Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: (A) NCI-H292 cells were stimulated with EGF (25 ng/mL) in the presence of varying concentrations of acetate, propionate, and butyrate for 24 hours, and they were subjected to ICC to determine MUC5AC protein expression. MUC5AC-positive cells were enumerated per field for total 8–11 high power fields. Data are shown as the mean ± SEM of 3 independent experiments compared with unstimulated cells. (B) Representative images of ICC are shown for inhibitory effects of SCFAs (1 mM) on EGF-induced MUC5AC expression. Arrows indicate MUC5AC-positive cells. (C) NCI-H292 cells were treated with EGF in the presence of SCFAs (0.5 mM) for 24 hours and examined for MUC5AC mRNA via real time polymerase chain reaction. PP1A was used to normalize levels of MUC5AC transcripts. Data are shown as the mean ± SEM of 3 independent experiments, compared with the EGF-stimulated MUC5AC mRNA level. (D) The inhibitory effect of butyrate on EGF-induced MUC5AC protein production was examined at a range of concentrations. Data are shown as the mean ± SEM of 2 to 3 independent experiments. (E) The inhibitory effect of butyrate (0.5 mM) on MUC5AC protein production induced by EGF, LPS (500 ng/mL), LysoPS (30 μM), PMA (20 ng/mL), IL-1β (20 ng/mL), and hydrogen peroxide (4 mM), were examined. Data are shown as the mean ± SEM of 2 to 3 independent experiments. (F) NHBE cells were examined for the effect of EGF and butyrate on MUC5AC mRNA expression. Data are shown as the mean ± SEM of 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Tukey’s post hoc test, and is indicated in Fig. 1A-F except for Fig. 1B, as * P < 0.05 and ** P < 0.01 based on the post hoc comparisons. SCFA, short-chain fatty acid; MUC5AC, mucin 5AC; EGF, epidermal growth factor; ICC, immunocytochemistry; SEM, standard error of the mean; LPS, lipopolysaccharide; LysoPS, lysophosphatidylserine; PMA, phorbol 12-myristate 13-acetate; IL-1β, interleukin-1β; NHBE, normal human bronchial epithelial; ANOVA, analysis of variance.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry

Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: (A) NCI-H292 cells were stimulated with EGF in the absence or presence of butyrate (0.5 mM) or VPA (10 μM) for 24 hours and examined for MUC5AC-postive cells. The mRNA levels of SPDEF , FOXA3 , and FOXA2 genes were analyzed by qRT-PCR. Data are shown as mean ± SEM of 3 to 4 independent experiments. The polymerase chain reaction products were also visualized on the gel following qRT-PCR, with the use of PP1A , a loading control, as seen in the upper panel. (B) NHBE cells were examined for the effect of EGF and butyrate on SPDEF , FOXA2 and FOXA3 mRNA expression, using cDNA prepared for . Data are shown as the mean ± SEM of 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Tukey’s post hoc test. MUC5AC, mucin 5AC; EGF, epidermal growth factor; VPA, valproic acid; SPDEF , SAM-pointed domain-containing ETS transcription factor; FOXA , forkhead box protein A; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; NHBE, normal human bronchial epithelial; ANOVA, analysis of variance. * P < 0.05, ** P < 0.01.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Quantitative RT-PCR, Polymerase Chain Reaction, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: (A) NCI-H292 cells were stimulated with EGF in the presence of butyrate (0.5 mM) and/or PTX (100 ng/mL) for 24 hours and examined for MUC5AC-positive cells. Data are shown as the mean ± SEM of 2 independent experiments. (B) NCI-H292 cells were pretreated in the presence of butyrate (0.5 mM), GPR43 agonist CFMB, GPR42 agonist AR420626 , or GPR109A agonist fumarate for 30 minutes, followed by EGF for 24 hours. Data are shown as the mean ± SEM of 2 to 3 independent experiments. (C) NCI-H292 cells were stimulated with EGF in the absence or presence of butyrate (0.5 mM) for indicated time intervals and determined for phosphorylation of EGFR and ERK1/2 via immunoblot analysis. Total EGFR and ERK were used as loading controls for p-EGFR and p-ERK, respectively. This result represents 1 of 2 independent experiments. EGF, epidermal growth factor; MUC5AC, mucin 5AC; EGFR, epidermal growth factor receptor; PTX, pertussis toxin; SEM, standard error of the mean; ERK, extracellular signal-regulated kinase; ANOVA, analysis of variance. ** P < 0.01.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Phospho-proteomics, Western Blot

Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: (A) NCI-H292 cells were stimulated with EGF for different time intervals and examined for MUC5AC mRNA via real-time polymerase chain reaction. Data are shown as the mean ± SEM of 4 independent experiments. (B) NCI-H292 cells were stimulated with EGF followed by simultaneous treatment (0 hour) or posttreatment with 0.5 mM butyrate at 30 minutes, 4, 8, and 16 hours after EGF stimulation, and they were analyzed for MUC5AC-positive cells at 24 hours. Data are shown as the mean ± SEM of 3 independent experiments. (C) NCI-H292 cells were stimulated with EGF in the presence of butyrate (0.5 mM), and the culture medium was removed at indicated times. The cultures were then washed with fresh culture medium and replaced with only EGF-containing medium at different time points (1, 2, 4, 8, and 16 hours). Cells were also stimulated with medium or EGF or EGF plus butyrate for 24 hours (arrow heads). Cells were fixed at 24 hours for immunocytochemistry of MUC5AC-positive cells. Data are shown as the mean ± SEM of 2 to 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Scheffé’s post hoc test. EGF, epidermal growth factor; MUC5AC, mucin 5AC; SEM, standard error of the mean; ANOVA, analysis of variance. * P < 0.05, ** P < 0.01.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Real-time Polymerase Chain Reaction, Immunocytochemistry

Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: NCI-H292 cells were stimulated with EGF in the presence of VPA (1 mM), TSA (500 nM), and butyrate (0.5 mM) for 24 hours and examined for MUC5AC mRNA and protein expression via real-time polymerase chain reaction and immunocytochemistry, respectively. Data are shown as the mean ± standard error of the mean of 3 to 4 independent experiments for MUC5AC mRNA and protein expression, respectively. Statistical significance was determined using 1-way ANOVA followed by Tukey’s post hoc test. VPA, valproic acid; TSA, trichostatin A; EGF, epidermal growth factor; MUC5AC, mucin 5AC; ANOVA, analysis of variance. * P < 0.05, ** P < 0.01.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunocytochemistry

Journal: Allergy, Asthma & Immunology Research

Article Title: Butyrate Silences MUC5AC in Airway Epithelial Cells Through Histone Deacetylation at Its Promoter Region

doi: 10.4168/aair.2026.18.2.204

Figure Lengend Snippet: (A) MUC5AC promoter is depicted on which some of functional cis -acting elements including 3 Sp1 binding sites are indicated, along with PCR amplicons at proximal and distal MUC5AC promoter regions for ChIP-PCR analysis. (B, C) NCI-H292 cells were stimulated with EGF in the presence of butyrate (0.5 mM), VPA, or Dex for 24 hours. ChIP assays were performed with anti-H3K27ac (B) and anti-Sp1 (C), and PCR was then conducted for indicated amplicons of 2 MUC5AC promoter regions and 3’-UTR. Data are shown as the mean ± SEM of 3 independent experiments. Statistical significance in (B) and (C) was determined using 1-way ANOVA followed by Scheffé’s post hoc test. (D) NCI-H292 cells were stimulated with EGF in the absence or presence of butyrate (0.5 mM) for 1, 8, and 24 hours, and ChIP-PCRs were performed for histone acetylation. Data are shown as the mean ± SEM of 2 independent experiments. Statistical significance was determined using repeated measure 1-way ANOVA followed by Scheffé’s post hoc test. (E) NCI-H292 cells were stimulated with EGF in the presence of butyrate (0.5 mM), VPA, or Dex for 24 hours. ChIP-PCR assays were performed with anti-H3K27ac and PCR for amplicons of the proximal promoter regions of SPDEF and FOXA2 . Data are shown as the mean ± SEM of 2 to 3 independent experiments. Statistical significance was determined using 1-way ANOVA followed by Scheffé’s post hoc test. EGF, epidermal growth factor; MUC5AC, mucin 5AC; PCR, polymerase chain reaction; ChIP, chromatin immunoprecipitation; VPA, valproic acid; Dex, dexamethasone; SEM, standard error of the mean; ANOVA, analysis of variance; SPDEF , SAM-pointed domain-containing ETS transcription factor; FOXA2 , forkhead box protein A2; NF-κB, Nuclear factor-κB; GRE, glucocorticoid response element; CRE, cAMP response element; N.S., not significant. * P < 0.05, ** P < 0.01.

Article Snippet: The NCI-H292 human lung mucoepidermoid carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Welgene, Gyeongsan, Korea) containing 10% fetal bovine serum (FBS), penicillin (100 units/mL), and HEPES (25 mM).

Techniques: Functional Assay, Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation