Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: GPR120/FFAR4 stimulation attenuates airway remodeling and suppresses IL-4- and IL-13-induced airway epithelial injury via inhibition of STAT6 and Akt.

doi: 10.1016/j.biopha.2023.115774

Figure Lengend Snippet: Fig. 1. GPR120 agonist alleviates allergic airway inflammation and mucin secretion in OVA-induced asthmatic mice. Schematic overview of OVA-induced asthma experiment. Mice were sensitized and challenged with OVA and were intraperitoneally treated with vehicle, 20 mg/kg/day of GSK137647A, a GPR120 agonist or 5 mg/kg/day of dexamethasone (DEX). (B) Hematoxylin and eosin (H&E) staining of lung tissue sections. Infiltration of inflammatory cells in peribronchiolar regions (arrowheads). Scale bar: 100 µm. (C) Histopathological scores of peribronchiolar inflammatory cell infiltration. (D) Inflammatory cell counts in the bronchoalveolar lavage fluid (BALF). (E) Spleen to body weight ratio. (F) Mucin production in bronchiolar epithelial goblet cells stained with Periodic Acid-Schiff (PAS). Scale bar: 100 µm. (G) Histopathological scores of PAS-stained mucin production in bronchiolar epithelial goblet cells. (H) Levels of MUC5AC secretion in BALF measured by ELISA. (I) Representative Western blot analysis images of GPR120 protein expression in lung tissue samples (3 independent sets of experiment). (J) Densitometry values of GPR120 expression in the lung tissue samples. (n = 3–6 per condition; *P < 0.05; **P < 0.01, compared with control. #P < 0.05; ##P < 0.01; ###P < 0.001, compared with OVA-challenged group; ANOVA with Bonferroni multiple comparison test.).

Article Snippet: MUC5AC levels in BALF were determined by ELISA (CSB-E11040m; Cusabio, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: GPR120/FFAR4 stimulation attenuates airway remodeling and suppresses IL-4- and IL-13-induced airway epithelial injury via inhibition of STAT6 and Akt.

doi: 10.1016/j.biopha.2023.115774

Figure Lengend Snippet: Fig. 2. GPR120 inhibits IL-4- and IL-13-induced production of MUC5AC and cytokines in 16HBE human bronchial epithelial cells. 16HBE monolayers were incu bated with 10 ng/mL each of IL-4 and IL-13 and co-treated with or without 1 µM of GSK137647A for 48 h. To confirm activation of GPR120, cells were pretreated with 100 µM of AH7614, GPR120 antagonist for 1 h before the experiment. (A) Representative Western blot analysis images of GPR120 expression (3 independent sets of experiment). (B) Expression of MUC5AC (green) as depicted by immunofluorescent staining. Scale bar: 50 µm. (C) Fluorescent intensity ratio (MUC5AC to nuclei). (D) mRNA expression of MUC5AC. (E) Expression of cytokine transcripts IL6, IL8, IL25, and TSLP. (ns, non-significant difference; *P < 0.05; **P < 0.01, ***P < 0.001; ANOVA with Bonferroni multiple comparison test. n = 6–7 per condition.).

Article Snippet: MUC5AC levels in BALF were determined by ELISA (CSB-E11040m; Cusabio, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Expressing, Staining, Comparison

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: GPR120/FFAR4 stimulation attenuates airway remodeling and suppresses IL-4- and IL-13-induced airway epithelial injury via inhibition of STAT6 and Akt.

doi: 10.1016/j.biopha.2023.115774

Figure Lengend Snippet: Fig. 7. Schematic diagram demonstrating mechanisms by which GPR120 stimulation suppresses cytokine-induced airway remodeling in asthmatic models. Acti vation of type-I IL-4R (IL4Rα/γ-chain dimerization) emanates signaling via activation of insulin receptor substrate (IRS)-phosphoinositide 3-kinase (PI3K)-Akt pathway and JAK-STAT6 pathway, while type-II IL-4R (IL-4Rα/IL-13Rα1 dimerization) activates JAK-STAT6 pathway. GPR120 stimulation by GSK137647A at tenuates airway epithelial remodeling hallmarks including increased MUC5AC, tight junction disruption, and upregulated fibrotic markers. GPR120 stimulation attenuates the epithelial disrupting effects of IL-4 and IL-13 signaling via inhibition of STAT6 and Akt.

Article Snippet: MUC5AC levels in BALF were determined by ELISA (CSB-E11040m; Cusabio, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Activation Assay, Disruption, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

doi: 10.3390/ijms23169405

Figure Lengend Snippet: HDM induces MUC5AC production in airway epithelial cells via TLR2/4-MyD88 signaling. ( A ) Expression of MUC5AC in 16HBE cells transfected with indicated siRNA and stimulated with HDM for 24 h. Results were normalized to the expression of ACTB (encoding β-actin) and control (siNC) cells. ( B ) Secretion of MUC5AC by 16HBE cells transduced as in ( A ) and stimulated with HDM. Data are representative of three independent experiments and presented as the mean ± SEM. ** p < 0.01.

Article Snippet: The collected BALF, lung tissue, or serum were assessed for expressions of IL-5, IL-13, and IgE levels using ELISA MAX Deluxe sets (Biolegend, San Diego, CA, USA) and a MUC5AC ELISA kit (Elabscience, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

doi: 10.3390/ijms23169405

Figure Lengend Snippet: Phosphorylation of sNASP regulates its interaction with TRAF6 and affects MUC5AC production following exposure to HDM. ( A ) The 16HBE cells were treated with HDM for different time periods and assessed by immunoblotting (IB) with antibodies against phosphorylated serine, NASP, or TRAF6 after immunoprecipitation (IP) with anti-NASP or by IB with anti-TRAF6 and anti-β-actin in total cell lysate (TCL). ( B ) RNA levels of MUC5AC in 16HBE cells transfected with the indicated siRNA and stimulated with HDM for 24 h. Results were normalized to the expression of ACTB (encoding β-actin) and control (siNC) cells. Data are presented as the mean ± SEM. ** p < 0.01 ( C ) Protein levels and ( D ) secretion of MUC5AC by 16HBE cells transduced as in ( B ) and stimulated with HDM. ( E ) The 16HBE cells were transfected with GFP-tagged wild-type (WT) sNASP or S158A and S164A mutants, followed by IB with antibody MUC5AC, GFP, or β-actin. Data are representative of three independent experiments.

Article Snippet: The collected BALF, lung tissue, or serum were assessed for expressions of IL-5, IL-13, and IgE levels using ELISA MAX Deluxe sets (Biolegend, San Diego, CA, USA) and a MUC5AC ELISA kit (Elabscience, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Transfection, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

doi: 10.3390/ijms23169405

Figure Lengend Snippet: The PEP-NASP peptide negatively regulates MUC5AC via NF-κB signaling. ( A ) The 16HBE cells were transfected with the siNC or siNASP, and then stimulated with HDM and assessed by an immunoblotting (IB) analysis with the indicated antibodies. ( B ) Luciferase activity in 16HBE cells transfected with a luciferase reporter vector driven by an NF-κB-responsive promoter, plus the siNC or NASP, and stimulated with HDM. Results were standardized to a control (siNC; set to 1). Data are the mean ± SEM for each group. ( C ) The 16HBE cells pretreated with the PEP-NASP or NASP were stimulated with HDM for and assessed by IB analysis with the indicated antibodies. ( D ) Luciferase activity of NF-κB ( E ) Secretion of MUC5AC by 16HBE cells transduced as described in ( C ). Data are representative of three independent experiments. * p < 0.05 and ** p < 0.01 (by a one-way ANOVA).

Article Snippet: The collected BALF, lung tissue, or serum were assessed for expressions of IL-5, IL-13, and IgE levels using ELISA MAX Deluxe sets (Biolegend, San Diego, CA, USA) and a MUC5AC ELISA kit (Elabscience, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: International Journal of Molecular Sciences

Article Title: Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

doi: 10.3390/ijms23169405

Figure Lengend Snippet: Effects of the PEP-NASP peptide on mRNA expression and secretion of inflammatory cytokines in BALF and lung tissues. ( A ) RNA expressions of IL-5, IL-13, and MUC5AC in lung tissues were determined by a RT-qPCR. ( B ) Levels of IL-5, IL-13, and MUC5AC in bronchoalveolar lavage fluid (BALF) were detected by an ELISA. n = 10, * p < 0.05 and ** p < 0.01 (by a one-way ANOVA).

