Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet: (A) Scheme of the lentivirus-based MPRA. Oligonucleotides with each candidate cis- regulatory sequence (CRS) were cloned into the lentiMPRA vector to create the plasmid library, which was sequenced to associate the sequence and its BCs. A lentivirus library prepared from the plasmid library was transduced into HBECs, which were ALI cultured and treated with or without IL-13. RNA and DNA were harvested and sequenced for BC and UMI counts. (B) Selection of 1,904 CRSs for the MPRA. (C) MPRA results for CRSs. RNA/DNA ratios observed in untreated (UT) cells were plotted against those in IL-13-stimulated cells (combined data from two donors). IL-13-responsive CRSs are shown in black (>1.5-fold increase or decrease in IL-13 versus control, RNA/DNA ratio > 95th percentile of 100 scrambled negative control sequences, FDR < 0.1). (D and E) Validation of 18 selected CRSs from the MPRA in HBECs. HBECs from a single donor were individually transduced with lentiviral GFP reporter constructs of CRSs differentiated in ALI culture without (gray) or with (black) IL-13 stimulation during the last 7 days of culture. Mean fluorescence intensity (MFI), measured by flow cytometry, was compared with RNA/DNA ratios from the MPRA. Pearson correlation coefficient (p < 0.0001). (F) Reporter expression of the top three IL-13-inducible CRSs was measured in basal, ciliated, and secretory cells using a flow cytometry panel. “Reporter IL-13 fold change” represents the overall IL-13 effect seen in all cells. Cell type selectivity was quantified by taking the ratio of MFI in relevant cell types and normalizing it to the ratio observed for the empty construct. (G and H) SPDEFe - and IGHEe -driven reporter expression in basal (nerve growth factor receptor [NGFR] + carcinoembryonic antigen-related cell adhesion molecule 6 [CEACAM6] − ), ciliated (acetylated α-tubulin [ac-α-Tub] + ), and secretory (CEACAM6 + NGFR − ) cells. Shown are representative histograms from one of four donors (G) and IL-13-induced fold changes in reporter expression (MFI) in each cell type (H). **p < 0.01, ***p < 0.001 for comparison between cell types by two-way ANOVA Tukey’s post-test; all other differences were not statistically significant. (I) Reporter activity in undifferentiated HBECs and two lung epithelial cell lines, BEAS-2B and A549. Representative histograms from at least three donors or experiments are shown. See also – and .

Article Snippet: Anti-ac-α-Tubulin (6-11B-1) [Alexa Fluor 647] (mouse monoclonal) , Santa Cruz Biotechnology , Cat# sc-23950AF647; RRID: AB_628409.

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Cell Culture, Selection, Negative Control, Transduction, Construct, Fluorescence, Flow Cytometry, Expressing, Activity Assay

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet: (A) Scheme of CRISPRi targeting of SPDEFe . (B–D) CRISPRi effects on gene expression. HBECs were transduced with lentiviruses driving expression of dCas9-KRAB and non-targeting (NT) control sgRNAs (gray/black) or sgRNAs targeting the SPDEF promoter (red), MUC5AC promoter (blue), or SPDEFe (orange). After differentiation, cells were left unstimulated or stimulated with IL-13 for 7 days, as indicated. Expression of SPDEF (B), FOXA3 (C), and MUC5AC (D) was measured by quantitative real-time PCR. mRNA levels are relative to IL-13-stimulated HBECs with NT control sgRNAs. (E) CRISPRi effects on intracellular MUC5AC were quantified by flow cytometry. Each point corresponds to a different gRNA targeting the indicated region, tested separately in a single culture well from the same donor (B–E). **p < 0.01, ****p < 0.0001 for comparison with IL-13-stimulated HBECs with NT control sgRNAs by one-way ANOVA with Dunnett’s post-test (B–E). (F) Effects of targeting SPDEFe and surrounding regions on IL-13-induced MUC5AC production. gRNAs used in (B)–(E) were compared with gRNAs targeting flanking regions ~2 and 4 kb away from SPDEFe (magenta) in a separate set of experiments (three donors, two replicates per donor). To combine results from two donors, values for MUC5AC-producing cells are shown as percentages of mean values for IL-13-stimulated cells with NT sgRNAs in the same donor. **p < 0.01, ****p < 0.0001 compared with IL-13-stimulated HBECs with NT-1 sgRNA by one-way ANOVA with Tukey’s post-test. p values for all comparisons are provided in . (G–J) CRISPRi effects on mucin staining and mucociliary transport. HBECs were treated as above using NT-2 gRNA, SPDEF -TSS(+34) gRNA, or SPDEFe (+87) gRNA. Sections (G) and extracellular mucus gels from whole-mount preparations (H) were stained for MUC5AC (cyan), MUC5B (red), and the ciliated cell marker ac-α-Tub (yellow). Nuclei were stained with DAPI (purple). Scale bars: 20 μm (G) and 100 μm (H). Images are representative of two experiments with different donors. Mucociliary transport rates were determined from trajectories of fluorescent microspheres placed on gels atop cells (I and J). Shown is superimposition of 10 images acquired at 1-s intervals; scale bars: 50 μm (I). Microsphere speeds were determined from three donors, one well per donor, four fields per well (J). Values represent median microsphere speed for each field. Boxes extend from the 25th to the 75th percentile, horizontal lines within the box indicate means, and whiskers represent minimum and maximum values. *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey’s post-test. See also and and .

