Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: PLoS Genetics

Article Title: A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development

doi: 10.1371/journal.pgen.1006951

Figure Lengend Snippet: (A) Pie-chart summarizing the results from the microarray analysis of E12.5 ureters explanted and treated with DMSO or 10 μM cyclopamine for 18 h filtered with an intensity (Int) threshold of 150 and a fold change (FC) cut-off of 2.0. (B) Table of the downregulated transcripts. Shown are average intensities of transcripts in control and cyclopamine treated ureters and average fold changes (FC) of RNA intensities between the pools in two independent experiments. (C) In situ hybridization analysis of expression of microarray candidates on proximal ureter sections of control, Tbx18 cre/+ ; Smo fl/fl ( Smo LOF ) and Tbx18 cre/+ ; R26 mTmG/SmoM2 ( Smo GOF ) ureters at E12.5 and E14.5.

Article Snippet: For pharmacological manipulation of SHH signaling cyclopamine (Selleckchem) and purmorphamine (Millipore) were used at a final concentration of 10 μM and 2 μM, respectively.

Techniques: Microarray, Control, In Situ Hybridization, Expressing

Journal: PLoS Genetics

Article Title: A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development

doi: 10.1371/journal.pgen.1006951

Figure Lengend Snippet: (A-U) Ureters were explanted from E12.5 wildtype, Axin2 creERT2/+ ; Hprt Foxf1/y , Tbx18 cre/+ ; Hprt Foxf1DN/y or Tbx18 cre/+ ; R26 mTmG/+ embryos and cultured for 6 d in the presence or absence of 10 μM cyclopamine, 100 ng/μl BMP4, 10 μg/ml NOGGIN, 2 μM Purmorphamine or solvent as indicated. Whole explants were documented by epifluorescence analysis (Q), or were sectioned and proximal regions analyzed by Haematoxylin and Eosin staining (A,E,I,M,R), by immunofluorescence (B-D,F-H,J-L,N-P,S-U) for the SMC marker ACTA2 together with the epithelial marker CDH1 (B,F,J,N,S), for the SMC marker TAGLN without (C,G,K,O) or with the lineage marker GFP (T), and for the urothelial markers ΔNP63/UPK1B (D,H,L,P,U).

Article Snippet: For pharmacological manipulation of SHH signaling cyclopamine (Selleckchem) and purmorphamine (Millipore) were used at a final concentration of 10 μM and 2 μM, respectively.

Techniques: Cell Culture, Solvent, Staining, Immunofluorescence, Marker

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: 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: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells

doi: 10.1007/s10815-012-9824-9

Figure Lengend Snippet: Representative pictures show double staining of OCT4 and NANOG in the human morula and blastocyst. a Immunostaining of OCT4 in the morula. b Immunostaining of NANOG in the same morula. c DAPI staining in the morula. d Staining of OCT4 in the blastocyst. Immunostaining is seen both in the inner cell mass and the trophoblast; arrows. e Staining of NANOG in the same blastocyst. Staining is seen only in the inner cell mass; arrow. f DAPI staining in the same blastocyst. g Exclusion of OCT4 antibody. h Exclusion of NANOG antibody. i DAPI staining of the embryo without primary antibodies present

Article Snippet: Primary antibodies—monoclonal mouse antibodies for OCT4 (sc-5279, Santa Cruz Biotechnologies, Santa Cruz, USA), diluted 1:80, and monoclonal goat antibody for NANOG (MAB1997, R&D systems, Minneapolis, MN USA), diluted 1:200—were added in 5 % blocking buffer overnight at 4 °C and washed three times with PBS to remove any unbound primary antibodies.

Techniques: Double Staining, Immunostaining, Staining

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells

doi: 10.1007/s10815-012-9824-9

Figure Lengend Snippet: In situ hybridization of NANOG in the human pre-implantation embryo. a 2-cell embryo, day 1. b 4-cell embryo, day 2. c 8-cell embryo, day 3. d morula, day 4. e late day 4 embryo. f blastocyst, day 5. g mouse ESCs hybridized with human NANOG primer

Article Snippet: Primary antibodies—monoclonal mouse antibodies for OCT4 (sc-5279, Santa Cruz Biotechnologies, Santa Cruz, USA), diluted 1:80, and monoclonal goat antibody for NANOG (MAB1997, R&D systems, Minneapolis, MN USA), diluted 1:200—were added in 5 % blocking buffer overnight at 4 °C and washed three times with PBS to remove any unbound primary antibodies.

Techniques: In Situ Hybridization

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells

doi: 10.1007/s10815-012-9824-9

Figure Lengend Snippet: Immunostaining of OCT4 and NANOG in human embryonic stem cells, and in situ hybridization of NANOG in embryonic stem cells. a Light microscopy of human embryonic stem cells. b NANOG immunostaining in embryonic stem cells. The feeder cells do not show staining for NANOG (red arrow). c Nuclear staining of embryonic stem cells and fibroblast cells. DAPI staining is seen in feeder cells (blue arrow). d & e In situ hybridization showing NANOG mRNA in embryonic stem cells. NANOG f Mouse embryonic stem cells hybridized with human NANOG primer. g OCT4 immunostaining of stem cell line HS426 (green). Blue colour shows nuclear DAPI staining

Article Snippet: Primary antibodies—monoclonal mouse antibodies for OCT4 (sc-5279, Santa Cruz Biotechnologies, Santa Cruz, USA), diluted 1:80, and monoclonal goat antibody for NANOG (MAB1997, R&D systems, Minneapolis, MN USA), diluted 1:200—were added in 5 % blocking buffer overnight at 4 °C and washed three times with PBS to remove any unbound primary antibodies.

