Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hypoxia-associated circRNA profiling and expression characteristics of Hsa_circ_0000566 in osteosarcoma (OS). (A) CircRNA microarray analysis reveals 35 upregulated and 23 downregulated circRNAs in OS cells under normoxic and hypoxic conditions. The black arrow represents Hsa_circ_0000566. (B) OS cells incubated under various oxygen concentrations. Total RNA extraction was performed for qRT-PCR assay. Western blotting was performed to determine the protein level of HIF-1α. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Scale bars, 200 μm. (C) Hsa_circ_0000566 expression is much higher in primary OS tissue than in chondroma tissue. Results are representative images according to three different experiments. (D) Quantitative real-time polymerase chain reaction (qRT-PCR) results comparing Hsa_circ_0000566 mRNA expression in 12 OS and chondroma samples. Results are reported as mean ± SD, *p < 0.05, n = 12. (E) Hsa_circ_0000566 expression levels in hFOB1.19 and various OS cell lines. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Schematic diagram showing Hsa_circ_0000566 back-spliced by exons 2-11 of the VRK1 gene and the corresponding Sanger sequencing. (G) RT-PCR results validating the presence of Hsa_circ_0000566 in 143B and HOS cells. Various primers amplified the Hsa_circ_0000566 region in cDNA but not in genomic DNA. β-actin was used as the negative control. Divergent primers are presented as the opposite direction of the arrowhead, and the convergent primers were shown as the face-to-face direction of the arrowhead. (H) RT-PCR results indicating Hsa_circ_0000566 and VRK1 mRNA expression in untreated 143B and HOS cells and in the cells subjected to treatment with RNase-R. (I) RNA fluorescence in situ hybridization (FISH) results revealing Hsa_circ_0000566 localized mainly in the cytoplasm. Hsa_circ_0000566 probes were labeled with cy3 and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (J) qRT-PCR determination of the main localization of Hsa_circ_0000566 in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Microarray, Incubation, RNA Extraction, Quantitative RT-PCR, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 contributes to in vitro osteosarcoma (OS) cell progression under hypoxic conditions. (A) Hsa_circ_0000566 overexpression and knockdown induced and repressed OS cell proliferation under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. Circ_0000566 represents Hsa_circ_0000566 overexpression, and si circ_0000566 represents Hsa_circ_0000566 knockdown. Vector and Si NC represents the negative control of Hsa_circ_0000566 overexpression and Hsa_circ_0000566 knockdown, respectively. (B) EdU exhibits the impact of Hsa_circ_0000566 on OS cell proliferation under hypoxia. Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI). Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (C) Colony formation experiment verifies Hsa_circ_0000566 functions in OS cells under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) Soft agar colony formation assay indicates the effects of Hsa_circ_0000566 on 143B and HOS cell colony forming capacity under hypoxia. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (E) OS cell migration capacity as determined by Transwell™ migration assays. Results are reported as mean ± SD, *p < 0.05, n = 3. Scale bars, 100 μm. (F) Flow cytometry verifies Hsa_circ_0000566 functions in OS cell apoptosis. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, Over Expression, Knockdown, Standard Deviation, Plasmid Preparation, Negative Control, Staining, Soft Agar Assay, Migration, Flow Cytometry

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 accelerates osteosarcoma (OS) glucose metabolism and regulates hypoxia-enhanced glycolysis. (A) Colors of the media indicate that Hsa_circ_0000566 silencing decreased lactate accumulation under hypoxia. (B-C) Quantitative real-time polymerase chain reaction (qRT-PCR) or western blots evaluating the expression levels of genes involved in glucose metabolism in 143B and HOS cells transfected with Hsa_circ_0000566-overexpressing, Hsa_circ_0000566 (shRNA), or vector plasmids. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (D) Hsa_circ_0000566 knockdown in OS cells with decreased lactate accumulation, while Hsa_circ_0000566 overexpression has increased lactate accumulation. Results are reported as mean ± SD, *p < 0.05, n = 3. (E) Extracellular acidification rate (ECAR) indicates glycolysis rate. ECAR decreases in response to Hsa_circ_0000566 knockdown and increases in response to Hsa_circ_0000566 overexpression. Oxygen consumption rate (OCR) represented mitochondrial respiratory capacity. OCR is enhanced in response to Hsa_circ_0000566 silencing and reduced in response to Hsa_circ_0000566 overexpression in OS cells. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Plasmid Preparation, Standard Deviation, Knockdown, Over Expression

