Journal: Allergy, asthma & immunology research

Article Title: TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFβ1/STAT3 Action.

doi: 10.4168/aair.2024.16.4.399

Figure Lengend Snippet: Fig. 6. Tnfrsf11a+/− alleviated HDM-induced asthma. (A) Strategy for HDM-induced asthma in Tnfrsf11a+/− mice (n = 6). (B) Effects of the Tnfrsf11a+/− on HDM- induced collagen I, vimentin, and N-cadherin verified by Western blot. (C-F) Relative protein level of (B). (G) BALF IL-4 and (H) Serum IgE detected by ELISA. (I) Effects of the Tnfrsf11a+/− on HDM-induced inflammation detected by HE staining. (J) Effects of the Tnfrsf11a+/− on HDM-induced airway remodeling detected by Masson staining. Magnification 100×, scale bar: 100 μm. Data are expressed as the means ± standard deviation of 3 independent experiments. TNFRSF11A, TNF receptor superfamily 11A; HDM, house dust mite; BALF, bronchoalveolar lavage fluid; IL, interleukin; Ig, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin–eosin. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Lentiviruses of human TNFSF11 shRNA (sc-29464-V), human TNFRSF11A shRNA (sc-42960-V), human TGFβ1 shRNA (sc-270322-V), and human TGFβR1 shRNA (sc-40222-V) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Standard Deviation

Journal: Allergy, asthma & immunology research

Article Title: TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFβ1/STAT3 Action.

doi: 10.4168/aair.2024.16.4.399

Figure Lengend Snippet: Fig. 7. STAT3 signaling involved in HDM-induced asthma in Tnfsf11+/− or Tnfrsf11a+/− mice (n = 6). (A) Volcano plots for screening differential expression proteins with thresholds of P < 0.05 and fold change > 2. (B) KEGG enrichment after HDM-induced Tnfsf11+/− vs. HDM-induced WT. (C, D) Enriched signaling pathway of JAK-STAT validated by Western blot. (E) p-STAT3/STAT3 ratio in mice with HDM-induced Tnfsf11+/− vs. HDM-induced WT. (F) p-STAT3/STAT3 ratio in mice with HDM-induced Tnfrsf11a+/− vs. HDM-induced WT. (G) The p-STAT3 and EPCAM around the airway epithelium in Tnfsf11+/− mice with HDM-induced asthma measured by double-labeling immunofluorescence. Magnification 200×, scale bar: 50 μm. (H) p-STAT3 intensity analysis. Data are expressed as the means ± standard deviation of 3 independent experiments. STAT3, signal transducer and activator of transcription 3; HDM, house dust mite; TNFSF11, TNF ligand superfamily member 11; TNFRSF11A, TNF receptor superfamily 11A; KEGG, Kyoto Encyclopedia of Genes and Genomes; WT, wild type. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Lentiviruses of human TNFSF11 shRNA (sc-29464-V), human TNFRSF11A shRNA (sc-42960-V), human TGFβ1 shRNA (sc-270322-V), and human TGFβR1 shRNA (sc-40222-V) were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative Proteomics, Western Blot, Labeling, Immunofluorescence, Standard Deviation

Journal: Allergy, asthma & immunology research

Article Title: TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFβ1/STAT3 Action.

doi: 10.4168/aair.2024.16.4.399

Figure Lengend Snippet: Fig. 8. Intervention on TNFSF11/TNFRSF11A axis alleviated HDM-induced remodeling proteins with HBE cells by STAT3 phosphorylation. (A) Strategy for cell models. (B-E) Effects of TNFSF11 shRNA on HDM-stimulated HE, p-STAT3, collagen I, and vimentin. (F-I) Further analysis on the effects of TNFRSF11A shRNA on HDM-stimulated HE, p-STAT3, fibronectin, and collagen I. Magnification 200×, scale bar: 50 μm. Data are expressed as the means ± standard deviation of 3 independent experiments. TNFSF11, TNF ligand superfamily member 11; TNFRSF11A, TNF receptor superfamily 11A; HDM, house dust mite; HBE, human bronchial epithelial; STAT3, signal transducer and activator of transcription 3; shRNA, short hairpin RNA; HE, hematoxylin–eosin; LV-NC, negative control lentivirus; NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Lentiviruses of human TNFSF11 shRNA (sc-29464-V), human TNFRSF11A shRNA (sc-42960-V), human TGFβ1 shRNA (sc-270322-V), and human TGFβR1 shRNA (sc-40222-V) were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, shRNA, Standard Deviation, Negative Control