Techniques: Immunostaining, In Situ Hybridization, Light Microscopy, Staining

Journal: Journal of Assisted Reproduction and Genetics

Article Title: Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells

doi: 10.1007/s10815-012-9824-9

Figure Lengend Snippet: Data from microarray analysis of human embryos and embryonic stem cells and fibroblasts. For Nanog, the average signal intensity from one probe set and for Oct4 the average number of three probe sets is shown

Article Snippet: Primary antibodies—monoclonal mouse antibodies for OCT4 (sc-5279, Santa Cruz Biotechnologies, Santa Cruz, USA), diluted 1:80, and monoclonal goat antibody for NANOG (MAB1997, R&D systems, Minneapolis, MN USA), diluted 1:200—were added in 5 % blocking buffer overnight at 4 °C and washed three times with PBS to remove any unbound primary antibodies.

Techniques: 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 ) 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: Nature Communications

Article Title: The oncogene AAMDC links PI3K-AKT-mTOR signaling with metabolic reprograming in estrogen receptor-positive breast cancer

doi: 10.1038/s41467-021-22101-7

Figure Lengend Snippet: a Analysis of somatic alterations of AAMDC using cancer genomic data sets and tools available from cBioPortal (see “Methods”). The frequency of amplification is shown as a percentage and the sample numbers are shown in brackets. METABRIC Molecular Taxonomy of Breast Cancer International Consortium, TCGA The Cancer Genome Atlas, BRCA Breast Cancer, INSERM Institut national de la santé et de la recherche médicale, MBC Metastatic Breast Cancer, NSCLC non-small-cell lung carcinoma, FHCRC Fred Hutchinson Cancer Research Center, NEPC National Environment Protection Council, PanCan Pan-Cancer. b Kaplan–Meier survival plots for patients with tumors expressing high (red) or low (green) levels of AAMDC mRNA. The lower left plots correspond to luminal B tumors treated with tamoxifen (see “Methods”). The p value shown for each plot is determined by the log-rank test. GEO Gene Expression Omnibus, GSE genomic spatial event, NSCLC non-small-cell lung carcinoma. c Localization of the AAMDC protein in tumors from a breast tissue microarray (TMA) assessed by immunohistochemistry (IHC). Representative IHC sections of normal breast tissue, estrogen receptor-negative (ER − ) tumor tissue, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) are shown. 0, 1+, 2+, 3+ indicate the staining intensity score. d Associations between AAMDC expression (IHC) and lymph node metastasis (LN + ) as well as tumor grade, tumor size (T3-4), and ER positivity (ER + ) by AAMDC localization from the same TMA. Statistical significance is indicated by Chi-square analysis with a one-tailed p -value relative to ER − tissue. For T3-4: * p = 0.03; for LN + : * p = 0.03; for ER + , from left to right: * p = 0.003, * p = 0.005, * p = 0.005. n = 60 biologically independent samples. Full details of the TMA are provided in Supplementary Table . e Frequency of AAMDC amplification/polysomy in a cohort of 119 luminal B breast cancer specimens. Representative fluorescence in situ hybridization (FISH) images are indicated, with specific probes for AAMDC (red) and Centromere enumeration 11 probe for chromosome 11 ( C11 , green). The full clinical and pathological features of these tumors are shown in Supplementary Data . f Real-time expression analyses (qRT-PCR) of AAMDC in luminal, non-luminal, and normal-like breast cells. Significance levels are determined relative to MCF-12A by Ordinary one-way ANOVA with Dunnett multiple comparison test. Data are presented as mean values ± SEM (* p = 0.0217, ** p = 0.0018, **** p < 0.0001). n = 3 biologically independent RNA extractions. Representative images of immunocytochemistry (ICC) and FISH of selected luminal cell lines are presented. HuMECs non-transformed human mammary epithelial cells.

Article Snippet: For experiments involving non-genetically manipulated human mammary epithelial cells (HuMECs) (Fig. ), human primary mammary epithelial cells were purchased from ATCC (HMEC, PCS-600-010).

Techniques: Amplification, Expressing, Gene Expression, Microarray, Immunohistochemistry, In Situ, Staining, One-tailed Test, Fluorescence, In Situ Hybridization, Quantitative RT-PCR, Comparison, Immunocytochemistry, Transformation Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 1 Regulation of PKN kinase activity. IP-kinase assays with WT and TM mutants of PKN1 (S916A) and PKN2 (T958A). Torin inhibited the PKN kinase activity to about the same extent as mutating the TM in both PKN isoforms. B, The PKN1 TM mutant S916A has reduced kinase activity toward multiple substrates. C, Deletion of the PKN N-terminus results in constitutive histone H3 phosphorylation in vitro and in cells. D and E, The PKN1 TM mutant S916A dramatically reduces autophosphorylation as well as Histone H3 and MARCKS phosphorylation

