Journal: Frontiers in Pharmacology

Article Title: Guishaozichuan granules can attenuate asthma in rats via the MUC5AC/EGFR signaling pathway

doi: 10.3389/fphar.2022.1011751

Figure Lengend Snippet: Effects of GSZC granules on lung MUC5AC (A) , EGFR (B) mRNA expression in rats suffering from asthma. Values are the mean ± SD, n = 6 for each group. ▲▲ p < .01 versus the normal group, ** p < .01 versus the model group.

Article Snippet: MUC5AC antibody was purchased from Boster Group (20180824; Wuhan, China).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Guishaozichuan granules can attenuate asthma in rats via the MUC5AC/EGFR signaling pathway

doi: 10.3389/fphar.2022.1011751

Figure Lengend Snippet: Effects of GSZC granules on lung MUC5AC (A) , EGFR (B) protein expression in rats suffering from asthma. Values are the mean ± SD, n = 6 for each group. ▲▲ p < .01 versus the normal group, * p < .05, ** p < .01 versus the model group.

Article Snippet: MUC5AC antibody was purchased from Boster Group (20180824; Wuhan, China).

Techniques: Expressing

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 for culturing primary human bronchial epithelial cells (HBECs) at an air-liquid interface (ALI) with IL-13 stimulation. On day 0, cells are predominantly basal cells (orange). ALI culture allows differentiation into ciliated (magenta) and secretory cells (cyan). The mucin 5B (MUC5B)-rich mucus gel layer (red) changes to a MUC5AC-rich gel (green) upon IL-13 stimulation. (B and C) Effects of IL-13 on secretory cells compared with basal cells (B) or ciliated cells (C). Each point represents a gene. Genes that were regulated differently between cell types are shown in black (log 2 fold change difference > 1 and FDR < 0.1 for interactions between cell type and cytokine effect), and other genes are shown in gray. R , Pearson correlation coefficient (p < 2.2 × 10 −16 for both comparisons). (D) Relative expression of 20 genes with the highest absolute IL-13-induced fold change in any of the three cell types. (E) Gene set enrichment analysis of IL-13-induced cell type-selective responses in HBECs. Relative enrichment coefficients were calculated for the full collection of 2,555 gene ontology biological process (GOBP) gene sets; results are shown for the five GOBP gene sets that were most highly induced by IL-13 in basal, ciliated, and secretory cells (p < 10 −5 in one cell type and enrichment coefficient < 0.5 in other cell types). (F) IL-13-induced genes in secretory cells are highly enriched for a previously defined set of goblet cell genes not included in GOBP. For comparison, unlabeled points represent values for the full collection of 2,555 GOBP gene sets. (G) Distribution of IL-13-enriched H3K27ac peaks (N = 387) by genomic region classification. (H) Bulk RNA-seq expression changes in genes closest to H3K27ac ChIP-seq peaks that were not significantly affected (p > 0.1) by IL-13 (no Δ, 26,875 peaks) or were significantly (FDR < 0.1) enriched (387 peaks) or depleted (215 peaks) after IL-13 stimulation. Shown are p values for comparison with unaffected peaks by two-sided Wilcoxon test. (I) The most highly enriched motif discovered in regions with enriched H3K27ac after IL-13 stimulation closely resembles a previously defined STAT6 binding motif. See also and and – .

Article Snippet: Either anti-MUC5AC-DL488 or anti-MUC5AC-DL405 (45M1, mouse IgG 1 ; Novus Biologicals, Centennial, CO) antibody was used.

Techniques: Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding 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: Either anti-MUC5AC-DL488 or anti-MUC5AC-DL405 (45M1, mouse IgG 1 ; Novus Biologicals, Centennial, CO) antibody was used.

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: (A) Scheme of IL-13-responsive, secretory cell-specific, SPDEFe -driven CRISPRi. minP, minimal promoter. (B–H) A lentivirus containing a KRAB-dCas9 transgene driven by SPDEFe (or other enhancers) and a second lentivirus driving expression of sgRNA targeting the SPDEF (SPDEF-TSS(+34)) or MUC5AC (MUC5AC-TSS(+134)) promoter were used in combination. Transduced cells were ALI cultured without cytokine stimulation or with IL-13 (B–E) or IL-1β (F–H) stimulation for the last 7 days of culture, as indicated. Changes in expression of dCas9 (B and F), SPDEF (C and G), and MUC5AC (D and H) were measured by quantitative real-time PCR. MUC5AC-producing cells (E) were quantitated by flow cytometry. mRNA levels and MUC5AC-producing cells are relative to mRNA levels and MUC5AC-producing cells from IL-13-stimulated cells with SV40e-KRAB-dCas9 and NT-2 sgRNA in the same donor. IL-13 data (B–E) are from four donors. The IL-1β data (F–H) are from cells from three donors, including two used for the IL-13 experiment. *p < 0.05, **p < 0.01, ****p < 0.0001 compared with the unstimulated empty vector control for NT gRNA and IL-13-stimulated empty vector control for SPDEF and MUC5AC gRNAs by one-way ANOVA Tukey’s post-test (B–H).

Article Snippet: Either anti-MUC5AC-DL488 or anti-MUC5AC-DL405 (45M1, mouse IgG 1 ; Novus Biologicals, Centennial, CO) antibody was used.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Flow Cytometry, Plasmid Preparation

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: Either anti-MUC5AC-DL488 or anti-MUC5AC-DL405 (45M1, mouse IgG 1 ; Novus Biologicals, Centennial, CO) antibody was used.

Techniques: Recombinant, SYBR Green Assay, Software

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-MUC5AC (45M1) [DyLight 488] (mouse monoclonal) , Novus Biologicals , Cat# NBP2-32732G.

Techniques: Recombinant, SYBR Green Assay, Software