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 establishes interactions with HIF-1α and confers protection against ubiquitination-mediating degradation. (A) Effects of Hsa_circ_0000566 knockdown and Hsa_circ_0000566 overexpression on mRNA and protein expression in 143B and HOS cells under hypoxia. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 3. (B) Western blotting results revealing the impact of bortezomib treatment on the changes occurring at HIF-1α protein level mediated by Hsa_circ_0000566 silencing and vector transfection. (C) Western blotting assessment of the impact of CHX treatment on the variations in HIF-1α protein levels affected by Hsa_circ_0000566 silencing and vectors. Results are reported as mean ± SD, *p < 0.05, n = 3. (D) The western blot illustrates the effects of Hsa_circ_0000566 knockdown in the Hyp564 HIF-1α protein levels in the presence or absence of bortezomib treatment. (E) Immunoprecipitation assessing the HIF-1α ubiquitination levels in Hsa_circ_0000566 silencing and Hsa_circ_0000566 overexpressing osteosarcoma (OS) cells under hypoxia. Culture media were supplemented with bortezomib (250 nM) for 6 h. (F) The combination of Hsa_circ_0000566 with HIF-1α confirmed by radioimmunoprecipitation (RIP). Results are reported as mean ± SD, *p < 0.05, n = 3. (G) Pulldown assay validation of the interaction between Hsa_circ_0000566 and HIF-1α. (H) A RIP assay of HIF-1α regions interacting with Hsa_circ_0000566. Schematic diagram shows HIF-1α protein fragments. Results are reported as mean ± SD, *p < 0.05, n = 3. (I) Interaction profile between Hsa_circ_0000566 and HIF-1α obtained from catRAPID (left). (J) Schematic diagram showing Hsa_circ_0000566 RNA fragments. Combinative regions between Hsa_circ_0000566 and HIF-1α were identified by RIP assay. Results are reported as mean ± SD, *p < 0.05, n = 3.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Over Expression, Expressing, Standard Deviation, Western Blot, Plasmid Preparation, Transfection, Immunoprecipitation, Biomarker Discovery

Journal: Aging and Disease

Article Title: Positive Feedback Regulation of Circular RNA Hsa_circ_0000566 and HIF-1α promotes Osteosarcoma Progression and Glycolysis Metabolism

doi: 10.14336/AD.2022.0826

Figure Lengend Snippet: Hsa_circ_0000566 promotes osteosarcoma (OS) glucose metabolism and tumorigenesis progression in vivo. (A) 143B cells stably transfected with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or empty vector plasmids. Nude mice were subcutaneously injected with 1 × 10 7 cells that were either stable negative controls or those with Hsa_circ_0000566 knockdown, HIF-1α overexpression, or Hsa_circ_0000566 knockdown. Thirty days after injection, the animals were euthanized, and their tumors dissected and photographed. (B) Tumor weight measurements on the same day the mice were euthanized. Results are reported as mean ± standard deviation (SD), *p < 0.05, n = 5. (C) Tumor volumes (ab2/2) were calculated every 6 d from the day after the mice were injected with stable OS cells. (D-E) Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) exhibit the expression levels of the genes involved in glycolysis metabolism. Results are reported as mean ± SD, *p < 0.05, n = 3. (F) Fluorescence in situ hybridization (FISH), hematoxylin and eosin (H&E) staining, and immunohistochemistry (IHC) analysis indicate the OS organization in mice and relative GLUT1, GLUT4, PDK1, PDK4, and LDHA protein levels in tumors from different groups. (G) In situ tumor formation experiment reveals that HIF-1α overexpression recovered Hsa_circ_0000566 knockdown-induced tumor attenuation. Results are reported as mean ± SD, *p < 0.05, n = 4. (H) Micro-computed tomography (CT) indicates the functions of HIF-1α and Hsa_circ_0000566 knockdown in bone loss. (I) H&E staining of lung metastasis. In mice injected in the tail vein with various stable 143B cells, lung metastasis was detected using an in vivo bioluminescence imaging system. Results are reported as mean ± SD, *p < 0.05, n = 5.