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, In Vitro

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 3 Torin and rapamycin sensitivity of PKN, AKT, and PKCα. A, Cells stably transduced with WT PKN1 were treated with a range of torin and rapamycin concentrations for 24 h, and analyzed by using pan- and phosphosite-specific antibodies. B, Cells were treated with torin and rapamycin during a time course up to 24 h and subsequently analyzed by using pan- and phosphosite- specific antibodies

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Stable Transfection, Transduction, Phospho-proteomics

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 2 PKN contains a TM phosphorylated by a torin- sensitive kinase. A, Alignment of TM sequences with the predicted phosphorylated residues indicated (bold). B, Transfection of PKN1 bearing mutations in the TM (S916A), activation loop (T774E) and ATP binding pocket (K644E) probed with antibodies specific for phos-S916 and phos-T774. Including nonphospho-TM peptide during the antibody incubation reduces the detection of non- phosphorylated PKN. C, IP-blot of WT PKN1 expressed in cells treated with torin and rapamycin

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Transfection, Activation Assay, Binding Assay, Incubation

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 4 Cell motility functions of PKN. A, Localization of Flag-tagged PKN1 (green) at the cleavage furrow during mitosis, imaged by confocal microscopy. B, Examples of binucleate cells generated in response to depletion of PKN1, PKN2, and Ect2 (positive control), indicative of cytokinesis failure. C, Quantification of cytokinesis failure data as a consequence of PKN1 and PKN2 depletion. D, Expression levels (immunoblotting) of PKN1 and PKN2 after siRNA depletion. E, Stable C4-2b cell lines showing that (E) ectopic expression and (F) knockdown increase and decrease, respectively, cell migration in a Boyden chamber assay (****P < 0.0001). G, Transient depletion of PKN1, PKN2, and the TORC2 subunit Rictor reduces cell invasion of PC-3 cells to a similar extent as torin treatment

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Confocal Microscopy, Generated, Positive Control, Expressing, Western Blot, Knockdown, Migration, Boyden Chamber Assay

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 5 Analysis of PKN isoform expression in human prostate cancer. A, Representative IHC showing PKN1 protein levels in normal, primary tumor, and lymph node metastasis. B, PKN1 and PKN2 expression (using microarray data from reference 47) in normal prostate, primary tumor, and metastases. C, RNA expression (using RNAseq data from TCGA) of PKN1-3 isoforms, PTEN, PKCα, AKT, and select mTOR components. **P < 0.01, ***P < 0.001, ****P < 0.0001

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Expressing, Microarray, RNA Expression

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 6 Pkn2 is required for embryonic development. A, Embryos from Pkn1 and Pkn2 lacZ reporter mice were stained for β- galactosidase activity, and are shown as whole mount images. Upper row: E10.5, E11.5, E11.5. Scale bars: 1.0 mm. Bottom row: E6.5, E8.5 (side and dorsal view), E9.5, E9.5. Scale bars: 0.2 mm, 0.5 mm, 1.0 mm. B, Whole mount images of Pkn2 heterozygotes and homozygous null embryos at E7.0, E7.75, and E9.5. Scale bars 0.2 mm (upper four panels) 1.0 mm. C, Whole mount images of wild-type and Pkn2 null embryos analyzed by whole mount in situ hybridization for Otx2 (E7.5) and Bra (E7.25) are shown. Scale bars: 0.2 mm

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining, Activity Assay, In Situ Hybridization

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 7 Analysis of PKN1 overexpression in prostate. A, Immunoblots showing transgenic expression of full-length (Tg-PKN1) and constitutively active (Tg-PKN1ΔN) proteins in anterior, dorsal, lateral, and ventral lobes (AP, DP, LP, VP). B-E, H&E stained images of sections through the ventral prostates from mice of the indicated genotypes are shown. The ages of the mice are as follows: WT, 53 weeks; Tg-PKN1, 58 weeks; Tg-PKN1ΔN, 58 weeks; Tg-AKT1, 52 weeks; Tg-AKT1;Tg-PKN1, 41 weeks; Tg-AKT1;Tg-PKN1ΔN, 52 weeks; TRAMP and TRAMP;Tg-PKN1, 16 weeks (showing HGPIN); TRAMP and TRAMP;Tg-PKN1, 17 weeks (showing small cell carcinoma). All images were captured at 200× magnification. Lower magnification views of the same samples are also provided (Supplemental Figure S3)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Over Expression, Western Blot, Transgenic Assay, Expressing, Staining

Journal: The Prostate

Article Title: The protein kinase C super-family member PKN is regulated by mTOR and influences differentiation during prostate cancer progression.

doi: 10.1002/pros.23400

Figure Lengend Snippet: FIGURE 8 Analysis of PKNs in Pten null prostate tumors. H&E stained images of sections through the prostates from mice of the indicated genotypes are shown. All images were captured at 200× magnification and are of the ventral prostate, except for the right-most image in panel D, which shows squamous differentiation from the anterior prostate. The ages of the mice (panels A–C) are as follows: Ptenr/r, 12 and 45 weeks; Ptenr/r;Tg-PKN1, 12 and 43 weeks; Ptenr/r;Pkn1r/r;Pkn2r/r, 26 and 45 weeks. D, The images of invasive cancer (left and middle) are from 53-week ventral prostates, the squamous differentiation shown to the right is from the anterior prostate of a 53-week animal. Lower magnification views of the same samples are also provided (Supplemental Figure S4)

Article Snippet: PKN1 (Homo sapiens transcript variant 2, Origene, Rockville, MD, TC118456) and PKN2 (Addgene, Cambridge, MA, #20587) were cloned into pcDNA3 (Thermo Fisher Scientific, Grand Island, NY) along with anN-terminal Flag tag.