Article Snippet: Anti-ac-α-Tubulin (6-11B-1) [Alexa Fluor 647] (mouse monoclonal) , Santa Cruz Biotechnology , Cat# sc-23950AF647; RRID: AB_628409.

Techniques: Expressing, Transduction, Real-time Polymerase Chain Reaction, Flow Cytometry, Staining, Marker

Journal: Cell genomics

Article Title: Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma

doi: 10.1016/j.xgen.2022.100229

Figure Lengend Snippet:

Article Snippet: Anti-ac-α-Tubulin (6-11B-1) [Alexa Fluor 647] (mouse monoclonal) , Santa Cruz Biotechnology , Cat# sc-23950AF647; RRID: AB_628409.

Techniques: Recombinant, SYBR Green Assay, Software

Journal: Scientific reports

Article Title: Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice.

doi: 10.1038/s41598-020-58568-5

Figure Lengend Snippet: Figure 1. Aortic calcification and smooth muscle phenotype changes in SM-specific Asah1 KO mice. Representative images of aortic sections stained by (A) Alizarin Red S (red color) and (C) Von Kossa (black color) staining showed that aorta of Asah1fl/fl/SMCre mice exhibited more calcification compared with littermates (Asah1fl/fl/SMwt and WT/WT). (B,D) Summarized data in the bar graph clearly revealed that lysosomal acid ceramidase (Asah1 gene) contribute to the development of AMC. Representative immunohistochemical images from the aorta and quantitative analysis shows that immunostaining of osteogenic markers. (E,F) OSP (brown stain) and (G,H) RUNX2 (brown stain) significantly increased in the aortic media of Vit D-treated Asah1fl/fl/SMCre mice compared to their littermates. Smooth muscle cell (SM); Osteopontin (OSP); Runt-related transcription factor 2 (RUNX2); AMC (arterial medial calcification). Data are shown as means ± SEM, (n = 6). *P < 0.05 vs. WT/WT Vehl; #P < 0.05 vs. WT/WT Vit D group by two-way ANOVA followed by Duncan’s test.

Article Snippet: Mouse monoclonal antibody against α-SMA (ab7817, Abcam, USA), rabbit polyclonal antibodies against acid ceramidase-α (ACα) (sc-292176, Santa cruz, USA), RUNX2 (ab23981, Abcam, USA), OSP (ab63856, Abcam, USA), SM22-α (ab14106, Abcam, USA), VPS16 (Cat. No.17776-1-AP, Protein biotech group, USA), Rab7 (ab137029, Abcam, USA), CD63 (ab216130, Abcam, USA), annexin-II (AnX2, ab41803, USA), and alkaline phosphatase (ALP, sc-28904, Santa Cruz, USA).

Techniques: Staining, Immunohistochemical staining, Immunostaining

Journal: Scientific reports

Article Title: Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice.

doi: 10.1038/s41598-020-58568-5

Figure Lengend Snippet: Figure 10. Schematic model demonstrating lysosomal Ac-mediated sEV secretion from SMCs during vascular calcification. Lysosomal Ac controlling ceramide metabolism involved in regulation of lysosomal TRPML1- mediated Ca2+ release and interactions of lysosome and MVBs which is associated with sEV excretion in arterial SMCs during vascular calcification. Ac: Acid ceramidase; SMCs: Smooth muscle cells.

Article Snippet: Mouse monoclonal antibody against α-SMA (ab7817, Abcam, USA), rabbit polyclonal antibodies against acid ceramidase-α (ACα) (sc-292176, Santa cruz, USA), RUNX2 (ab23981, Abcam, USA), OSP (ab63856, Abcam, USA), SM22-α (ab14106, Abcam, USA), VPS16 (Cat. No.17776-1-AP, Protein biotech group, USA), Rab7 (ab137029, Abcam, USA), CD63 (ab216130, Abcam, USA), annexin-II (AnX2, ab41803, USA), and alkaline phosphatase (ALP, sc-28904, Santa Cruz, USA).

Techniques: