 by the Student t test. I , The CSC frequency was determined from a limiting dilution assay performed with HCC cells depleting HNRNPM from the third transplant recipient mice (n = 6). The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Over Expression, Stable Transfection, CCK-8 Assay, Control, Flow Cytometry, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM drives HCC tumorigenesis and maintains CSC properties. A-B , Sphere formation and limiting dilution assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. C-D , Cell cycle detected by flow cytometry when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. E-F , Colony formation assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05; ∗∗ P < .01 by the Student t test. G-H , Cell migration assay when overexpressed HNRNPM in MHCC97L and HepG2 cells. ∗ P < .05 by the Student t test. I , Cell invasion assays when overexpressed HNRNPM in MHCC97L and HepG2 cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Flow Cytometry, Colony Assay, Cell Migration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The oncofetal properties of HNRNPM in hepatocyte differentiation model. A , The model scheme in hepatocyte differentiation model. B , The expression of OCT4, E2F1, SOX2, and HNRNPM in different stages from hepatocyte differentiation model. C , The correlation analysis between HNRNPM and E2F1 from TCGA databases. D , The potential binding site for E2F1 to HNRNPM promoter. E , E2F1 directly bind with HNRNPM promoter by ChIP assays. Data were from 3 independent experiments. ∗∗ P < .01 by the Student t test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM is required for tumorigenesis of HCC cells. A , The mRNA and protein levels of HNRNPM in MHCC97H cells stably depleting HNRNPM. B , The protein levels of HNRNPM by immunofluorence stably depleting HNRNPM. C , Sphere formation and limiting dilution assays when depleting HNRNPM in MHCC97H cells. The number of spheroids formed as a fraction of the number of cells seeded per well is given. Data are from 3 independent experiments. ∗∗ P < .01 by the Student t test. D , The cell proliferation by CCK-8 assays stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. E , The cell apotosis by flow cytometry stably depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. F , Cell cycle detected by flow cytometry when depleting HNRNPM in MHCC97H cells. G , Colony formation assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗ P < .05; ∗∗ P < .01 by the Student t test. H , Cell migration assay when depleting HNRNPM in MHCC97H cells. Results are presented as mean ± standard error of the mean, n = 3. ∗∗∗ P < .001 by the Student t test. I , Cell invasion assays when depleting HNRNPM in MHCC97H cells. J-K , The in vivo effects in BALB/c nude mice (n = 6 per group) when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. L-M , The number of liver metastasis in BALB/c nude mice when overexpressed and depleted HNRNPM. Results are presented as mean ± standard error of the mean, n = 6. ∗ P < .05; ∗∗ P < .01 by the Student t test. N , The CSC frequency was determined from a limiting dilution assay performed with HCC cells from the third transplant recipient mice. The ELDA web tool was used to calculate the frequency of CSCs.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Stable Transfection, CCK-8 Assay, Flow Cytometry, Colony Assay, Cell Migration Assay, In Vivo, Limiting Dilution Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The genome-wide landscape and global alternative splicing of HNRNPM. A , Kyoto Encyclopedia of Genes and Genomes analysis of HNRNPM-targeted splicing events. B , Quantification of the different AS events regulated by HNRNPM. A3SS , alternative 3′ splicing site; A5SS , alternative 5′ splicing site; MXE , mutually exclusive exon; RI , retained intron; SE , skipped exon. ∗∗∗ P < .001 by the Student t test. C-D , The quantification of significant AS events regulated by HNRNPM ( P < .05). E , HNRNPM-RIP-seq peaks were enriched in 5′UTR, promoter and 3′ UTR. All RIP-seq peaks were categorized according to the distribution on different genomic elements andcompared with the genomic background. F , De novo motif analysis identifying GU-repeat motif as the only enriched motif within the top HNRNPM RIP-seqpeaks. G , Schematic diagram of MBD2 molecular model. H , The RIP experiment showed HNRNPM directly binded with MBD2. I , The shift of MBD2a and MBD2c between HNRNPM overexpressed stably transduced and control MHCC97H cells. J , The shift of MBD2a and MBD2c between HNRNPM shRNA stably transduced and control MHCC97H cells. K , The RMMs of HNRNPM bind to MBD2 by RIP experiments. L , The potential binding of HNRNPM to MBD2 pre-mRNA by CLIP assay.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Genome Wide, Alternative Splicing, Stable Transfection, Control, shRNA, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Significant Alternative Splicing Events by Comparing Depletion of HNRNPM With Wild-type HCC Cells

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Alternative Splicing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Results of HNRNPM-RIP Analysis

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The Intersection Results of HNRNPM-RIP Analysis and Transcriptomic Sequencing

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: DNA methylation controls MBD2-mediated FZD3 transcription. A , The shematic diagram of HNRNPM domains. B , The specific binding site for MBD2 with HNRNPM by CLIP assay. C , The luciferase assay for FZD3 transcription activity when overexpressing MBD2a or MBD2a and MBD2c. Data were from three independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. D-H , qPCR analysis of MBD2a, MBD2c, FZD3, β-catenin, and Snail1 mRNA transcripts in MHCC97H cells stably expressing NC, shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2. Immunoblot analysis showed the knockdown efficiency of shRNAs targeting HDAC1, HDAC2, RBBP7, or MTA2 in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I-J , β-catenin promotes the expression of OCT4 ( I ) and SOX2 ( J ) by binding its promoter. Data were from 3 independent experiments. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: DNA Methylation Assay, Binding Assay, Luciferase, Activity Assay, Comparison, Stable Transfection, Expressing, Western Blot, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: MBD2a induces, whereas MBD2c represses, HCC tumorigenesis and CSC properties. A , Sphere formation and limiting dilution assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. B , The cell proliferation by CCK-8 assays when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. C , Cell migration and migration assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. D , Colony formation assay when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. E , The cell apotosis by flow cytometry when overexpressing MBD2a or with HNRNPM depletion, MBD2c in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. F , The protein expression of HNRNPM, MBD2a, MBD2c when downregulating SOX2, OCT4, and together with overexpressing HNRNPM by Western blot experiments. G , The in vivo effects in BALB/c nude mice when overexpressing MBD2a (n = 5) or with HNRNPM depletion (n = 5), MBD2c (n = 5). ns, Non-significant. ∗ P < .05, ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: CCK-8 Assay, Migration, Colony Assay, Flow Cytometry, Expressing, Western Blot, In Vivo, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The coregulated genes by MBD2a and MBD2c in HCC cells. A-B , Venn diagram of the RNA-seq data showing the genes commonly regulated by MBD2a and MBD2c. C-D , Gene Ontology (GO) enrichment analysis. The top 5 GO terms in the indicated categories with the lowest P values are shown. E , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing shRNAs targeting MBD2a, and in MHCC97H cells stably expressing MBD2c. Data were from 3 independent experiments. ∗ P < .05 as compared with controls. F , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when silencing MBD2a or overexpressing MBD2c. Data were from 3 independent experiments. ∗ P < .05; ∗∗ P < .01 by the Student t test. G , The expression of Snail1, OCT4, SOX2 mRNA, and proteins was measured by qPCR and Western blot in MHCC97H cells expressing HNRNPM and shRNA targeting MBD2a. Data were from 3 independent experiments. ∗∗∗ P < .001 as compared with controls; ns, Not significant; P > .05. H , The expression of β-catenin by nuclear/cytoplasmic protein fractionation and TOP/FOP-flash reporter assays when overexpressing HNRNPM and silencing MBD2a. Data were from 3 independent experiments. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: RNA Sequencing, Expressing, Western Blot, Stable Transfection, Fractionation, shRNA, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: A , The relative expression of MBD2a, MBD2c in fetal liver, adult liver, HCC, and adjacent noncancerous liver tissues. ∗∗∗ P < .001 by the Student t test. B-C , The Kaplan-Meier analyses of the correlations between MBD2a ( B ), MBD2c ( C ) level and overall survival of n = 100 patients with HCC. The median MBD2a or MBD2c level was used as the cutoff. D , The multivariate analysis for MBD2a and MBD2c in patients with HCC. E , The correlation analysis between HNRNPM and MBD2a in patients with HCC (n = 30) by IHC experiments. F , FZD3 and HNRNPM expression in protein levels in HCC tissues with strong or weak HNRNPM staining intensity. The median HNRNPM staining intensity was used as the cutoff (n = 60 HCC tissues). ∗∗∗ P < .001. G-H , The correlation between the expression of HNRNPM and FZD3 ( G ), β-catenin ( H ) from TCGA datasets.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: The effects of HNRNPM-specific ASO for HCC in vivo and in vitro. A , The expression of HNRNPM was significantly correlated with MBD2a. B , The IC50 of ASO-2 for MHCC97H cells. C , The protein expression of HNRNPM, MBD2a, FZD3, OCT4, SOX2, and β-catenin related assays when treated with ASO-2 in HCC cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. D , The CSC markers expression by ASO treatment. E , The CCK-8 experiment when treated with HNRNPM-specific ASO in MHCC97H cells. F , Sphere formation and limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. G , Limiting dilution assays when treated with HNRNPM-specific ASO in MHCC97H cells. H , Colony formation assay when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05. I-J , Invasion assay ( I ) and cell migration ( J ) and when treated with HNRNPM-specific ASO in MHCC97H cells. Data were from 3 independent experiments. ∗ P < .05 by the Student t test. K , The HCC cell apoptosis changes by ASO treatment. Data were from 3 experiments. ∗∗ P < .01 by the Student t test. L , The schematic diagram of ASO-2 treating nude mice when inoculating the tumor cells. M , The effects of HNRNPM-specific ASO when treated ASO I.P by 25 mg/kg (n = 5). ∗∗∗ P < .001 by the Student t test. N ,. The HNRNPM expression in tumors when treating HNRNPM-ASO by IHC experiments.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: In Vivo, In Vitro, Expressing, CCK-8 Assay, Colony Assay, Invasion Assay, Migration