Techniques: Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and immunostaining of the Samd7−/− retina. (A) In situ hybridization analysis of Samd7 in developing and adult mouse retinas. No Samd7 signal was detected at E17.5, but weak Samd7 expression was observed in the neuroblastic layer at P1. P6 and P9 retinas exhibited Samd7 signals in the prospective photoreceptor layer, and P14 and adult (4 wk, 4W) retinas express Samd7 in the photoreceptor layer. (B) Immunostaining of a P4 WT retinal section using anti-Samd7 (red) and anti-Thrb2 (a cone photoreceptor marker; green) antibodies. Cell nuclei were stained with DAPI. The Samd7 signals did not substantially overlap with Thrb2-positive cone photoreceptor cells (arrows). Dotted lines indicate heterochromatin regions. (C) Samd7 immunostained signals (green) were mainly observed in DAPI (blue)-negative euchromatin regions in the P12 retina. The photoreceptor nuclear membrane was immunostained with the anti-lamin B antibody (red). Dotted lines indicate heterochromatin regions. (D) Retinal sections from WT and Samd7−/− mice at P9 were immunostained using the anti-Samd7 antibody (green) with DAPI (blue). The Samd7 signal in the photoreceptor layer disappeared in the Samd7−/− mice. (E) Retinal sections from adult WT and Samd7−/− mice were immunostained with anti–S-opsin (red) and anti-rhodopsin antibodies (green) with DAPI (blue). Ectopic expression of S-opsin in rod outer segments was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; NBL, neuroblastic layer; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Immunostaining, In Situ Hybridization, Marker, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 expression and generation of the Samd7−/− allele. (A) Northern blot analysis of mouse Samd7 in developing and adult retinas. Northern blot analysis of Samd7 transcripts was performed using mRNAs purified from retinas of mice between P1 and 4 wk of age. (Upper) A major band of ≈2.4 kb Samd7 mRNA was detected. (Lower) Ethidium bromide staining of RNAs. (B) Immunostaining of P6 WT mouse retinal sections using antibodies against Samd7 (red), Thrb2 (a cone precursor marker, green), and S-opsin (an S-cone marker, blue). Dotted lines indicate Thrb2-positive cone photoreceptor nuclei. The Samd7 signal is obviously weaker both in S-opsin–positive S-cones (arrows) and in S-opsin–negative M-cones (arrowheads) than in rods. (C) Diagram of the targeting vector and the Samd7−/− allele. Removal of exons 4–6 is predicted to result in a translational frame shift and a complete loss of Samd7 function. (D) RT-PCR analysis of the Samd7 transcript in the Samd7−/− retina. An approximately 500-bp fragment of Samd7 was amplified from cDNA prepared from the WT retina. No Samd7 transcript was detected in the Samd7−/− retina. β-Actin was used as a loading control. (E) Western blot analysis of the Samd7 protein in the Samd7−/− retina. An approximately 56-kDa Samd7 band was detected in the WT retina. No Samd7 band was detected in the Samd7−/− retina. β-Actin was used as a loading control.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Northern Blot, Purification, Staining, Immunostaining, Marker, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Immunohistochemical analysis of the Samd7−/− retina. (A) Retinal sections from 2-mo-old WT and Samd7−/− mice were immunostained with the anti–M-opsin antibody (a marker for cone outer segments, green) and PNA (a cone photoreceptor outer and inner segment marker, red). No obvious difference was observed between WT and Samd7−/− retinas. (B) The distribution of S-opsin (red) and rhodopsin (green) signals was normal in the Samd7+/− retina at 2 mo. (C) At P9, ectopic S-opsin signals in premature rod photoreceptor outer segments were observed in the Samd7−/− retina. (D) Retinal sections from dorsal and ventral regions of 2-mo-old WT control and Samd7−/− mice were immunostained using anti–S-opsin (red) and anti-rhodopsin (green) antibodies with DAPI (blue). There were fewer S-opsin–positive cones in the dorsal region than in the ventral region of the control retina. Ectopic S-opsin expression in rod outer segments was similar in the dorsal and ventral regions of the Samd7−/− retina. (E) Flat-mount immunostaining of dorsal and ventral regions of WT control and Samd7−/− retinas. M-opsin (green) and Rhodamine-PNA (red) were used for visualizing cone photoreceptor cells. The numbers of M-opsin and PNA double-positive cells decreased in the ventral region in both WT control and Samd7−/− retinas. (F) Retinal sections from WT and Samd7−/− mice were immunostained with anti-Pikachurin (a marker of photoreceptor synaptic clefts, red) and anti-Ctbp2 (a marker of synaptic ribbons, green) antibodies. No obvious difference was observed between WT and Samd7−/− retinas at 2 mo. (G) Immunofluorescent examination of WT and Samd7−/− retinal sections from 2-mo-old mice using anti-Calbindin (a marker for amacrine and horizontal cells, red), anti-S100b (a maker for Müller glia, green), anti-Chx10 (a marker for bipolar cells, red), and anti-Pax6 (a marker for amacrine and ganglion cells, green) antibodies showed no obvious difference between WT and Samd7−/− retinas. (H) Retinal sections from WT and Samd7−/− mice at 12M were stained with toluidine blue. The thickness of the ONL of WT and Samd7−/− retinas was measured. Average layer thickness in the WT retina was set to 100%. No significant change in ONL thickness was observed in the Samd7−/− retina. GCL, ganglion cell layer; INL, inner nuclear layer; n.s., not significant; ONL, outer nuclear layer; OS, outer segments.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Immunohistochemical staining, Marker, Expressing, Immunostaining, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. ERGs were recorded from WT (n = 5) and Samd7−/− (n = 5) mice at 12 wk. (A and B) Representative scotopic (A) and photopic (B) ERGs in WT and Samd7−/− mice elicited by white light. (C) The amplitude of the scotopic ERG a-wave as a function of the stimulus intensity (1.0 log cd⋅s⋅m−2) was significantly reduced in Samd7−/− mice. (D) The amplitude of the scotopic ERG b-wave as a function of the stimulus intensity (−1.4 log cd⋅s⋅m−2) decreased significantly in Samd7−/− mice. Error bars show the SD. **P < 0.03.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: ERG analysis of Samd7−/− mice. (A) The a-wave amplitudes of scotopic ERGs from WT (n = 5) and Samd7−/− (n = 5) mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2) at 12 wk. The amplitude of the a-wave at +1.0 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (B) The b-wave amplitudes of scotopic ERG from WT and Samd7−/− mice elicited by seven different white light stimuli (−6.2, −5.0, −3.8, −2.6, −1.4, −0.2, and 1.0 log cd⋅s⋅m−2). The amplitudes of the b-wave at −2.6 and −1.4 log cd⋅s⋅m−2 significantly decreased in Samd7−/− mice. (C) The b-wave amplitudes of photopic ERG from WT and Samd7−/− mice elicited by four different white light stimuli (−0.8, −0.2, 0.4, 1.0 log cd⋅s⋅m−2). No obvious change in amplitude was observed between WT and Samd7−/− mice. Error bars show the SD.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Global change of retinal expression profile in the Samd7−/− retina. (A) Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and cone-enriched (red circle) and rod-enriched (yellow circle) genes. Microarray analysis was performed using mRNAs from the WT and Samd7−/− retinas at P12. One hundred sixty-three genes were up-regulated (signal log ratio greater than +1.0, blue circle), and 251 genes were down-regulated (signal log ratio less than −0.5, green circle) in the Samd7−/− retina compared with those in the WT retina. (B) Lists of the 33 genes that overlap between cone-enriched genes and up-regulated genes in the Samd7−/− retina (Left) and the 23 genes that overlap between rod-enriched genes and down-regulated genes in the Samd7−/− retina (Right). Cone- and rod-enriched genes (more than fourfold FPKM value) were identified using RNA-seq data from a previous study (22). (C and D) The expression levels of the selected genes were measured by qRT-PCR using mRNAs of WT and Samd7−/− retinas at P12. Up-regulation of nonrod genes (C) and down-regulation of rod genes (D) in the Samd7−/− retina by microarray analysis were confirmed. (E) Nonrod genes are ectopically expressed in the ONL of the Samd7−/− retina. In situ hybridization analysis of WT and Samd7−/− retinal sections at P12 was performed using probes for the up-regulated genes in the Samd7−/− retina: Cacna1h (a bipolar cell gene), Gngt2, Cnga3, and S-opsin (cone-specific genes). The vertical brackets indicate the extent of the ONL. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Error bars show the SD (n = 4). **P < 0.03. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing, Microarray, RNA Sequencing Assay, Quantitative RT-PCR, In Situ Hybridization

