Journal: Oncotarget

Article Title: Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis

doi:

Figure Lengend Snippet: (A) number of differentially expressed genes in rats treated with NMBA at weeks 6 (blue circle) and week 29 (red circle); (B) the heat map descripts the increased expression of Defb4 in control and NMBA-treated animals from weeks 6 to 29; (C) the biofunction network of Defb4 at early stage of NMBA-induced carcinogenesis (week 6); and (D) the biofunction network of Defb4 at late stage of NMBA-induced carcinogenesis (week 29). Green color indicates that genes are downregulated and red color indicates that genes are upregulated during NMBA-induced esophageal carcinogenesis. Genes without green or red color are selected from literatures, which have been reported to be associated with the differentially expressed genes identified in this study. The straight lines indicate the direct interactions and dash lines indicate the indirect interactions between genes based on the data mining using IPA.

Article Snippet: The expression of Defb4 was determined using Taqman assays: Defb4 (Rn02532184_g1), using GAPDH as reference gene (Rn01775763_g1).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis

doi:

Figure Lengend Snippet: (A) Defb4 mRNA expression (left panel) and HBD-2 protein expression (right panel) in rats treated with NMBA compared to normal animals from weeks 6 to 29. The numbers under each HBD-2 blot are intensity of the blot relative to that of normal animal; (B) overexpression of IL-1β, IL-6 and TNF-α mRNA in rat esophagus during tumorigenesis from weeks 6 to 29; and (C) immunohistochemistry staining of HBD-2 in rat esophagus (magnification × 200). The values are expressed as mean; bars , ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The expression of Defb4 was determined using Taqman assays: Defb4 (Rn02532184_g1), using GAPDH as reference gene (Rn01775763_g1).

Techniques: Expressing, Over Expression, Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis

doi:

Figure Lengend Snippet: (A) HBD-2 protein expression in non-malignant cell HET-1A and esophageal SCC cells KYSE-410, KYSE-270, KYSE-150 and KYSE-70; (B) HBD-2 protein expression in KYSE-150 cells that were transfected with Defb4 siRNA (siRNA) or scrambled siRNA (NC); (C) WST-1 analysis of the growth of KYSE-150 cells that were transfected with Defb4 siRNA or scrambled siRNA; (D) left panel, representative photographs of colonies formed in the presence and absence of Defb4 . Purple spots represent KYSE-150 cell colonies; and right panel, quantitative analysis of number of colonies; (E) scratch wound healing assay to examine the effect of Defb4 gene silencing on KYSE-150 cell mobility; and (F) left panel, representative images of migratory cells that were transfected with Defb4 siRNA or scrambled siRNA; and right panel, quantitative analysis of number of migratory cells. All the experiments were performed in triplicate. The values are expressed as mean; bars , ± SE. ** P < 0.01; *** P < 0.001.

Article Snippet: The expression of Defb4 was determined using Taqman assays: Defb4 (Rn02532184_g1), using GAPDH as reference gene (Rn01775763_g1).

Techniques: Expressing, Transfection, Wound Healing Assay

Journal: Oncotarget

Article Title: Overexpression of human β-defensin 2 promotes growth and invasion during esophageal carcinogenesis

doi:

Figure Lengend Snippet: (A) the expression levels of Defb4 mRNA in rats fed with celecoxib + PBIT, 5% BRB or control diet. The values are expressed as mean; bars , ± SE; and (B) the expression levels of HBD-2 protein in KYSE-150 cells treated with different doses of PBIT, celecoxib, MK2206, BEZ235, C3G or C3R. The numbers under each HBD-2 blot are intensity of the blot relative to that of untreated control.

Article Snippet: The expression of Defb4 was determined using Taqman assays: Defb4 (Rn02532184_g1), using GAPDH as reference gene (Rn01775763_g1).