Article Snippet: The collected BALF, lung tissue, or serum were assessed for expressions of IL-5, IL-13, and IgE levels using ELISA MAX Deluxe sets (Biolegend, San Diego, CA, USA) and a MUC5AC ELISA kit (Elabscience, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

doi: 10.3390/ijms23169405

Figure Lengend Snippet: Effects of the PEP-NASP peptide on the phosphorylation of sNASP in lung tissues. ( A ) Phospho-NASP-stained lung sections from each group were highlighted using immunohistochemical (IHC) staining by anti-pSerine158-sNASP. Scale bar = 100 μm ( B ) Lung from each group was analyzed by Western blotting with pSerine158-sNASP, sNASP, p-NF-κB p65, NF-κB p65, MUC5AC and β-actin antibodies. ( C ) Densitometric analysis of the Western blot shown below. Data represent the mean ± SEM of eight to twelve experiments (** p < 0.001 compared to PEP-treated control mice).

Article Snippet: The collected BALF, lung tissue, or serum were assessed for expressions of IL-5, IL-13, and IgE levels using ELISA MAX Deluxe sets (Biolegend, San Diego, CA, USA) and a MUC5AC ELISA kit (Elabscience, Houston, TX, USA), according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Staining, Immunohistochemical staining, Immunohistochemistry, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: LysoPS induces MUC5AC production in airway epithelial cells. ( A ) NCI-H292 cells were treated with LysoPS (30 μM), EGF (25 ng/mL), or S100A9 (200 ng/mL) plus ATP (1 mM) for 24 h. ICC was performed for quantitative measurement of MUC5AC-positive cells. MUC5AC-positive cells were indicated as representative images (left panel), and enumerated in 9 HPFs (right panel). Data are shown as mean ± SEM of three independent experiments (** p < 0.01). Scale bars represent 100 μm. ( B ) MUC5AC mRNA were determined by real time qPCR in NCI-H292 cells treated with either LysoPS or EGF for the indicated time intervals (left panel, n = 4) or with different concentrations of LysoPS for 8 h (right panel, n = 3). The expression levels are displayed as fold increases relative to untreated cells. Data are shown as means ± SEM. ( C ) Cell viability was determined by WST-8 assays with cells treated with varying concentrations of the indicated lysophospholipids or EGF for 24 h. Cell viability was expressed as %, in which the OD value of cells cultured with medium was set to 100%. The results are shown as the mean ± SEM of three independent experiments performed in duplicates. ( D ) NHBE cells were treated with LysoPS for eight hours and analyzed for MUC5AC mRNA and MUC5AC protein expression. MUC5AC mRNA were determined by real time PCR ( n = 4, * p < 0.05) and normalized by PP1A . MUC5AC protein levels in the supernatant were measured by ELISA ( n = 4, * p < 0.05). ( E ) NCI-H292 cells were treated with varying concentrations of lysophopholipids and EGF for 24 h and then analyzed for MUC5AC-positive cells by ICC. This result is representative of three independent experiments and expressed as the mean ± SEM of MUC5AC-positive cells enumerated in 9 HPFs (* p < 0.05 and ** p < 0.01 compared with unstimulated cells).

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: LysoPS-induced MUC5AC is abolished by inhibitors of TACE, EGFR, and ERK, but not by inhibitors of NF-κB and caspase-1. NCI-H292 cells were pretreated with inhibitors for 30 min and then treated with LysoPS, EGF or S100A9 plus ATP for 24 h for MUC5AC production determined by ICC. MUC5AC-positive cells were enumerated in 9 HPFs and shown as the mean ± SEM of 2–7 independent experiments (* p < 0.05, ** p < 0.01). The following inhibitors were used: TAPI2 (10 μM), AG1478 (10 μM), U0126 (10 μM), SB203580 (10 μM), SP600125 (20 μM), Ac-YVAD-cmk (10 μM), Z-VAD-fmk (10 μM), BAY11-7082 (10 μM), dexamethasone (1 μg/mL), and EGFR neutralizing antibody (10 μg/mL).

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: The TACE-EGFR-ERK pathway at the late phase is required for MUC5AC production. The indicated inhibitors were added to NCI-H292 cells at either 30 min before or one, three, and eight h after treatment with LysoPS. MUC5AC-positive cells were evaluated 24 h after treatment with LysoPS. MUC5AC positive cells were enumerated in 8–9 HPFs and expressed as the mean ± SEM of two independent experiments.

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: Knockdown of TACE gene expression reduces LysoPS-dependent EGFR phosphorylation and MUC5AC production. NCI-H292 cells were treated with siRNAs (40 nM) or Control siRNA (NC, 40 nM) for 48 h. ( A ) RT-PCR was performed to validate the effects of siRNAs against TACE mRNA. PP1A was used as a loading control. ( B ) Cells were treated with LysoPS for 8 h and examined for EGFR phosphorylation. The blot was reprobed with anti-EGFR for a loading control. ( C ) 24 h after LysoPS treatment, MUC5AC production determined by ICC. MUC5AC-positive cells were enumerated in 9 HPFs and expressed as the mean ± SEM of two independent experiments (** p < 0.01).