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 interacts with Phc proteins in PRC1 and a rod TF Nr2e3. (A) Yeast two-hybrid assay using full-length Samd7 as the bait. Proteins containing SAM domains with high similarity to Samd7 (Samd7/11, Phc1/2/3, and L3mbtl3) were identified as Samd7 interactor proteins. Growth on selective plates lacking histidine (−His) and adenine indicates a physical interaction between the bait and prey constructs. (B) Immunoprecipitation analysis of Samd7 and Phc2. A plasmid expressing FLAG-Samd7 or FLAG-Phc2 was transfected with a plasmid expressing GFP-Samd7 into HEK293 cells. FLAG-tagged proteins were immunoprecipitated using an anti-FLAG antibody. GFP-Samd7 was coimmunoprecipitated with FLAG-Samd7 or FLAG-Phc2. (C) Immunoprecipitation was performed using mouse retinal lysate from 2-mo-old mice using an anti-Phc2 antibody. Immunoprecipitated Samd7 with Phc2 was detected by Western blot analysis using the anti-Samd7 antibody. (D) A 3D structural model of the EH and ML surfaces of the Samd7 SAM domain. The putative 3D structure of the Samd7 SAM domain was obtained by MODELLAR (https://salilab.org/modeller/) using the Phc3 SAM domain as a template [Protein Data Bank (PDB) ID code 4PZN]. Two molecules of the Samd7 SAM domain (green and blue) are shown. Amino acids essential for interaction on the EH surface (L372) and the ML surface (L358/H363) are indicated in red. (E) Immunoprecipitation of FLAG-Samd7-WT with GFP-Samd7-WT or GFP-Samd7-LR (with the L372R mutation on EH surface of the SAM domain). Reduced interaction between FLAG-Samd7-WT or FLAG-Samd7-LR and GFP-Samd7-LR was observed compared with that between FLAG-Samd7-WT and GFP-Samd7-WT. (F) FSEC analysis of the Samd7 protein. Plasmids expressing GFP-fused WT Samd7 (GFP-Samd7-WT) and Samd7 with a mutation on the EH surface of the SAM domain (GFP-Samd7-L372R) were transfected into HEK293 cells, and the fluorescent signals in lysates were analyzed by FSEC. In GFP-Samd7-WT lysates, a putative monomer peak between the 44- and 158-kDa size markers and a broad peak likely corresponding to polymers larger than 669 kDa (arrowhead) were observed. Putative monomer and oligomer peaks but no obvious peaks at a higher molecular size were detected in GFP-Samd7-L372R lysates. (G) Yeast two-hybrid assay using Samd7 with mutations in the SAM domain ML (L358R/H363R, HR) and/or EH (L372R, LR) surfaces as baits. The interaction of Samd7 constructs with SAM domain proteins (Samd7/11, Phc1/2/3, L3mbtl3) was analyzed. Samd7 with mutations both on the ML and EH surfaces (L372R/L358R/H363R, LRHR) showed no interaction with any of the SAM domain proteins tested (Samd7/11, Phc1/2/3, L3mbtl3). (H) Immunoprecipitation analysis of Samd7 with rod photoreceptor transcription factors. A plasmid expressing FLAG-Samd7 was transfected with a plasmid expressing HA-Nr2e3 or HA-Nrl into HEK293 cells. FLAG-tagged Samd7 was immunoprecipitated using an anti-FLAG antibody. The interaction of Samd7 with Nr2e3 was observed, whereas Samd7 showed no substantial interaction with Nrl.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Y2H Assay, Construct, Immunoprecipitation, Plasmid Preparation, Expressing, Transfection, Western Blot, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Partial colocalization of Samd7 and Phc2 in rod photoreceptor nuclei. Retinal sections at P6, P12, and 1 mo (A and B) and at 2 mo (A and C) were immunostained with the anti-Samd7 (red) and anti-Phc2 (green) antibodies. Small points of Samd7 and Phc2 partially colocalized (arrowheads in A) surrounding heterochromatin regions of rod photoreceptor nuclei stained with DAPI (blue). Subnuclear localization of Phc2 was unchanged in the photoreceptor nuclei in the Samd7−/− retina compared with that in the control retina (C).

