Journal: Nature protocols

Article Title: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

doi: 10.1038/s41596-021-00577-3

Figure Lengend Snippet: a, (Steps 1-7) Production of the substrate begins using a ccDNA with the oligonucleotide-binding sequence of interest. (Steps 8-21) A nick is introduced into the plasmid on the strand that will modified with a site-specific DNA lesion. (Steps 22-29) The nicked strand is digested using Exo III to generate circular ssDNA. (Steps 30-33) An oligonucleotide containing the lesion of interest is annealed to the ssDNA. (Steps 34-42) The annealed plasmid:oligo duplex is incubated with T4 Polymerase and T4 Ligase produce ccDNA containing the lesion of interest. (Steps 42-69) Pure closed circular product is obtained through incubation with T5 Exo followed by Proteinase K digest, DNA extraction and precipitation, and dissolution in TE buffer. Note, procedures in steps 59-68 result in linearized plasmids. All yields refer to reporter plasmids generated using the oligonucleotide extension methodology. (Steps 70-101) substrates are subjected to a battery of in vitro and in vivo QC steps. b, Representative agarose gel electrophoretic analysis of starting ccDNA (Lane 2), ocDNA (Lane 3), circular ssDNA (Lane 4) and the final purified product (Lane 5) are shown. c, Representative gel analysis showing evidence of incomplete digest of ccDNA with nicking enzyme (Lane 3) and incomplete digest of nicked DNA with Exo III (Lane 5). d, Overexposed representative gel analysis showing evidence of residual ssDNA due to incomplete extension when the molar ratio of oligonucleotide to ssDNA is too low (1:1, Lane 5 and 1:2 Lane 6) and an absence of ssDNA when the ratio is optimal (4:1, Lane 7). e, Representative gel analysis showing accumulation of open circular DNA in a preparation where the proteinase K step was excluded (Lane 5), and the near absence of nicked product when proteinase K is included (Lane 6). In panels b-e, the relevant steps of the protocol are indicated below the gel.

Article Snippet: In a 1.8 mL microcentrifuge tube, prepare an annealing reaction between the custom 5’-phosporylated oligo and the ssDNA, using a 4:1 molar ratio of phosphorylated oligo to single stranded DNA with 1X NEBuffer 2.1 as shown in the example below: Example: To anneal phosphorylated oligo to 100 μg (~91 pmol) ssDNA Add 50 μL single stranded DNA (initial concentration 2000 ng/μL; final concentration 833 ng/μL) Add 12 μL 10X NEBuffer 2.1 (1X) Add 3.6 μL 100μM custom oligonucleotide (360 pmol; 16 μM) Add 54 μL DEPC-treated water (up to a total volume of 120 μL) Mix thoroughly by pipetting.

Techniques: Binding Assay, Sequencing, Plasmid Preparation, Modification, Incubation, DNA Extraction, Generated, In Vitro, In Vivo, Agarose Gel Electrophoresis, Purification