Techniques: Expressing, Control

Journal: PLoS Pathogens

Article Title: Vitamin D Induces Interleukin-1β Expression: Paracrine Macrophage Epithelial Signaling Controls M. tuberculosis Infection

doi: 10.1371/journal.ppat.1003407

Figure Lengend Snippet: ( A ) Expression of DEFB4 and CAMP in SAECs as measured by RT/qPCR. Cells were incubated with IL-1β (10 ng/ml) or 1,25D (100 nM) for 24 h. ( B ) Media supernatants from cells in (A) were tested for DEFB4 and LL-37 protein secretion by ELISA. ( C ) and ( D ) Expression of DEFB4 (C) and CAMP (D) genes in SAECs incubated with conditioned media from uninfected (NI) or H37Rv-infected (I) THP-1 cells treated with vehicle or 100 nM 1,25D (+D) for 24 hours. Media was treated with neutralizing antibody against IL-1β (α-IL-1β), or normal serum IgG, as indicated, for 30 minutes prior to incubation with cells. Values are expressed as a fold of the NI control. All data are from one experiment and representative of three independent experiments using separate donors of SAECs (n = 3, mean, s.d.). *P<0.05, **P<0.01 as determined by Student's t-test relative untreated (A, B) or respective IgG (C) control.

Article Snippet: ELISAs for DEFB4 (Abnova) and LL-37 (Hycult Biotech) were performed in accordance with the manufacturer's instructions.

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

Journal: PLoS Pathogens

Article Title: Vitamin D Induces Interleukin-1β Expression: Paracrine Macrophage Epithelial Signaling Controls M. tuberculosis Infection

doi: 10.1371/journal.ppat.1003407

Figure Lengend Snippet: ( A ) CFU of macrophages (Mφ) infected with H37Rv at an MOI of 5 for 4 hours and treated with vehicle control or 100 nM 1,25D (+D), with and without the additional presence of SAECs in transwell co-culture (CC). Data are from three experimental replicates (mean and SD) and representative of three independent experiments using different donors of primary cells. Statistical significance was determined by one-way ANOVA. (*P<0.05). ( B ) Validation of siRNA-mediated knockdown of IL1R1 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siIL1R1. Data are from three experimental replicates (mean and SD), **P<0.01 as determined by student's T-test relative to respective siCTL controls. ( C ) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 . 1,25D (D) was added as indicated. Data are from three experimental replicates (mean and SD) and representative of two independent experiments. Statistical significance was determined by one-way ANOVA. (**P<0.01). ( D ) Validation of siRNA-mediated knockdown of DEFB4 expression in SAECs. RNA was extracted from SAECs cells 3 days after the initiation of co-culture and 4.5 days after transfection of control siRNA (siCTL) or siDEFB4. Data are from three experimental replicates (mean and SD) and representative to two independent replicates. **P<0.01 as determined by student's T-test relative to siCTL control. ( E ) CFU quantification of Mtb in THP-1 cells infected at an MOI of 5 for 4 hours after 72 hours of co-culture with SAEC cells transfected with control siRNA (siCTL) or siRNA specific to IL1R1 or DEFB4 . Data are from three experimental replicates (mean and SD) and representative of two independent experiments using separate donors of SAECs. Statistical significance was determined by one-way ANOVA. (*P<0.05).

Article Snippet: ELISAs for DEFB4 (Abnova) and LL-37 (Hycult Biotech) were performed in accordance with the manufacturer's instructions.

Techniques: Infection, Control, Co-Culture Assay, Biomarker Discovery, Knockdown, Expressing, Transfection

Journal: PLoS Pathogens

Article Title: Vitamin D Induces Interleukin-1β Expression: Paracrine Macrophage Epithelial Signaling Controls M. tuberculosis Infection

doi: 10.1371/journal.ppat.1003407

Figure Lengend Snippet: IL1B gene expression is driven by infection and 1,25D, and pro-IL-1β maturation is dependent on the NLRP3 inflammasome. Released IL-1β signals via IL1R1 on adjacent epithelial cells to induce expression of DEFB4 . The release of DEFB4 induced by IL-1β, in combination with 1,25D, leads to control of mycobacterial proliferation in the macrophage.

Article Snippet: ELISAs for DEFB4 (Abnova) and LL-37 (Hycult Biotech) were performed in accordance with the manufacturer's instructions.

Techniques: Gene Expression, Infection, Expressing, Control

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: PCR Primers.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: (A) HBE1 cells were transfected with Act1 siRNA or a non-targeting (NT) siRNA control. 48h post transfection, cells were treated with media only (-IL-17A) or 100 ng/mL IL-17A (+IL-17A) for an additional 17h, then harvested for RNA extraction and real-time PCR analysis of DEFB4 expression. C(t) values were normalized to a housekeeping gene, GAPDH , and calibrated to NT siRNA/-IL-17A. Error bars represent SEM of 3 independent experiments. *p<0.05 compared to NT siRNA/-IL-17A samples, **p<0.05 compared to NT siRNA/+IL-17A samples. (B) HBE1 cells were transfected with increasing amounts of Act1-FLAG plasmid. Total transfected DNA was normalized using p3XFLAG-CMV-14. 24h post-transfection, cells were treated with media only or 100 ng/mL IL-17A for an additional 17h, then harvested for RNA extraction and real-time PCR analysis of DEFB4 expression. *p<0.05 compared to 0 ng transfected (mock transfection) (C) To confirm increasing Act1-FLAG protein expression, 30 μg whole cell extracts from transfected cells were analyzed by Western blot for Act1-FLAG. β-actin was used as a loading control. (D) DEFB4 protein was quantitatively measured in cell culture supernatant by ELISA (n = 3). *p<0.05 compared to empty vector (EV) transfected cells.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Transfection, Control, RNA Extraction, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: (A) HBE1 cells were transfected with Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of various IL-17 target genes. (B) A549 or NHBE cells were transfected with empty vector (EV) or Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of DEFB4 expression. (C) HBE1 cells were transfected with empty vector, Act1-FLAG or untagged Act1 and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of DEFB4 expression. C(t) values were normalized to GAPDH and calibrated to empty vector controls. Error bars represent SEM of 3 independent experiments. *p<0.05 compared to empty vector (EV) transfected cells.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Transfection, RNA Extraction, Real-time Polymerase Chain Reaction, Plasmid Preparation, Expressing

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: (A) HBE1 cells were transiently co-transfected with DEFB4 promoter-luciferase constructs, pRL-TK and either empty vector or Act1-FLAG (n = 6) and lysed 24 hours post-transfection. *p<0.05 compared to empty vector (EV) transfected cells. (B) HBE1 cells were co-transfected with a CCL20 promoter-luciferase construct (CCL20-LUC), pRL-TK and either empty vector or Act1-FLAG (n = 4) and lysed 24 hours post-transfection. *p<0.05 compared to empty vector (EV) transfected cells. (C) HBE1 cells were co-transfected with a DEFB4 promoter-luciferase construct in which all 3 NF-κB binding sites are mutated (DEFB4-mut-LUC), pRL-TK and either empty vector or Act1-FLAG. 24 hours post-transfection, cells were left unstimulated (-IL-17A) or stimulated with 100 ng/mL IL-17A for 6 hours (n = 4). Lysate was measured for firefly and Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity and calibrated to empty vector transfected controls. *p<0.05 compared to empty vector (EV) transfected cells. NS: not significant when IL-17A treated cells (+IL-17) were compared to non-treated cells (-IL-17). (D) Lysate was separated by SDS-PAGE and immunoblotted to confirm Act1-FLAG expression. β-actin was used as a loading control.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Transfection, Luciferase, Construct, Plasmid Preparation, Binding Assay, Activity Assay, SDS Page, Expressing, Control

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: (A) Schematic representation of various FLAG-tagged Act1 derivatives. Numbers represent amino acid residue location. (B) HBE1 cells were transiently transfected with empty vector or various Act1 derivatives. 24h post-transfection, cells were lysed for RNA extraction and real-time PCR analysis of DEFB4 gene expression. C(t) values were normalized to GAPDH and calibrated to the empty vector control. Error bars represent SEM of 3 independent experiments. * p< 0.05 compared to Act1-FL (C) 30 μg whole cell lysate was immunoblotted with anti-FLAG antibody to confirm expression of FLAG-tagged Act1 derivative. β-actin was used as a loading control.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Residue, Transfection, Plasmid Preparation, RNA Extraction, Real-time Polymerase Chain Reaction, Gene Expression, Control, Expressing

Journal: PLoS ONE

Article Title: A Novel Nuclear Function for the Interleukin-17 Signaling Adaptor Protein Act1

doi: 10.1371/journal.pone.0163323

Figure Lengend Snippet: HBE1 cells were transfected with Act1-FLAG and fixed 24h post-transfection. Isolated nuclei were sonicated and chromatin immunoprecipitation (chIP) was performed using an anti-FLAG antibody or non-specific mouse IgG. PCR was used to detect DEFB4 promoters in the immunoprecipitated material. The data was presented as % of the input as described in Materials and Methods.

Article Snippet: DEFB4 ELISA construction kit was purchased from Antigenix America and performed according to the manufacturer’s suggested protocol.

Techniques: Transfection, Isolation, Sonication, Chromatin Immunoprecipitation, Immunoprecipitation