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Analysis of Samd7 function in transcriptional regulation. (A) Samd7 did not affect transcriptional activation of the Rhodopsin promoter by Crx and Nrl. A luciferase reporter construct driven by the Rhodopsin promoter was cotransfected with Crx and Nrl expression plasmids into HEK293 cells, and luciferase activities of cell lysates were measured at 48 h after transfection. Cotransfection of the Samd7 expression plasmid did not affect the transactivation of the Rhodopsin promoter by Crx and Nrl. (B) Localization of Samd7 in Polycomb bodies. Phc2-dependent colocalization of Samd7 with Ring1B in Polycomb bodies in HEK293 cells. A plasmid expressing GFP-Samd7-WT was transfected with a plasmid expressing FLAG-Phc2 into HEK293 cells. Cells were stained with anti-FLAG and anti-Ring1B antibodies. Puncta of GFP-Samd7 (green) colocalized with the Ring1B signals (blue, a marker for Polycomb bodies) were observed in Polycomb bodies (arrowheads) in a Phc2-dependent manner (red). (C) Localization of Samd7 in Polycomb bodies. Subcellular localization of various Samd7-mutant proteins in U2OS cells was analyzed. U2OS cells were transfected with FLAG-tagged Samd7-mutant protein expression plasmid and were immunostained with anti-FLAG and anti-Ring1B antibodies with DAPI (blue). Puncta of the FLAG signal (green) colocalized with Ring1B signals (red) in Polycomb bodies of cells transfected with Samd7-WT or Samd7-del2 expression plasmids (arrowheads). In contrast, FLAG signals did not colocalize with the Ring1B signal in Polycomb bodies of cells transfected with the Samd7-del1, -del3, or -L372R expression plasmid. In cells with a high-level expression of Samd7-WT, -del1, or -del2 proteins, Polycomb bodies are not formed, likely due to Ring1B depletion. Dotted lines indicate cell nuclei. (D) Schematic diagrams showing the structure of Samd7-mutant proteins. Two highly conserved homology domains (HD1 and HD2) in the Samd7 protein, based on amino acid residue homology among species from mouse to zebrafish, were identified. Samd7-del1, -del2, and -del3 constructs lack the HD1, HD2, and SAM domain, respectively. The Samd7-L372R construct has a point mutation in the SAM domain disrupting its oligomerization activity. (E) Indirect physical interaction between Samd7 and Ring1B through Phc2. The immunoprecipitation assay was performed using HEK293 cells transfected with FLAG-Samd7, Myc-Ring1B, and HA-Phc2 expression plasmids. Myc-Ring1B was coimmunoprecipitated with FLAG-Samd7 in a Phc2-dependent manner. (F) Partial colocalization of Samd7 with Suz12, H2A119ub, and H3K27me3 but not with H3K4me3 marks in U2OS cells. The plasmid expressing FLAG-Samd7-WT was transfected into U2OS cells and was immunostained with anti-Suz12 (a component of PRC2), anti-H2A119ub, anti-H3K27me3, and anti-H3K4me3 antibodies. Samd7 colocalized with Suz12, H3K27me3, and H2AK119ub in some of Polycomb bodies (arrowheads). In contrast, the active histone mark H3K4me3 did not colocalize with Samd7-positive puncta in cell nuclei.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Activation Assay, Luciferase, Construct, Expressing, Transfection, Cotransfection, Plasmid Preparation, Staining, Marker, Mutagenesis, Activity Assay, Immunoprecipitation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Samd7 regulates H3K27me3 in the retina. (A and B) ChIP-seq profiles of Cnga3 (A) and Cacna1h (B) loci for H3K27me3 in developing rod photoreceptor cells at three different stages (P2, P10, and P28). H3K27me3 levels at Cnga3 and Cacna1h genes, which were up-regulated in the Samd7−/− retina, increased along with rod photoreceptor development. The ChIP-seq profiles were analyzed using previous ChIP-seq data (21). (C) A Venn diagram of up-regulated (blue circle) and down-regulated (green circle) genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in the developing rods (red circle). Thirty-one genes overlapped between the up-regulated genes in the Samd7−/− retina and the genes with increased H3K27me3 marks in developing rods. (D–F) ChIP-qPCR analysis of H3K27me3 (D) and H2AK119ub (E) on the selected up-regulated genes in Samd7−/− retinas. H3K27me3 levels of the selected genes in WT and Samd7−/− retinas were analyzed by ChIP-qPCR. The H3K27me3 levels of the selected up-regulated genes (Cacna1h, Cnga3, Gngt2, En2, S-opsin, and Rxrg) in WT and Samd7−/− retinas are indicated. No significant change in H3K27me3 levels was observed for the Hoxc13 and Gnat2 promoters. The H2AK119ub levels of the selected up-regulated genes in the Samd7−/− retina (Cacna1h, Cnga3, En2, and S-opsin) significantly decreased in the Samd7−/− retina. (F) ChIP with IgG was performed as a negative control. Error bars show the SD (n = 3). **P < 0.03, *P < 0.05. n.s., not significant.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: ChIP-sequencing, Negative Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: A proposed model of the Samd7 function in rod photoreceptor cells. (Left) In WT rod photoreceptor cells the Samd7-PRC1 complex induces chromatin condensation together with PRC2 and increases H3K27me3 marks on the S-opsin promoter. Increased H3K27me3 represses S-opsin expression in rod photoreceptor cells. (Right) In the Samd7−/− retina, Samd7 deletion causes a reduction of H3K27me3 and H2AK119ub on the S-opsin promoter, resulting in the ectopic expression of S-opsin in rod photoreceptor cells.