Article Snippet: Human hFOB1.19 osteoblasts, HEK-293, and various osteosarcoma cell lines, including 143B, HOS, MG-63, and U2OS, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Stable Transfection, Transfection, Knockdown, Over Expression, Plasmid Preparation, Injection, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Fluorescence, In Situ Hybridization, Staining, Immunohistochemistry, In Situ, Micro-CT, Imaging

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: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are <1. f) Total MEG3:MEG3 hybrid count against predicted free energy of hybridization (dG) for MEG3 interactions ( red lncRNA:MEG3, blue mRNA:MEG3, green MEG3:antisense, purple snoRNA:MEG3) with free hybridization energy cutoff at dG<-10 kcal mol -1 , as captured by EZH2-FLASH-seq ( i ) vs. IgG control ( ii ) .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, Expressing, Microarray, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: ChIP-sequencing, Microarray, RNA Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, ChIP-sequencing, Gene Expression, Binding Assay, Incubation, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Control, Expressing, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Measure of cell migratory capacity using ECIS functional analysis in ECs treated with control or A-395 (5µM, 24h) inhibitor. Experiments were performed in duplicates (technical replicates) and four experiments were run for migration assay and six for adhesion (biological replicates). The data showing ECIS trace (left hand side) is mean ±SD as calculated by the ECIS. The graph on the right is mean±SEM with N =6, data was compared using ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. b. Adhesion to Fibronectin, FN (20µg/ml) was used to coat the culture plates and assess adhesion of endothelial cells within 3h of ECIS assay, following cell pre-treatment with A-395, 24h. The difference in resistance change was calculated over 3h. c. Subcutaneous Matrigel plug injection (200µl) into mice ( N =5) treated with DMSO (control, left flange) and A-395 (1mg/ml, right flange) was done for 2 weeks. Matrigel plugs were collected and processed for histology. Staining for H3K27me3 was done, displaying nuclear positivity with strong intensity in control (<0.02% DMSO in water) and the A-395 treatment decreased total H3K27me3 staining, as compared by t-test. d. Staining for arterioles was performed to assess vessel growth as angiogenesis and data was compared using Student’s t-test. The data shows increased area of staining for Isolectin B4 (Iso-B4) dye in A-395 vs. DMSO treated Matrigel plugs with increased neovascularization, P<0.05. e. A-395 has increased the percentage of vessels positive for ITGA4 (red) within the Isolectin B4 positive cells, compared with the DMSO using t -test. f. Graphical abstract. 1 Maternally Expressed Gene–MEG3 is highly expressed with hypoxia and bound to EZH2 in endothelial cells (EC) affected by ischaemic insult. 2 Such MEG3:EZH2 complex assembles onto the target genes to 3 direct the EZH2 activity to “write” H3K27me3 trimethylation repressive mark and block expression of target gene i.e. integrin alpha 4 (ITGA4) and its ability to dimerise with integrin beta 1 (ITGB1), leading to 4 reduced EC function as measured by adhesion and migration. Hence 5 targeted disruptions of MEG3:EZH2 interaction, or inhibition of EZH2 activity could increase EC function under ischaemia.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Functional Assay, Control, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: TaqMan gene expression assays used in the study (Thermo-Fisher Scientific Cat Number 4331182).

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Gene Expression

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: RNA-Seq analysis of the PEAC cohort illustrates the differential correlation of the FDC genes associated with early perivascular and late mature developmental stages in the RA synovium. Strong positive correlations of the pericyte/fibroblast markers NG2, THY1 and αSMA with the PDGFR-β/PDGF-BB axis (A-I) , NG2 and FDC-CNA.42/FBXO2 (A-II) , and NG2, THY1 and αSMA (A-III) in RA. (B) Correlations of PDGF-BB and TNFα/LTβ with the expression of each other's receptors and early FDC developmental genes. PDGF-BB positively correlates with its receptor and the TNFα/LTβ receptors (B-I) , TNFα and LTβ negatively correlate with PDGFR-β expression and the early FDC markers NG2 and αSMA (B-II) . (C) Converse correlations of the PDGF-BB/PDGFR-β and the TNF-α/LT-β axes with the expression of mature FDC markers. PDGF-BB/PDGFR-β and TNF-α/LT-β differently correlate with the mature FDC related genes CXCL13 (B cell chemoattractant), BAFF (B cell survival factor), and antigen display and presentation to B cells namely complement receptors (CR1/CD35, CR2/CD21), and Fcg receptors (FcγRIIA/CD32A, FcγRIIB/CD32A). (D) Correlation of the RA synovial pathotypes with the expression of PDGF-BB, PDGFR-β, TNF-α, and LT-β. Person correlation coefficient (r) and adjusted p -values are shown with the corresponding plots and tables.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: RNA Sequencing, Expressing

