Journal: Scientific Reports

Article Title: Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

doi: 10.1038/s41598-018-36387-z

Figure Lengend Snippet: Constitutive expression of HBD1 is decreased in colon cancer. Transcription of the HBD1 gene in non-tumor and tumor specimens. Box plots are presented on a linear scale for the cohorts of patients GSE6988 (South Korea, n = 77), GSE40967 (France, n = 585), GSE44076 (Spain, n = 196) and GSE44861 (USA, n = 111). *P < 0,001 evaluated by Welch t test.

Article Snippet: We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

doi: 10.1038/s41598-018-36387-z

Figure Lengend Snippet: EGFR inhibition increases the constitutive expression of HBD1 in vitro and ex vivo . ( A ) Transcription of the HBD1 and IL8 genes in human colonic TC7 cells treated for 48 h with 1 μM of EGFR inhibitors AG1478, Afatinib, Erlotinib, Gefitinib and Osimertinib, or 100 nM Cetuximab. Values are presented on a logarithmic scale as the ratio of gene expression in treated cells compared with non-treated cells. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 5 biological replicates). ( B ) ELISA dosage of the HBD1 and IL8 peptides secreted by TC7 cells treated for 48 h with 1 μM of EGFR inhibitors AG1478, Afatinib, Erlotinib, Gefitinib and Osimertinib, or 100 nM Cetuximab. Values are presented on a linear scale in picogram of peptide per milliliter. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 5 biological replicates). ( C ) Transcription of the HBD1 and IL8 genes in human colonic organoids treated for 48 h with 1 μM AG1478, Afatinib, or Gefitinib. Values are presented on a logarithmic scale as the ratio of gene expression in treated organoids compared with non-treated organoids. NT, non-treated organoids. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 5 biological replicates).

Article Snippet: We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier.

Techniques: Inhibition, Expressing, In Vitro, Ex Vivo, Gene Expression, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

doi: 10.1038/s41598-018-36387-z

Figure Lengend Snippet: EGF decreases the constitutive expression of HBD1. ( A ) Transcription of the HBD1 and IL8 genes in TC7 cells treated for 48 h with 200 ng/mL EGF. Values are presented on a logarithmic scale as the ratio of gene expression in treated cells compared with non-treated cells. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 7 biological replicates). ( B ) ELISA dosage of the HBD1 and IL8 peptides in supernatants of TC7 cells treated for 48 h with 200 ng/mL EGF. Values are presented on a linear scale in picogram of peptide per milliliter. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 7 biological replicates). ( C ) Transcription of the HBD1 and IL8 genes in TC7 cells treated for 48 h with 1 μM Gefitinib, 200 ng/mL EGF, or 1 μM Gefitinib + 200 ng/mL EGF. Values are presented on a logarithmic scale as the ratio of gene expression in treated cells compared with non-treated cells. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 4 biological replicates). ( D ) ELISA dosage of the HBD1 and IL8 peptides secreted by TC7 cells treated for 48 h with 1 μM Gefitinib, 200 ng/mL EGF, or 1 μM Gefitinib + 200 ng/mL EGF. Values are presented on a linear scale in picogram of peptide per milliliter. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 4 biological replicates).

Article Snippet: We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier.

Techniques: Expressing, Gene Expression, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

doi: 10.1038/s41598-018-36387-z

Figure Lengend Snippet: The ERK signaling pathway mediates the EGFR-dependent repression of HBD1. ( A ) Transcription of the HBD1 gene in TC7 cells treated for 48 h with 10 μM of the MEKK1/2 inhibitor (PD184352), 10 μM of the MEKK1/2 inhibitor + 1 μM Afatinib, 10 μM of the MEKK1/2 inhibitor + 1 μM Gefitinib, or 10 μM of the MEKK1/2 inhibitor + 200 ng/mL EGF. Values are presented on a logarithmic scale as the ratio of gene expression in treated cells compared with non-treated cells. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test; ns, not significant. Data are represented as mean ± SD (n = 5 biological replicates). ( B ) Transcription of the HBD1 gene in human colonic organoids treated for 48 h with 10 μM of the MEKK1/2 inhibitor (PD184352). Values are presented on a logarithmic scale as the ratio of gene expression in treated organoids compared with non-treated organoids. NT, non-treated organoids. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 4 biological replicates). (C) Immunoblot analysis of ERK1/2 and phosphorylated ERK1/2 in TC7 cells treated or not with 1 μM Gefitinib, 10 μM of the MEKK1/2 inhibitor (PD184352), or 200 ng/mL EGF. After lysis of cells at the indicated time points, Western blots were performed using specific antibodies directed against proteins or phosphorylated proteins (representative of 3 biological replicates). “P” prefix, phosphorylation. NT, non-treated cells.

Article Snippet: We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier.

Techniques: Gene Expression, Two Tailed Test, MANN-WHITNEY, Western Blot, Lysis, Phospho-proteomics

Journal: Scientific Reports

Article Title: Expression of the human antimicrobial peptide β-defensin-1 is repressed by the EGFR-ERK-MYC axis in colonic epithelial cells

doi: 10.1038/s41598-018-36387-z

Figure Lengend Snippet: MYC mediates the EGFR-dependent repression of HBD1. ( A ) Promoter sequence of the human HBD1 gene. E-box DNA binding sites for MYC are indicated by grey boxes. ( B ) Transcription of the HBD1 gene in TC7 cells treated for 48 h with 500 nM of the MYC inhibitor (+)-JQ1, 500 nM of the MYC inhibitor (+)-JQ1 + 1 μM Afatinib, or 500 nM of the MYC inhibitor (+)-JQ1 + 1 μM Gefitinib. Values are presented on a logarithmic scale as the ratio of gene expression in treated cells compared with non-treated cells. NT, non-treated cells. *P < 0,05 evaluated by two-tailed Mann-Whitney u test; ns, not significant. Data are represented as mean ± SD (n = 5 biological replicates). ( C – E ) ChIP and ChIP-re-ChIP analysis of MYC ( C ), MIZ1 ( D ), and the MYC-MIZ1 complex ( E ) recruitment at the HBD1 promoter in cells treated with 1 μM Gefitinib (black bars), 200 ng/mL EGF (grey bars), or non-treated cells (white bars). Enrichment in chromatin was detected using MYC and/or MIZ1 specific antibodies, or rabbit IgG as control, and quantified by qRT-PCR using specific primers matching the HBD1 promoter at E-box sites (I, II, III). Values are presented as the percentage of signal relative to the histone H3 protein. *P < 0,05 evaluated by two-tailed Mann-Whitney u test. Data are represented as mean ± SD (n = 4 biological replicates).

Article Snippet: We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier.

Techniques: Sequencing, Binding Assay, Gene Expression, Two Tailed Test, MANN-WHITNEY, Control, Quantitative RT-PCR