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

doi: 10.1073/pnas.1707021114

Figure Lengend Snippet: Primer sequences

Article Snippet: The GST-tagged Samd7 fusion protein was expressed in Escherichia coli strain BL21-DE3 and was purified with Glutathione Sepharose 4B (GE Healthcare).

Techniques: In Situ

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and endothelial cell marker CD31 in the blood vessels of human nasal turbinates using six different anti-ACE2 antibodies and anti-CD31. b Double immunofluorescence staining of ACE2 and type II pneumocyte marker mucin 1 (MUC1) in the human lung using six different anti-ACE2 antibodies and anti-MUC1. Abcam ab15348 clone yielded the most robust staining of pneumocytes, while the other clones showed negligible or less specific membrane staining. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Marker, Staining, Clone Assay, Membrane

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: Representative images of human tissues on a tissue microarray (TMA) stained by chromogenic immunohistochemistry using antibodies targeting the ACE2 protein (brown) and counterstained with hematoxylin (blue). Highest ACE2 expression was observed in the villi of the intestinal tract (jejunum), renal tubules, testis, and glandular cells in the seminal vesicle. Minimal to no/non-specific staining can be seen in the heart, stomach, spleen, skin, and liver. Staining of lung pneumocytes was observed using Abcam ab15348, and less specifically with Sigma HPA000288 (Fig. 2b; Supplementary Table 1 ). Scale bars: 100 μm.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Microarray, Staining, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative double immunofluorescence staining of ACE2 and acetylated α-tubulin (ACTUB) on normal human nasal turbinate, ethmoid sinus, uncinate process (sinus), trachea, and bronchus, using anti-ACE2 and anti-ACTUB antibodies, respectively. b Representative double immunofluorescence staining of ACE2 and ACTUB on normal C57BL/6J mouse nasal turbinate and trachea. c Immunofluorescent staining of (top panel) ACE2, cilia marker ADP-ribosylation factor-like protein 13B (ARL13B), and cilia centrosome marker FGFR1 oncogene partner (FOP); (bottom panel) ACE2, and cilia markers ACTUB and ARL13B in a ciliated mouse cell line, IMCD3. d Immunofluorescent staining of ACE2 in the primary cilia of IMCD3 cells transiently transfected with human ACE2 (yellow outline) compared to endogenous mouse ACE2 (blue outline). e Quantified percentages of endogenous ACE2-positive cilia (34.67 ± 13.58%; control (Ctrl)) versus cilia with overexpressed human ACE2 (82.67 ± 4.73%). Ciliated cells were identified by staining of ARL13B. Error bars represent mean ± SD. ( n = 100 cells examined per experiment over three independent experiments). (Two-tailed Student’s t test, ** p = 0.004). f Representative multiplexed images of in situ hybridization against the SARS-CoV-2 Spike mRNA, in combination with immunofluorescence staining of ACE2 and the differentiated epithelial cell marker cytokeratin 8 (KRT8). SARS-CoV-2 Spike mRNA expression (red) was detected within ciliated epithelial cells containing motile cilia positive for ACE2 (green). The nuclei were stained using DAPI (blue) as a counterstain. Scale bars: 20 μm ( a , b top panels; f large panels); 5 μm ( a , b bottom panels; f small panels); 2 μm ( c , d ).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Double Immunofluorescence Staining, Staining, Marker, Transfection, Control, Two Tailed Test, In Situ Hybridization, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Representative immunofluorescence double staining of ACE2 and mucin 5AC (MUC5AC) reveals absence of co-localization of ACE2 within secretory goblet cells in the human nasal turbinate, uncinate process, and bronchus. b Representative in situ hybridization using an ACE2 probe in combination with an anti-MUC5AC antibody. ACE2 mRNA expression (red dots) was not detected within goblet cells marked by MUC5AC in the nasal turbinate, uncinate process, and trachea. Nuclei were stained using DAPI. Scale bars: 20 μm (top) and 5 μm (bottom).