Journal: Nature protocols

Article Title: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

doi: 10.1038/s41596-021-00577-3

Figure Lengend Snippet: Gel electrophoretic analysis and flow cytometric validation of GFP_Hx, mPlum_A-8oxoG, and mOrange_8oxoG-C reporter plasmids. a, Analytical digest and flow cytometric validation of GFP_Hx plasmid. (Lane 1) NEB 1kb MW Ladder (Lane 2) pMax_GFP_C289T ccDNA (Lane 3) HIA overnight extension reaction for GFP_Hx (Lane 4) pMax_GFP_C289T ccDNA (Lane 5) pMax_GFP_C289T after 45 min ApaLI digest at 37°C, which cleaves 2 restriction sites, resulting in 2 linear DNA fragments (Lane 6) GFP_Hx ccDNA after T5 Exo and PEG purification steps (Lane 7) GFP_Hx after 45 min ApaLI digest at 37°C, in which the Hx lesion blocks ApaLI cleavage of 1 restriction site, leaving a single linearized fragment. At right: Flow cytometric quantitation of % reporter expression and normalized relative reporter expression in WT HAP cells compared to MPG−/− HAP cells. b, Analytical digest and flow cytometric validation of mPlum_A-8oxoG plasmid. (Lane 1) HIA overnight extension reaction for mPlum_A-8oxoG (Lane 2) pMax_mPlum ccDNA (Lane 3) pMax_mPlum after 45 min Fpg endonuclease digest at 37°C (Lane 4) mPlum_A-8oxoG after T5 Exo and PEG purification steps (Lane 5) mPlum_A-8oxoG after 45 min Fpg endonuclease digest at 37°C, resulting in plasmid nicking and upward mobility shift. At right: Flow cytometric quantitation of % reporter expression and normalized relative reporter expression in WT HAP cells compared to MUTYH−/− HAP cells. c, Analytical digest and flow cytometric validation of mOrange_8oxoG-C plasmid. (Lane 1) NEB 1kb MW Ladder (Lane 2) pMax_mOrange_A215C ssDNA (Lane 3) pMax_mOrange_A215C ocDNA (Lane 4) pMax_mOrange_A215C ccDNA (Lane 5) pMax_mOrange_A215C after 45 min Fpg endonuclease digest at 37°C (Lane 6) mOrange_8oxoG-C ccDNA (Lane 7) mOrange_8oxoG-C after 45 min Fpg endonuclease digest at 37°C, which introduces a nick at the 8oxoG lesion, resulting in upward mobility shift. At Right: Flow cytometric quantitation of % reporter expression and normalized relative reporter expression in WT MEF cells compared to OGG1−/− MEF cells. Error bars represent SEM from 3-4 biological replicates, differences of statistical significance (P < 0.05 *, P < 0.005 **, P < 0.005 ***, P < 0.0001 ****) were determined by unpaired two-tail t test.

Article Snippet: In a 1.8 mL microcentrifuge tube, prepare an annealing reaction between the custom 5’-phosporylated oligo and the ssDNA, using a 4:1 molar ratio of phosphorylated oligo to single stranded DNA with 1X NEBuffer 2.1 as shown in the example below: Example: To anneal phosphorylated oligo to 100 μg (~91 pmol) ssDNA Add 50 μL single stranded DNA (initial concentration 2000 ng/μL; final concentration 833 ng/μL) Add 12 μL 10X NEBuffer 2.1 (1X) Add 3.6 μL 100μM custom oligonucleotide (360 pmol; 16 μM) Add 54 μL DEPC-treated water (up to a total volume of 120 μL) Mix thoroughly by pipetting.

Techniques: Plasmid Preparation, Purification, Quantitation Assay, Expressing, Mobility Shift

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

Journal: Cell

Article Title: Repressive gene regulation synchronizes development with cellular metabolism

doi: 10.1016/j.cell.2019.06.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A list of all mutants and transgenics used in this study is in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-Prospero Developmental Studies Hybridoma Bank MR1A MAb Rat monoclonal anti-Elav Developmental Studies Hybridoma Bank 7E8A10 MAb Goat anti-mouse Alexa488 Invitrogen A-11001 Goat anti-rat Alexa546 Invitrogen A-11081 Goat anti-mouse Alexa546 Invitrogen A-11030 Goat anti-rat Alexa633 Invitrogen A-21094 Mouse monoclonal anti-ATP5A (MAb15H4C4) Abcam ab14748 Bacterial and Virus Strains N/A Biological Samples N/A Chemicals, Peptides, and Recombinant Proteins Paraformaldehyde (powder) Polysciences 00380–1 Triton X-100 Sigma Aldrich T9284–500ML VectaShield Vector Labs H-1000 4′,6-diamidino-2-phenylindole (DAPI) Life Technologies D1306 Critical Commercial Assays N/A Deposited Data Data of all model simulations This paper https://arch.library.northwestern.edu/concern/generic_works/n296wz31t Fluorescence data for Yan-YFP and His2Av-mRFP in eye cells This paper https://arch.library.northwestern.edu/concern/generic_works/n296wz31t Fluorescence data for sfGFP-Sens in wing cells This paper https://www.dropbox.com/sh/1m9silkrs76rvpr/AADunGcGUylP2rQ9WRo5bXVha?dl=0 Experimental Models: Cell Lines N/A Experimental Models: Organisms/Strains D. melanogaster: mir-9a E39 Swap of the pre-miRNA sequence with white + .

Techniques: Virus, Recombinant, Plasmid Preparation, Fluorescence, Sequencing, Mutagenesis, Residue, Full Display Name, Transgenic Assay, Phospho-proteomics, Binding Assay, Control, Construct, Software