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: Activation of sorted tonsillar stromal cell subsets with PDGF-BB and TNF-α/LT-β induces early and mature FDC markers in vitro . (A) The CD45 − tonsillar stromal subsets were sorted using combinations of NG2/αSMA, NG2/CNA.42, CNA.42/αSMA and CNA.42/CR2 Abs. (B) Type-1 Pericytes [NG2 + /αSMA + ; indicated by red * in (A,B) ], early FDCs; (CNA.42 + /NG2 + , CNA.42 + /αSMA + , CNA.42 + /CR2 − ; indicated in A and B by blue, magenta, and green *, respectively) and mature FDCs [CNA.42 + /CR2 + , indicated by brown * in (A,B) ] were treated with 300 ng/ml PDGF-BB or 100 ng/ml TNF-α+ 100 ng/ml LT-αβ and the fold change in FBXO2 (CNA.42), αSMA, Collagen 1, CR2, and FcγRIIB gene expression compared to untreated cells was calculated by Livak's DD equation and shown in bar graphs. (C) Treatment of the NG2 + /αSMA + type-1 pericyte subset with PDGF-BB in slide cultures for 6 days induced the expression of the FDC marker CNA.42 compared to untreated cells as demonstrated by Immunocytochemistry. Image J quantification of the mean fluorescence intensity (MFI) of CNA-42 of the different conditions is shown in the histogram. Data is representative of three different experiments and is expressed as the mean ± SEM.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Activation Assay, In Vitro, Gene Expression, Expressing, Marker, Immunocytochemistry, Fluorescence

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: Correlation of IL-6 expression with synovial pathotypes and FDC markers. (A) Boxplots displaying the correlation of synovial IL-6, JAK2, STAT1, and blood IL-6 expression with synovial pathotypes. (B,C) Correlation blots of synovial IL-6, IL-6R, JAK1, and JAK2 expression with the FDC markers complement receptor 1 (CR1/CD35) and CXCL13, respectively. Correlation of STAT3 and STAT1 with CR1 and CXCL13 respectively are also shown. (D) Correlation of synovial IL-6 expression with the markers associated with early FDC differentiation including NG2 (pericytes), αSMA (myofibroblasts), and FBXO2 (CNA.42). (E) IL-6 release from synovial organ and fibroblast cultures stimulated with 300 ng/ml PDGF-BB for 24 hrs and 6 days respectively. (E-I) Synovial organ culture showing a piece of synovial tissue placed in cell culture inserts mounted in 24-well plates (Upper). Diagrammatic representation of the synovial organ culture setup (S = Synovial Tissue, M = Culture Medium, F = Filter Device). (E-II) Rheumatoid arthritis synovial fibroblasts (RASFs) at base line (Day 1 = D1) and after 6-day (D6) stimulation with PDGF-BB. IL-6 levels at baseline and after stimulation are shown in (E-III) . Cultures were run in triplicates and data is expressed as the mean ± SEM. Significance was calculated using 2-tailed unpaired student T test and the p -value between baseline and PDGF-BB stimulation is shown.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Expressing, Organ Culture, Cell Culture

Journal: Frontiers in Medicine

Article Title: Follicular dendritic cell differentiation is associated with distinct synovial pathotype signatures in rheumatoid arthritis

doi: 10.3389/fmed.2022.1013660

Figure Lengend Snippet: The FDC mAb CNA.42 recognizes FBXO2. (A) Immunoprecipitation (IP) and characterization of the CNA-42 binding protein. (A-I) Western blotting of total cell lysate (a), negative control (agarose beads only) and the CNA.42-immunoprecipitated proteins (c and d, 1.5 and 6 uls/lane respectively) from tonsillar single cell suspension probed with CNA-42. A single band is detectable at 120 Kd. (A-II) the reactivity of the CNA.42 mAb on the HuProt™ human proteome microarray showing subarray 9-1 of array 1300017931 (used for the CNA.42) with fluorescence detection at 633 nm excitation (a) and 543 nm excitation (b). (a) Staining with biotinylated anti-GST and Streptavidin-647. Rows 1-28 show generic staining of the GST-tagged immobilized human proteins, among them FBXO2 in row 11. (b) Probing with CNA.42 and Cy3 labeled anti-mouse IgM shows one hit, the human protein FBXO2 in the subarray. (A-III) Western blotting of tonsillar lysates with FBXO2 and CNA-42-specific antibodies recognize 120 Kd bands in the lysates [CNA.42 BP = CNA.42 binding protein]. (B) In situ hybridization of FBXO2 mRNA (green) showing intracellular signal in tonsillar CD21 + FDC reticula (red). (C) Western blotting of lysates from the CAN.42 expressing CEM cell line using mAb CAN.42 and anti FBXO2. CEM were untreated or treated either with Accell human FBXO2 siRNA (1 uM), or non-targeting control (NTC). GAPDH is used as a loading control. Compared to untreated cells, densitometric analysis with Image J indicates that FBXO2 siRNA-treated cells expressed 50% (*) and 35% (**) less FBXO2 and CNA.42, respectively.