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: Expression of HNRNPM correlated with immune checkpoint in human HCC. A-E , Expression correlation between HNRNPM and immune checkpoint gene RNA amounts in the TCGA HCC database, n = 370, HNRNPM (HNRNPM), PD-L1 (CD274), B7-H3 (CD276), B7-H4 (VTCN1), LAG-3 (LAG3), and TIM-3 (HAVCR2). B , Pearson correlation analysis of HNRNPM and CD276 immune checkpoint expressions in human HCC tissue microarray based on the IHC results, n = 240.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Expressing, Microarray

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: HNRNPM inhibition curbs immune escape and enhances PD-1 blockade by promoting CD8+ T cells activation phenotype. A , Schematic diagram of Hep1-6-OVA cells co-cultured with OTI cells. B , The flow cytometry analysis of IFN-γ+ or granzyme B+ CD8+ T cells between control and shHNRNPM groups. Data were from 3 independent experiments. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. C , Schematic diagram of ASO and anti-PD-1 therapy in C57/BJ6 mice. D , Tumor inhibition by IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. E , Survival analysis of IgG (n = 6), HNRNPM-ASO (n = 6), anti-PD-1 (n = 6), or combination therapy (n = 6) in C57/BJ6 mice. ∗ P < .05. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. F , The profiles of immune cells in tumors by HNRNPM-ASO, anti-PD-1 or combination therapy. G , CD8+ T cells infiltration in HNRNPM-ASO, anti-PD-1 or combination therapy groups. ∗∗ P < .01. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. H , The changes of Treg, IFNG+, GMZB+ CD8+ T cells in control, HNRNPM-ASO, anti-PD-1 or combination therapy groups in tumor-bearing C57/BJ6 mice. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. I , The immune cells infiltration landscape of spleen in control, HNRNPM-ASO, anti-PD-1, or combination therapy groups in tumor-bearing C57/BJ6 mice. J , The mice weight between controls and HNRNPM-ASO group. ns , Non-significant. K , The relative expression of β-catenin in HNRNPM-ASO, anti-PD-1, or combination therapy groups. ∗∗∗ P < .001. P values were calculated using 1-way analysis of variance and Dunnett’s multiple comparison test. L-M , The distribution of CTNNB1 mutation in PD-1 responders or non-responders. N , The study model diagram.

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: Inhibition, Activation Assay, Cell Culture, Flow Cytometry, Control, Comparison, Expressing, Mutagenesis

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Antibodies Used in This Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

doi: 10.1016/j.jcmgh.2022.02.006

Figure Lengend Snippet: List of Primers Sequences and shRNA Sequences Used in this Research

Article Snippet: IHC was performed with rabbit anti-human HNRNPM (1:50; sc-20002, 1D8, SANTA CRUZ).

Techniques: shRNA, Sequencing, Control, Negative Control