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Gene Expression, Phospho-proteomics, Control, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: LysoPS induces TGF-α secretion in a TACE-dependent manner. NCI-H292 cells were treated with LysoPS (30 μM) for different time intervals ( A ) or with different concentrations of LysoPS for 8 h ( B ) or pretreated with TAPI2 for 30 min and then stimulated with LysoPS for 8 h ( C ). TGF-α in the culture supernatant was measured by ELISA. n = 2–5 for A, n = 5 for B and C, * p < 0.05, ** p < 0.01. ( D ) NCI-H292 cells were pretreated with TGF-α neutralizing antibody (2 μg/mL) for 30 min before determining LysoPS- triggered MUC5AC producing cells. MUC5AC-positive cells were enumerated in 9 HPFs and expressed as the mean ± SEM of three independent experiments.

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: None of the known receptors for LysoPS is engaged in LysoPS-induced MUC5AC production. ( A ) The expression of G2A, GPR34, GPR174, and P2Y10 on the surface of NCI-H292 cells ware analyzed by FACS. Staining with specific antibodies (solid lines) and isotype control antibodies (gray) are shown. ( B ) NCI-H292 cells were pretreated with suramin (10 μM) or A438079 (10 μM) for 30 min and then stimulated for 24 h with LysoPS or EGF. MUC5AC-positive cells were enumerated in nine HPFs and expressed as the mean ± SEM of two independent experiments. ( C ) NCI-H292 cells were treated for 10 min (for immunoblot analysis) or 24 h (for ICC) with LysoPS, LPA (30 μM), or EGF in the absence or presence of PTX (100 ng/mL). MUC5AC positive cells were enumerated in 9 HPFs and expressed as the mean ± SEM of two to three independent experiments (** p < 0.01 and NS = not significant). ERK phosphorylation and total ERK was analyzed by immunoblot analysis. ( D ) Total cell lysate of NCI-H292 cells that had been treated with LysoPS or EGF for 10 min were subjected to immunoblot analysis against TLR2 and ERK. Lane 1 for size marker, lane 2 for medium, lane 3 for LysoPS, and lane 4 for EGF. ( E , F ) NCI-H292 cells were treated with LysoPS, EGF, Pam2csk4 (1 μg/mL), or Pam3csk4 (1 μg/mL) in the absence or presence of C29 (40 μM) for 24 h (for ICC) or for 10 min and 8 h (for immunoblot analysis). MUC5AC-positive cells were enumerated in 9 HPFs and expressed as the mean ± SEM of two independent experiments (** p < 0.01 and NS = not significant). ERK phosphorylation and total ERK were analyzed by immunoblot analysis. Lanes 1–4 in F indicate that NCI-H292 cells were treated LysoPS for 8 h in the absence or presence of C29 (40 μM), while lane 5 indicates that C29 was added to the culture 1 h after treatment with LysoPS and incubated for an additional 7 h. These results are representative of three independent experiments.

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Staining, Control, Western Blot, Phospho-proteomics, Marker, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: ROS is dispensable for LysoPS-induced MUC5AC production ( A ) In the absence or presence of DPI (5 μM) or NAC (5 mM), NCI-H292 cells were treated for 1h with EGF or stimulated with LysoPS and TBHP (100 μM). ROS production was measured by DCFDA fluorescence intensity ( n = 2–4, * p < 0.05, ** p < 0.01, NS = not significant). ( B ) NCI-H292 cells were treated for 24 h with LysoPS in the absence or presence of DPI or NAC. MUC5AC-positive cells were enumerated in 9 HPFs and expressed as the mean ± SEM of two independent experiments.

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Lysophosphatidylserine Induces MUC5AC Production via the Feedforward Regulation of the TACE-EGFR-ERK Pathway in Airway Epithelial Cells in a Receptor-Independent Manner

doi: 10.3390/ijms23073866

Figure Lengend Snippet: Schematic of LysoPS-induced MUC5AC production. In the early phase (black arrows), LysoPS activates the membrane-associated TACE, leading to maturation of a pre-existing small pool of pro-TGF-α. This process appears to be receptor-independent but dependent on the amphipathic nature of LysoPS that causes membrane perturbation. The resulting mature TGF-α signals through EGFR, which activates ERK in the early phase, followed by the induction of new TGF-α. In the late phase (red arrows), a large amount of newly synthesized TGF-α promotes the prolonged activation of ERK via EGFR and drives ERK-dependent MUC5AC production in airway epithelial cells.