Article Snippet: The membranes were probed overnight at 4°C with 2 μg/mL of CNA.42 or 1:200 FBXO2 rabbit polyclonal antibody (Proteintech).

Techniques: Immunoprecipitation, Binding Assay, Western Blot, Negative Control, Suspension, Microarray, Fluorescence, Staining, Labeling, In Situ Hybridization, Expressing, Control

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Graphical representation of Esrp1 (blue) and Esrp2 (red) expression in mouse tissues and cells (BioGPS) . Relatively overlapping expression patterns for Esrp1 and Esrp2, except in the Liver where Esrp2 is expressed and Esrp1 is not. ( B ) (Panel A ) Array of whole-body sections with e10.5 (#1–#3), e12.5 (#4 and #5), e15.5 (#6 and #7) and postnatal (#8 and #9) mice following staining with cresyl violet. (Panel B ) X-ray film autoradiography detection of Esrp2 mRNA, seen as bright labeling. The labeling is clearly detectable at stage e15.5 in the skin. Pronounced signal is detectable in postnatal mice skin, olfactory neuroepithelium, esophagus, stomach and rectum. Moderate labeling is seen in submaxillary gland, liver, lung and kidney. (Panel C ) Control (sense) hybridization in an adjacent section comparable to (panel B ). ( C ) (Panel A ) Whole-body sections of adult mouse (male) following staining with cresyl violet. (Panel B ) X-ray film autoradiography detection of Esrp2 mRNA. The expression pattern encompasses skin, stomach, intestine and gut-associated salivary glands and liver. In the stomach, Esrp2 is expressed in glandular epithelium, but not in non-glandular epithelium. Non-specific labeling is seen in bone (*) and thymus (**). (Panel C ) Control (sense) hybridization. Abbreviations: AT—adipose tissue; Br—brain; C —colon; Es—esophagus; H—heart; K—kidney; Li—liver; Lu—lung; Mu—skeletal muscles; NC—nasal chamber; ONE—olfactory neuroepithelium; Pa—pancreas; R(embryo)/Re(adult)—rectum; Re (embryo)—retina; Sm—submaxillary gland; St—stomach; Tc—telencephalon; Th—thymus; UB—urinary bladder; (as)—antisense; (s)—sense (Magnification: 2.4×). ( D ) Digoxigenin-UTP-labeled in situ hybridization of Esrp1 in P0 epidermis of WT ( Esrp1 +/+ , Esrp2 +/+ ) and KO ( Esrp1 −/− , Esrp2 +/+ ). Esrp1 expression in restricted to the basal keratinocyte (including epithelial cells of the hair follicle) to the granular layer of the epidermis, and absence of signal in the Esrp1 KO epidermis. E: Epidermis, D: Dermis, HF: Hair follicle. ( E ) Expression of Esrp1 and Esrp2 from published microarrays. Esrp1 and Esrp2 are enriched in the epithelial compartments of the skin and associated appendages: Epidermis, Matrix, Outer Root Sheath (ORS), Bulge cells, and Hair Germ (HG) while absent in the non-epithelial cells: Dermis, Dermal Papilla (DP), and Melanocytes. Graphs from left to right: ( , GSE10773), ( , GSE3142), and ( , GSE15185) represent Esrp1 and Esrp2 expression from publically available microarray data. DOI: http://dx.doi.org/10.7554/eLife.08954.004

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Expressing, Staining, Autoradiography, Labeling, Control, Hybridization, Muscles, In Situ Hybridization, Microarray