Journal: Allergy, asthma & immunology research

Article Title: TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFβ1/STAT3 Action.

doi: 10.4168/aair.2024.16.4.399

Figure Lengend Snippet: Fig. 10. Effects of denosumab (anti-human TNFSF11) on HDM-induced humanized HSC-NOG-EXL murine. (A) Strategy for HDM-induced asthma in HSC-NOG-EXL murine (n = 6). (B) Western blot analysis of TNFSF11, TNFRSF11A, IL-4, IL-5, and EMBP. (C) TNFSF11/GAPDH ratio. (D) TNFRSF11A/GAPDH ratio. (E) IL-4/GAPDH ratio. (F) IL-5/GAPDH ratio. (G) EMBP/GAPDH ratio. (H) HE, (I) PAS, (J) Masson staining to examine the effects of denosumab on HSC-NOG-EXL mice with HDM- induced asthma. (K) Mouse lungs stained with TGFβ1 by immunofluorescence. (L) Mouse lungs stained with p-STAT3 by immunofluorescence. (M-Q) Intensity analysis of (H-L). Magnification 100×, scale bar: 100 μm. Magnification 200×, scale bar: 50 μm. Data are expressed as the means ± standard deviation of 3 independent experiments. TNFSF11, TNF ligand superfamily member 11; HDM, house dust mite; TNFRSF11A, TNF receptor superfamily 11A; IL, interleukin; EMBP, eosinophil major basic protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HE, hematoxylin–eosin; PAS, periodic acid–Schiff; TGFβ1, transforming growth factor β1; STAT3, signal transducer and activator of transcription 3; NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Lentiviruses of human TNFSF11 shRNA (sc-29464-V), human TNFRSF11A shRNA (sc-42960-V), human TGFβ1 shRNA (sc-270322-V), and human TGFβR1 shRNA (sc-40222-V) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Staining, Immunofluorescence, Standard Deviation

Journal: Allergy, asthma & immunology research

Article Title: TNFSF11/TNFRSF11A Axis Amplifies HDM-Induced Airway Remodeling by Strengthening TGFβ1/STAT3 Action.

doi: 10.4168/aair.2024.16.4.399

Figure Lengend Snippet: Fig. 11. Structure of TNFSF11/TNFRSF11A axis aggravated airway remodeling by strengthening TGFβ1/STAT3 signaling in HDM-induced asthma. TNFSF11, TNF ligand superfamily member 11; TNFRSF11A, TNF receptor superfamily 11A; TNFRSF11B, TNF receptor superfamily 11B; TGFβ1, transforming growth factor β1; TGFβR1, transforming growth factor β receptor type 1; STAT3, signal transducer and activator of transcription 3; HDM, house dust mite.

Article Snippet: Lentiviruses of human TNFSF11 shRNA (sc-29464-V), human TNFRSF11A shRNA (sc-42960-V), human TGFβ1 shRNA (sc-270322-V), and human TGFβR1 shRNA (sc-40222-V) were purchased from Santa Cruz Biotechnology.

Techniques:

Journal: iScience

Article Title: A rationally designed optochemogenetic switch for activating canonical Wnt signaling

