Journal: Genes

Article Title: Identification and Validation of Three m6A Regulators: FTO , HNRNPC , and HNRNPA2B1 as Potential Biomarkers for Endometriosis.

doi: 10.3390/genes14010086

Figure Lengend Snippet: Figure 8. Immune cell infiltration and pyroptosis-related genes of m6A patterns in endometriosis. (A) The relationship between 23 kinds of immune cells and the candidate genes. (B) Variation and difference in the immune cell infiltration between low and high FTO expression groups. (C) Difference and variation in the immune cell infiltration between low and high HNRNPC expression groups. (D) Difference and variation in the immune cell infiltration between low and high HNRNPA2B1 expression groups. (E) The correlation between the three m6A patterns and the infiltration of 23 immune cells (F) The relationship between pyroptosis-related genes and the three m6A patterns. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Then, membranes were probed with primary antibodies against FTO (1:2000, Abclonal, Wuhan, China), HNRNPC (1:1000, Abclonal, Wuhan, China), or HNRNPA2B1 (1:1000, Abclonal, Wuhan, China) overnight.

Techniques: Expressing

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: (A) Schematic of in vitro interactome capture from protein interactors of biotinylated IVT SARS-CoV-2 FSE RNA. (B) Host proteins identified from HCoV-OC43-infected H460 cells and HCoV-229E-infected Huh7 cells. (C) RNA pull-down assays. hnRNPC proteins were detected by immunoblotting in Huh7 cell lysates. (D-F) Graphic illustration of RNA immunoprecipitation (RIP) experiments ( D ) using anti-hnRNPC ( E ) or anti-hnRNPC2 ( F ) antibody to capture IVT FSE RNA. Representative immunoblot showing hnRNPC protein in input and immunoprecipitation (IP) samples for RIP assay (left panel). SARS-CoV-2 FSE RNA was quantified by qRT-PCR after enrichment from RIP (right panel). (G) SPR analysis of affinity binding between hnRNPC and RNA. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test .

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: In Vitro, Infection, Pull Down Assay, Western Blot, RNA Immunoprecipitation, Immunoprecipitation, Quantitative RT-PCR, Analysis, Binding Assay, Two Tailed Test, Polymerase Chain Reaction, Gel Extraction, Mass Spectrometry, Biotinylation, RNA Binding Assay

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: (A) Domain mapping of Flag-tagged full-length (FL) and truncated hnRNPC. (B) RNA binding activity of FL and truncated hnRNPC with SARS-CoV-2 FSE RNA in Huh7 cells. (C) Binding mode between SARS-CoV-2 FSE RNA and hnRNPC predicted by molecular docking. (D) Schematic representation of wild-type and mutated hnRNPC proteins. (E) RNA binding activity of WT and mutated hnRNPC with SARS-CoV-2 FSE RNA in Huh7 cells. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post hoc comparison test.

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: RNA Binding Assay, Activity Assay, Binding Assay, Docking Assay, Comparison, RNA Immunoprecipitation

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: ( A ) Schematic view of FL and deleted fragments of SARS-CoV-2 FSE RNA. ( B ) Two-dimensional representations of the SARS-CoV-2 FSE RNA pseudoknot. ( C ) Representative immunoblot examining hnRNPC protein in input and immunoprecipitation (IP) samples for RNA immunoprecipitation (RIP) assay (left panel). FL and deleted SARS-CoV-2 FSE RNA quantified by qRT-PCR after enrichment from RIP (right panel). ( D ) RNA pull-down assay showing the binding activity of hnRNPC with FL or deleted SARS-CoV-2 FSE RNA. ( E ) SPR assays monitoring hnRNPC binding to RNA. U-rich single-stranded RNA served as the positive RNA control. For the RNA sequences used in the SPR assays, see also S4 Table. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post hoc comparison test.