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. ( B ) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. ( C ) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. ( D ) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. ( E ) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented. DOI: http://dx.doi.org/10.7554/eLife.08954.005

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Knock-In, Southern Blot, Biomarker Discovery, Injection, Sequencing, Construct, Generated, Knock-Out

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: Sagittal sections were generated from WT, KO, and DKO E15.5 embryos. Indicated organs were evaluated for gross morphological defects by H&E staining of sections. Esrp1/Esrp2 DKO embryos do not form lungs (*) and salivary glands (arrow). Thoracic, lung, and kidney images were taken at 4×. Thymus and salivary images were taken at 10× (except for the DKO salivary section for orientation and clear indication of salivary gland agenesis) (n = 3 for each genetic group). DOI: http://dx.doi.org/10.7554/eLife.08954.007

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Generated, Staining

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Representative H&E stained sections of dorsal skin from control Esrp1 +/+ , Esrp2 −/− and Esrp DKO ( Esrp1 −/− , Esrp2 −/− ) E18.5 embryos used in B – E . ( B – D ) Metrics of epidermal thickness measured from basal keratinocyte layer to granular layer ( B ), hair follicle number measured over 9 fields of view ( C ), and hair follicle stages from Esrp DKO (n = 6) and control (CON) (n = 8) littermates. Two-tailed Student's t -test was used for B , C and 2way ANOVA multiple comparisons test for D . ( E ) Immunofluorescence of skin differentiation markers for basal keratinocytes (Krt14 and p63), spinous layer (K10), cornified layer (Loricrin (Lor)), and granular layer (Filaggrin (Fil)). β-catenin and its transcriptional target Lef1 are markers of developing hair follicles (n = 3). DOI: http://dx.doi.org/10.7554/eLife.08954.008

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Staining, Control, Two Tailed Test, Immunofluorescence

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) qRT-PCR expression of Esrp1 and Esrp2 in purified E18.5 epidermis from the designated genetics of Esrp1 and Esrp2 KO embryos (n = 3). Western blot confirmation of Esrp1 and Esrp2 KO in purified epidermis from E18.5 embryos (n = 2). ( B ) Graphical representation of the epithelial -IIIb exon inclusion rates for Fgfr1 , Fgfr2 , and Fgfr3 in epidermis (n = 3). ( C ) Esrp regulated splicing events in Enah and Arhgef11. Graphical representation of Percent Spliced in (PSI) are presented (n = 3). Two-way ANOVA multiple comparisons tests statistical analysis was used and all groups were compared to Esrp1 +/+ , Esrp2 +/+ (WT). Statistical indications for p-values, *<0.05, **<0.01, ***<0.001, ****<0.0001. DOI: http://dx.doi.org/10.7554/eLife.08954.010

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: Quantitative RT-PCR, Expressing, Purification, Western Blot

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Heatmap representing the predicted ΔPSI values for skipped exon (SE) events from KO, KH, and DKO epidermis compared to WT. Summary table of total detected splicing events in KO, KH, and DKO strand-specific RNA-seq. Venn diagram depicting overlap in detected SE events in KO, KH, and DKO datasets. Splicing events detected by rMATS at a FDR < 5% and |deltaPSI| ≥ 5% are depicted here. ( B ) Graphs for PSI for six Esrp targets are shown. Representative radioactive RT-PCR PAGE gels are presented. All events measured are from three independent biological samples. Two-way ANOVA multiple comparisons tests statistical analysis was used and all groups were compared to Esrp1 +/+ , Esrp2 +/+ (WT). ( C ) Representative UCSC custom genome browser snapshots of Esrp1 KO full switch ( Fam213b ), gradual ( Lsm14b ), and DKO only ( Arhgef10l ) SE splicing events. Negative strand transcripts are shown in faded colors compared to bold colored positive strand transcripts. ( D ) Graphical representation of the predicted deltaPSI of 25 ssRNA-seq targets from WT vs DKO rMATS analysis, compared to the RT-PCR validated deltaPSI. Pierson Correlation with r- and p-values is indicated. DOI: http://dx.doi.org/10.7554/eLife.08954.011 10.7554/eLife.08954.012 Figure 4—source data 1. rMATS analysis of Esrp deficient epidermis. DOI: http://dx.doi.org/10.7554/eLife.08954.012 10.7554/eLife.08954.013 Figure 4—source data 2. RNAseq and RT-PCR validated SE splicing events. DOI: http://dx.doi.org/10.7554/eLife.08954.013