Article Snippet: To measure the production of MUC5AC, NHBE cells were incubated with LysoPS for 8 h, and the culture supernatants were analyzed by a MUC5AC ELISA kit (Novus Biologicals, Littleton, CO, USA).

Techniques: Membrane, Synthesized, Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: BMAL1/p53 mediating bronchial epithelial cell autophagy contributes to PM2.5-aggravated asthma

doi: 10.1186/s12964-023-01057-9

Figure Lengend Snippet: BMAL1 participated in carbon particles/PM2.5-induced airway remodeling in vitro and in vivo. A – F After transfection with control vector or BMAL1 expression vector for 72 h, HBEs were treated with carbon particles (20 μg/ml) or PM2.5 (200 μg/ml) for 24 h or 48 h. A , B mRNA changes of collagen-I, α-SMA and MUC5AC in HBEs. n = 3. C , D Representative blot images of collagen-I and fibronectin in HBEs. E , F Representative images of immunofluorescence staining of MUC5AC in HBEs stimulated by carbon particles/PM2.5. Scale bars: 100 μm. G – J After transfection with control or specific siRNA against BMAL1 for 48 h, HBEs were incubated with 20 μg/ml carbon particles or 200 μg/ml PM2.5, after which expression levels of related mRNA and protein were measured by the RT-qPCR (24 h) and western blotting (48 h). (G, H) mRNA changes of collagen-I, α-SMA and MUC5AC in HBEs. n = 3. I , J Representative blot images of collagen-I, fibronectin and α-SMA in HBEs. Bmal1 −/− mice and Bmal1 wt/wt mice were sensitized and challenged with OVA as described in the “Methods”. Lungs of mice were harvested at day 60. K – M Representative pictures of lung tissue sections stained with Masson staining, PAS staining and immunohistochemistry staining of MUC5AC. Scale bars: 50 μm. N Collagen deposition area (%) of proximal airways in each group (n = 5). O The quantification histograms of PAS staining in proximal airways (n = 5). P The quantification histograms of IHC MUC5AC staining in proximal airways (n = 5). Data are mean ± SEM of n individual experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 between indicated conditions (One-way ANOVA followed by the Bonferroni’s test)

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to measure BALF levels of IL-4 (Mouse IL-4 ELISA Kit; Dakewe), IL-5 (Mouse IL-5 ELISA Kit; Dakewe), IL6 (Mouse IL-6 ELISA Kit; Dakewe), IL-13 (Mouse IL-13 ELISA Kit; Dakewe) and MUC5AC (Mouse MUC5AC ELISA Kit, Dakewe).

Techniques: In Vitro, In Vivo, Transfection, Control, Plasmid Preparation, Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: BMAL1/p53 mediating bronchial epithelial cell autophagy contributes to PM2.5-aggravated asthma

doi: 10.1186/s12964-023-01057-9

Figure Lengend Snippet: p53 siRNA prevented PM2.5/carbon particle-induced collagen-I expression and cell autophagy in bronchial epithelial cells . HBEs were incubated for 48 h after transfection with control or specific siRNA against p53 and treated with carbon particles (20 μg/ml) or PM2.5 (200 μg/ml) for 48 h prior to harvest of cell lysates, after which expression levels of protein were measured by qRT-PCR and Western blots. A , B mRNA changes of collagen-I, α-SMA and MUC5AC in HBEs. n = 6. C , E Representative images of Western blots of p53, fibronectin, collagen-I, α-SMA and LC3B-II were shown. D , F The changes in relative density of p53, fibronectin, collagen-I, α-SMA and LC3B-II to GAPDH were presented. n = 3 ( C ); n = 6 (p53 and collagen-I in D ), n = 4 (fibronectin, α-SMA and LC3B-II in D ). Data are mean ± SEM of n individual experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 between indicated conditions (One-way ANOVA followed by the Bonferroni’s test)

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits were used to measure BALF levels of IL-4 (Mouse IL-4 ELISA Kit; Dakewe), IL-5 (Mouse IL-5 ELISA Kit; Dakewe), IL6 (Mouse IL-6 ELISA Kit; Dakewe), IL-13 (Mouse IL-13 ELISA Kit; Dakewe) and MUC5AC (Mouse MUC5AC ELISA Kit, Dakewe).

Techniques: Expressing, Incubation, Transfection, Control, Quantitative RT-PCR, Western Blot