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Immunofluorescence, Double Staining, In Situ Hybridization, Expressing, Staining

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a No statistically significant changes in ACE2 expression was detected among patients less than or greater than 65 years of age, males versus females, and patients with varying smoking history. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). b No statistically significant difference in ACE2 expression was observed between healthy controls and patients with chronic rhinosinusitis with polyps (CRSwNP) or without polyps (CRSsNP). (Kruskal–Wallis test, p > 0.05). c No statistically significant difference in ACE2 expression was noted between distinct human nasal tissue sites/regions. (Two-tailed Mann–Whitney test or Kruskal–Wallis test, p > 0.05). UNC uncinate process, Turb nasal turbinates, Eth ethmoid sinus, NP benign nasal polyps. The bottom and top of the box plots represent the 25th and 75th percentiles, respectively. The bands within the box show the median value, and the whiskers extending from both ends of the boxes are minimum and maximum values. Each dot represents one patient.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: a Quantification of ACE2 in controls and patients taking ARBs and ACEI. In the Stanford cohort, ACE2 is slightly but statistically significantly lower in patients taking ACEI (0.19 ± 0.02) compared to controls (0.26 ± 0.06). (Kruskal–Wallis test p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). There were no statistically significant differences in ACE2 expression between patients taking ARBs and controls in the Stanford, National Taiwan University Hospital (NTUH), and China Medical University Hospital (CMUH) cohorts. b In the Stanford cohort, when including only controls with hypertension (HTN) on other medications (“HTN w/o ARBs/ACEI”), ACE2 expression was statistically different between the groups (Kruskal–Wallis test, p = 0.044) but Dunn’s multiple comparison post-hoc test did not reveal any statistical significance between the three groups. No statistically significant differences were seen among patients taking ARBs compared to controls. c When cohorts from all three institutions were normalized by Z -score and integrated, patients taking ACEI (−0.72 ± 0.42) had a lower ACE2 expression compared to controls with hypertension (0.41 ± 1.07). (Kruskal–Wallis test, p = 0.032; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.043). Patients taking ARBs (−0.15 ± 0.95) showed a trend towards lower ACE2 compared to controls with hypertension, but this was not statistically significant. d ACE2 expression among patients of older (≥65 years) and younger (<65 years) age taking ARBs or ACEI was not statistically divergent from control patients of the same age group. (Kruskal–Wallis test, p > 0.05). e ACE2 expression among male and female patients on ARBs or ACEI trended comparably or lower than same-sex controls except for males taking ARBs in the CMUH group who showed a trend towards higher ACE2 expression. No statistically significant differences were observed. (Kruskal–Wallis test, p > 0.05). f Among non-smokers, there was a statistically significant trend towards lower ACE2 expression in patients taking ACEI compared to controls in the Stanford group (Kruskal–Wallis test, p = 0.021; Dunn’s multiple comparison post-hoc test, *adjusted p = 0.035). No statistical significance was observed with the non-smokers on ARBs. All data are noted as mean ± SD. Kruskal–Wallis test was used for three group comparisons and two-tailed Mann–Whitney test was used for two-group comparisons. Box plots are similar in format to Fig. .

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: Comparison, Expressing, Medications, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

doi: 10.1038/s41467-020-19145-6

Figure Lengend Snippet: The luminal differentiated airway epithelial cells consist of ciliated columnar cells (~80%) and secretory goblet cells (~20%). Club cells are infrequently found in the human upper airway. The basal cell layer, which faces the lamina propria, is comprised of both basal and suprabasal cells, which are considered multipotent progenitors capable of renewing the airway epithelium. This schematic depicts how SARS-CoV-2 may bind to ACE2 expressed on the cilia of the nasal cavity following exposure to respiratory droplets or airborne particles.

Article Snippet: Amplification of the ISH probes was performed the next day according to manufacturer’s protocol (323100, Bio-Techne), with the final deposition of Cyanine 3 for ACE2 mRNA probe targets (NEL744001KT, Akoya Biosciences).

Techniques: