anti rh1  (Developmental Studies Hybridoma Bank)

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: m 6 A‐modified circTACC3 is up‐regulated in MASH‐related HCC. (A) Schematic representation of m 6 A‐circRNA epitranscriptomic microarray assay. (B) Hierarchical clustering heatmap of differentially m 6 A modified circRNAs from MASH‐related HCC tumor and paired peritumoral normal tissues (m 6 A‐circRNA epitranscriptomic microarray assay; absolute m 6 A modification quantity shown, n = 5). (C) Venn diagram of overlapping circRNAs in MASH‐related HCC tumors with concurrent increases in absolute m 6 A modification quantity, relative m 6 A modification rate, and relative expression levels. (D‐E) The circTACC3 level in MASLD tissues, MASH‐related HCC tumor tissues, and paired peritumoral normal tissues ( n = 62) determined by ISH assay (D) and corresponding expression score analysis (E). ** P < 0.01; *** P < 0.001; NS , not significant. (F) MeRIP assay shows the enrichment of m 6 A‐modified circTACC3 in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues ( n = 3). (G) 3D‐FISH performed on MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids. Left upper panel shows circTACC3 (red) and DAPI (blue). Left lower panel shows the merge in 3D view. Right upper panel shows representative z‐stack layer capture. Right lower panel shows depth coding. (H) Nuclear‐cytoplasmic fractionation assay determined circTACC3 expression in nuclear and cytoplasmic fractionation, respectively ( n = 4). ** P < 0.01; *** P < 0.001; NS , not significant. (I) Schematic representation of exon 4 back‐splicing, circTACC3 forming, and the design of indicated primers. (J) Electrophoresis of RT‐PCR product amplified from cDNA or gDNA. (K) The expression of circTACC3 and TACC3 homologous mRNA from RNA with or without RNase R treatment. (L) Levels of circTACC3 and TACC3 mRNA in indicated cells that were treated with or without actinomycin D ( n = 4). *** P < 0.001. Abbreviations: m 6 A, N6‐methyladenosine; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; ISH, in situ hybridization‐ immunofluorescence; MeRIP, methylated RNA immunoprecipitation; 3D, three dimensions; FISH, fluorescence in situ hybridization; NAS, non‐alcoholic fatty liver disease activity score; RT‐PCR, reverse transcription‐PCR.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Modification, Microarray, Expressing, In Situ Hybridization, Derivative Assay, Fractionation, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Methylation, RNA Immunoprecipitation, Fluorescence, Activity Assay, Reverse Transcription

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop formation is regulated by lipid overload and m 6 A modification. (A) 3D‐distribution of circTACC3 (glow) and S9.6‐stained R loops (spectrum) in nuclei (blue) of MASH‐HCC tissue derived organoids after indicated treatment. (B‐C) DRIP assay shows the enrichment of circTACC3 in R loop structure following m 6 A modification interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 3). * P < 0.05; ** P < 0.01. (D) Representative fluorescence images show S9.6‐stained R loops (green) in PA + OA induced HCC cells transfected with the RNase H1‐Tet‐On system after treatment with or without Dox. (E) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (F‐G) Representative fluorescence pictures (F) and peak graphs of the linear ROI (G) demonstrating the colocalization of circTACC3 (red), S9.6‐indicated R loops (green), and NONO/p54 nrb (yellow) in nuclei (blue) with or without Dox‐inducible RNase H1 expression in PA and OA treated HCCLM3 and HepG2 cells. Abbreviations: MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; PA, palmitic acid; OA, oleic acid; T, tumor tissue; PT, peritumoral normal tissue; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; DRIP, DNA‐RNA immunoprecipitation; Dox, Doxycycline; ROI, region of interest.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Modification, Staining, Derivative Assay, Fluorescence, Transfection, Fractionation, Expressing, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loops aggregated to promote the inter‐TADs contact. (A) After DRIP‐ChIRP sequencing, the reads distributions across peaks of all independent biological replicates are presented. (B) Genome‐wide distribution of the circTACC3‐R Loop‐located genes positively correlated with PA + OA induction or negatively correlated with m 6 A modification intervention. (C) Representative fluorescence images of the colocalization of indicated molecules in nuclei of HCC cells treated with or without PA and OA following RNase R treatment. (D) Schematic representation shows combination of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq to analyze the distribution of the DSB‐circTACC3‐R Loop structures in the genome. (E) Genome‐wide distribution of the DSB‐circTACC3‐R loop located genes in PA + OA induced HepG2 cells. (F) Top four enriched DSB‐circTACC3‐R loop‐binding motifs based on de novo motif analysis. (G) The dynamic clustering of paraspeckles (indicated by NONO/p54 nrb ‐mCherry) were filmed using STELLARIS Dynamic Signal Enhancement 24 h after PA + OA induction at 5‐min intervals for a duration of 1.5 h. Examples (from 50 min to 85 min) of fusions of several NONO/p54 nrb ‐mCherry foci are shown (time points indicated in minutes). (H) Heatmap depicting the fold change(log 2 ) in Hi‐C contact frequencies between PA + OA‐treated and control cells throughout chromosome 7. Interactions that increase in PA + OA group (red) or decrease in Mock group (blue) are evident. Profile of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq are shown on the top. TADs that had higher inter‐TADs contact frequencies (named “contact‐elevated TADs”) in both long‐range (green box) and between adjacent TADs (red box) are marked. DSB‐circTACC3‐R loops are marked with red arrow. (I) Hi‐C maps around the human STX6 locus that formed DSB‐circTACC3‐R loop structure are shown. DSB‐circTACC3‐R loops are marked with red arrow. Abbreviations: DRIP, DNA‐RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; PA, palmitic acid; OA, oleic acid; Hi‐C, high‐throughput/resolution chromosome conformation capture; DSB, DNA double‐strand breaks; STX6, Syntaxin 6.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Sequencing, Genome Wide, Modification, Fluorescence, Binding Assay, Hi-C, Control, RNA Immunoprecipitation, Isolation, Purification, Residue, Variant Assay, Immunofluorescence, Hybridization, High Throughput Screening Assay

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loop‐localized genes are selectively activated. (A) List of DSB‐circTACC3‐R loop‐localized genes. (B) DSB‐circTACC3‐loop‐localized genes expression in HepG2 cells with/without lipid overload induction ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (C‐D) DRIP‐ChIRP‐seq (C) and γH2AX CUT&Tag‐seq (D) RPKM analysis of DSB‐circTACC3‐loop‐localized genes to compare circTACC3‐R Loop enrichment within the “contact‐elevated TADs” ( n = 26) or not within the “contact‐elevated TADs” ( n = 12). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (E‐F) Representative fluorescence images (E) and peaks graphs of the linear ROI (F) show the colocalization of indicated molecules with or without Dox‐inducible RNase H1 expression. (G) NONO/p54 nrb ‐mCherry HepG2 cells with or without Dox‐inducible RNase H1 expression were filmed 24 h after PA + OA induction at 5‐min intervals. Abbreviations: PA, palmitic acid; OA, oleic acid; TAD, topologically associated domain; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; RPKM, reads per kilobase per million mapped reads; Dox, Doxycycline; ROI, region of interest.

Article Snippet: To generate stable cell lines with the inducible overexpression of RNase H1, HepG2 and HCCLM3 cells were infected with lentivirus expressing TetIIP‐RNase H1‐3FLG‐Ubi‐TetR‐IRES‐Puromysin and selected with puromycin (# T19978 , TargetMol, MA, US, final concentration 2 μg/mL) for 1‐2 weeks.

Techniques: Expressing, Fluorescence, Residue, Variant Assay