doi: 10.1016/j.isci.2023.106233

Figure Lengend Snippet: Rational design of a dual-triggered Wnt signaling activator (A) Schematic diagram depicting a simple workflow of the Wnt signaling activator known as optochemoWnt. Canonical Wnt signaling can be activated by the clustering of LRP6c, c-terminal cytosolic part of the Wnt co-receptor. The clustering of LRP6c as a final output is modulated by dual-triggers based on an AND logic gate. (B and C) Construct configurations and schematic workflow of (B) cytosolic localizing and (C) membrane anchoring optochemoWnt. The only difference between the two prototypes was the absence or presence of a transmembrane module within the N-terminus of the first component. The first component contains the optical actuator of CRY2 and one of the chemical linkers, FRB. The second includes another chemical linker, FKBP, and LRP6c. The light-induced oligomerization of CRY2olig is transmitted into the clustering of LRP6c in the presence of rapamycin-dependent heterodimerization. (D) Four-days scheduled experimental time-line for optochemoWnt. Seeding of HEK293T cells (Day 1), transfection of plasmid vectors (Day 2), optical and chemical stimulation (Day 3), and SEAP-based reporter assay (Day 4) were done at 24-h intervals. (E) Blue light- and rapamycin-dependent Wnt activation of cytosolic localizing and membrane anchoring optochemoWnt. Wnt activity of optochemoWnt was monitored using a SEAP-based assay. Membrane anchoring optochemoWnt shows a higher efficiency of Wnt activation and a clearer AND-gated pattern compared with the cytosolic localizing construct. For activation of optochemoWnt, a 4 h illumination (duty cycle: 2 s on/58 s off) with blue light (470 nm) and treatment with 100 nM rapamycin was carried out. Data were collected from three biologically independent experiments and are presented as means ± S.D. The white circles represent the measured value of individual experiments. Statistical analysis was done using a two-way ANOVA followed by Tukey’s multiple comparisons test (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: The CRY2PHR-mCh-LRP6c plasmid vector was obtained from Addgene (42960) and the coding sequences of FKBP and FRB were synthesized (Cosmo Genetech, Republic of Korea).

Techniques: Construct, Transfection, Plasmid Preparation, Reporter Assay, Activation Assay, Activity Assay, SEAP Assay

Journal: iScience

Article Title: A rationally designed optochemogenetic switch for activating canonical Wnt signaling

doi: 10.1016/j.isci.2023.106233

Figure Lengend Snippet: Engineering and optimization of optochemoWnt (A) Graphical illustration showing the light-independent, nonspecific clustering of LRP6c resulting from overcrowded optochemoWnt onto with planar arrangements. (B) Caged optochemoWnt by integrating the light-induced photo switch, iLID. Folded and closed conformation of DARK-stated iLID minimizes external accessibility of LRP6c (left), whereas blue light-induced opening of iLID maximizes external accessibility of LRP6c (right). (C) Plasmid configuration of opened (upper; NES-FKBP-LRP6c) and caged optochemoWnt (lower; NES-FKBP-iLID-LRP6c). (D) Optochemogenetically induced SEAP expression of opened (upper) and caged (lower) optochemoWnt in HEK293T cells. Summary graph showing increased fold-change of caged optochemoWnt activity (19.0x) compared with the previous one (8.7x). The white circles represent individual measurements from three independent experiments. The data are presented as means ± S.D. Two-way ANOVA followed by Tukey’s multiple comparisons test. (∗∗∗∗p<0.0001). (E) OptochemoWnt variants with n- or c-termini truncated LRP6c (Δ1-64, Δ1-98, Δ208-218, and Δ193,218) based on the spatial distribution of LRP6c motifs (motif A, B, C, D, and E). (F) Optochemogenetic SEAP expression of five optochemoWnt truncated variants including WT. Summary graph shows optochemogenetically induced folds of five optochemoWnt variants. Loss of any motif of optochemoWnt generates a significant reduction in Wnt activity, whereas N-terminal trimming of LRP6c (Δ1-64) without deleting the motif exhibits a higher fold (39.9x) change compared with the WT (17.2x). The measured values were collected from three biologically independent samples. The white circles represent individual measurements. The data are presented as means ± S.D. from three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess the significance of the differences between the blue light/rapamycin group and the other groups. ∗p < 0.05, ∗∗∗∗p < 0.0001.

Article Snippet: The CRY2PHR-mCh-LRP6c plasmid vector was obtained from Addgene (42960) and the coding sequences of FKBP and FRB were synthesized (Cosmo Genetech, Republic of Korea).

Techniques: Plasmid Preparation, Expressing, Activity Assay