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: eLife

Article Title: The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development

doi: 10.7554/eLife.08954

Figure Lengend Snippet: ( A ) Strand-specific RNA-seq custom UCSC genome browser snapshots of Fgfr1- , Fgfr2- , and Fgfr3-IIIb and -IIIc mutually exclusive splicing events. These tracks confirm the variable sensitivity of this family of mutually exclusive splicing events to loss of Esrp1 and Esrp2. All three Fgfr transcripts include the mesenchymally expressed -IIIc isoform when both Esrp1 and Esrp2 are knocked out. Fgfr2 shows no switch in splicing until both Esrps are ablated, whereas both Fgfr1 and Fgfr3 show variable sensitivity to Esrp1 and Esrp2 loss. Negative strand transcripts are shown as faded colors compared to bold colored positive strand transcripts. ( B ) Strand-specific RNA-seq custom UCSC genome browser snapshot of the Cd44 gene. Inclusion of the variable region (10 exons) results in the multiple isoforms termed the Cd44v which are expressed in epithelial cells. KO of Esrp1 and Esrp2 results in full conversion of Cd44v to the mesenchymally expressed Cd44s (short) isoform. This confirms the Esrps also regulate the complex splicing of 10 consecutive exons in Cd44 in vivo. Representative gel of an ethidium bromide stained agarose gel of a Cd44 RT-PCR is shown (n = 3). DOI: http://dx.doi.org/10.7554/eLife.08954.014

Article Snippet: Real-time analysis of Esrp expression was evaluated using Taqman probes for Esrp1 (Mm01220936_g1), Esrp2 (Mm00616290_m1), and Gapdh (Mm99999915_g1) (LifeTechnologies) using a 7500 Fast Realtime machine (AppliedBiosystems).

Techniques: RNA Sequencing, In Vivo, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Journal: Free radical biology & medicine

Article Title: Targeting SLC3A2 subunit of system X C - is essential for m 6 A reader YTHDC2 to be an endogenous ferroptosis inducer in lung adenocarcinoma.

doi: 10.1016/j.freeradbiomed.2021.03.023

Figure Lengend Snippet: Fig. 8. Schematic presentation of the study. Briefly, the m6A reader YTHDC2 is an endogenous ferroptosis inducer and its function is to suppress system XC −. In addition to what we’ve previously reported that the mRNA stability of SLC7A11 is suppressed by YTHDC2, in the present study we also found that SLC3A2, another subunit of system XC − can also be suppressed by YTHDC2 via targeting HOXA13-mediated transcription of SLC3A2, and the m6A methylation and subsequent destabilization of HOXA13 mRNA is a prerequisite for this process.

Article Snippet: Abbreviations YTHDC2 YT521-B homology containing 2 LUAD lung adenocarcinoma SLC7A11 solute carrier 7A11 OS overall survival IB immunoblotting RT-qPCR reverse transcription-quantitative PCR IHC immunohistochemistry ELISA enzyme-linked immunosorbent assay Dox Doxycycline ROS reactive oxygen species EMSA electrophoretic mobility shift assay ChIP chromatin immunoprecipitation RIP RNA immunoprecipitation PAR-CLIP photoactivatable ribonucleotide crosslinking and immunoprecipitation SLC3A2 solute carrier 3A2 HOXA13 Homeo box A13 3′UTR 3′ untranslated region GSH glutathione GPX4 glutathione peroxidase RSL3 RAS-selective lethal 3 m6A N6-methyladenosine W writers E erasers R readers KP KrasG12D and p53R172H (HEK)-293T Human embryonic kidney BME Cultrex Basement Membrane Extract DFO Deferoxamine NAC N-acetyl-cysteine CHX cycloheximide 3-MA 3-Methyladenine ActD Actinomycin D Fer-1 Ferrostatin-1 DAA 3-Deazaadenosine Sh shRNA Sg single-guide RNA CST Cell Signaling Technology TMA tissue microarray assay 4-HNE 4-hydroxynonenal A adenosine C cytosine FISH Fluorescence in situ hybridization MDA malondialdehyde MeRIP-seq Methylated RNA Immunoprecipitation Sequening TSS transcription start site TF transcription factor POLR2L (poly II) RNA polymerase II, I and subunit L. L. Ma et al. Free Radical Biology and Medicine 168 (2021) 25–43

Techniques: Methylation