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: Western Blot, Immunoprecipitation, RNA Immunoprecipitation, Quantitative RT-PCR, Pull Down Assay, Binding Assay, Activity Assay, Control, Comparison, Co-Immunoprecipitation Assay, SPR Assay

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: (A-B) Protein levels measured by immunoblotting in whole cell, nuclear and cytoplasmic fractions of H460 cells infected with HCoV-OC43 (MOI = 0.01) at 48 hpi ( A ) and 72 hpi (B) . GAPDH and Lamin B1 served as cytoplasmic and nuclear endogenous controls, respectively. (C) Co-localization image assessed with specific probes against HCoV-OC43 FSE RNA (green) and the specific antibody against hnRNPC (red). Scale bar, 10 μm. Nuclei were stained with DAPI (blue). Fluorescence intensity was calculated using ImageJ software. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using an unpaired, two-tailed Student’s t-test.

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: Western Blot, Infection, Staining, Fluorescence, Software, Two Tailed Test, Immunofluorescence

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: ( A ) Representative images of mCherry-FSE CoV-2 -EGFP reporters in Huh7 cells transfected with protein overexpression plasmid. Scale bar, 100 μm. ( B ) Effects of protein overexpression on mCherry-FSE CoV-2 -EGFP reporters in Huh7 cells detected by fluorescence. ( C-E ) Protein levels measured by immunoblotting in Huh7 cells transfected with mCherry-FSE CoV-2 -EGFP ( C ), mCherry-FSE OC43 -EGFP ( D ), and mCherry-FSE 229E -EGFP reporters ( E ), respectively. ( F ) Frameshifting reporter RNAs were translated in RRL in the presence of increasing concentrations of hnRNPC ranging from 0 to 4 µM and measured by immunoblotting. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post hoc comparison test.

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Fluorescence, Western Blot, Comparison

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: (A) Schematic representation of the high-throughput compound screening procedure. (B) Binding sites and LibDockScores of compound with hnRNPC. (C) Binding affinity between hnRNPC and Elbasvir analyzed by SPR. (D) Binding mode between Elbasvir and hnRNPC predicted by molecular docking. (E) Antiviral efficacy of Elbasvir against HCoV-OC43 and HCoV-229E detected by CCK-8 assay at 72 hours of drug treatment. MOI = 1. Molnupiravir was served as a positive drug control. CC 50 , half-maximal cytotoxic concentration. EC 50 , half-maximal effective concentration. SI, selection index (CC 50 / EC 50 ). (F-H) Elbasvir reduced the efficacy of –1 PRF reporters for SARS-CoV-2 (F) , HCoV-OC43 (G) , and HCoV-229E ( H ) in Huh7 cells detected by immunoblotting. Merafloxacin (MER) and Carrimycin (CAM) were served as positive drug controls. (I-K) FSE RNA levels of SARS-CoV-2 (I) , HCoV-OC43 (J) , and HCoV-229E ( K ) quantified by qRT-PCR after enrichment from RIP in Huh7 cells treated with Elbasvir. Data were shown as mean ± s.d. with individual data points. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s post hoc comparison test.

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: Sample Throughput, Screening, Binding Assay, Docking Assay, CCK-8 Assay, Control, Concentration Assay, Selection, Western Blot, Quantitative RT-PCR, Comparison, SPR Assay, Cell Viability Assay, Co-Immunoprecipitation Assay

Journal: PLOS Pathogens

Article Title: hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA

doi: 10.1371/journal.ppat.1014491

Figure Lengend Snippet: hnRNPC directly binds to the SARS-CoV-2 FSE RNA to promote SARS-CoV-2 –1 PRF and enhance viral replication (Left). Elbasvir inhibits SARS-CoV-2 replication by directly binding to hnRNPC and blocking the interaction between hnRNPC and the viral FSE RNA (Right).

Article Snippet: The 3D structures of hnRNPC (PDB ID: 2MXY) and SARS-CoV-2 FSE RNA (PDB ID: 8VCI) were obtained from the RCSB database, while the commercial compound libraries L1000 and L6000 were provided by TargetMol (Shanghai, China).

Techniques: Binding Assay, Blocking Assay