Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: TNFα promoted HE4 secretion through the NF-kB signaling pathway (A) A2780 cells were incubated with TPCA-1 (25 μM, an IKK2 inhibitor), BIRB-769 (1 μM, a p38 inhibitor), JNK-IN-8 (1 μM, a JNK inhibitor), or CI-1040 (20 μM, a MEK inhibitor) for 1 hour, after which cells were subjected to TNFα treatment (30 ng/mL) for 15 minutes. Subsequently, protein expression and phosphorylation were assessed via immunoblot analysis using target-specific antibodies, with α-tubulin serving as a loading control. (B) A2780 cells underwent the same inhibitor treatment as in (A), after which cells were treated with TNFα (30 ng/mL) for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA. (C) A2780 cells were transfected with a construct encoding luciferase under the control of an NF-κB response element or HE4 promoter construct (HE4-652), and luciferase activity was assessed following TNFα (30 ng/mL) or IL-1β (10 ng/mL) treatment for 7 hours. (D) Same as (C), but HCH-1 cells were employed.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Incubation, Expressing, Phospho-proteomics, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Transfection, Construct, Luciferase, Activity Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) Expression of constitutively active IKK2 mutant (IKK2-EE) led to the phosphorylation of Iκβα. 2008 cells were transfected with either IKK2-EE or an empty vector for 24 hours. Cells were lysed, and protein expression was assessed via immunoblot analysis. Phosphorylated Iκβα levels were quantified relative to total Iκβα. (B) IKK2 inhibitor TPCA-1 reduced HE4 expression. 2008 cells were treated with TPCA-1 at a range of concentrations for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right) (C-E) Expression of p65 and IKK2-EE promoted HE4 expression in ovarian cancer cell lines. A2780, OVCAR-3, and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 72 hours. Afterward, HE4 levels in culture medium were determined using HE4 ELISA.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Expressing, Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, MTS Assay

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: A2780 cells were treated with TNFα (30 ng/mL) for 24 hours. OVCAR-3 and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 24 hours. Cells were lysed, and isolated RNAs were analyzed for HE4 (A) or TNF (B) via quantitative reverse transcription PCR, as described in “Materials and Methods”.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Transfection, Plasmid Preparation, Isolation, Reverse Transcription

Journal: PLOS ONE

Article Title: The NF-κB-HE4 axis: A novel regulator of HE4 secretion in ovarian cancer

doi: 10.1371/journal.pone.0314564

Figure Lengend Snippet: (A) 2008 cells were co-transfected with IKK2-EE expression vector and HE4 siRNAs (sourced from two different vendors) for 48 hours. Cells were lysed, and protein expression of putative NF-kB targets was measured via immunoblot analysis. Heat map represents the relative expression of each NF-kB target evaluated by densitometric analysis. GAPDH served as a loading control for normalization. (B) 2008 cells were transfected with HE4 siRNAs from three different vendors (48 or 72 hours). Expression of HE4, α 5 -integrin, and α-tubulin was measured via immunoblot analysis. (C)The effects of HE4 knockdown on adhesion molecules. 2008 cells were transfected with HE4 siRNA. Protein extracts were prepared and subjected to immunoblot analysis against the proteins as indicated. (D) as in (C) but other ovarian (HCH-1 and Caov-3) and non-ovarian (BxPC-3, pancreatic) cancer cell lines were employed. (E) HE4 knockdown inhibited cell adhesion to fibronectin. Cell adhesion of 2008 cells transfected with HE4 siRNA (72 hours) was determined in fibronectin- or collagen-coated cell culture plate as described in “Materials and Methods.” (F) HE4 knockdown inhibited cell migration. 2008 cells were transfected with either control or HE4 siRNA and cultured until confluence. A scratch was made using a P1000 pipette tip, and images were captured at 0, 6, 12, and 18 hours (scale bar: 500 μm). Right panel: The average width of the gap (y-axis, in μm) was calculated as described in the “Materials and Methods” section.

Article Snippet: Santa Cruz Biotechnology siRNAs for control (cat. No. sc-37007) and HE4 (cat. No. sc-43826); Vectors: empty (addgene, cat. No. 24165), p65 (addgene, cat. No. 21966), IKK2-EE (addgene, cat. No.11105); antibodies: p53 (Santa Cruz Biotechnology cat. No. sc-126), HE4 (OriGene cat. No. UM870019), p50 (BioLegend cat. No. 603901), P-c-Jun (BioLegend cat. No. 605551).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Knockdown, Cell Culture, Migration, Transferring

Journal: Science Advances

Article Title: Three modes of viral adaption by the heart

doi: 10.1126/sciadv.adp6303

Figure Lengend Snippet: ( A ) Differential changes in inflammatory hallmarks for CVB3-expressing AC16 clones engineered with constitutively active IKK2-EE or dominant-negative IκBα-SR. The NES was calculated for each inflammatory hallmark pathway relative to empty vector control. The hallmark pathways are ordered as in . ( B ) Reversion of ARE-Down and ARE-Up states in AC16 clones engineered with constitutively active MKK6-EE or treated with the p38 inhibitor (p38i) BIRB796 (5 μM). The distribution of DE genes with or without AREs for each adaption was compared as in . ( C ) PC paths between centroids of adapted states. RNA-seq data from empty vector controls of each clone are shown and organized by adaption. ( D and E ) Displacement of adapted states with IKK2-EE, and convergence toward ARE-Down with IκBα-SR. ( F and G ) Convergence toward ARE-Down with p38i. Clones ( n = 3 per adaption) were acutely transduced or treated, quiesced, and profiled by RNA-seq. For (D) and (E), the relocalization of perturbed centroids (arrowheads) toward the empty vector controls of other states (circles) was assessed by paired two-sided t test. Bonferroni-corrected P values are colored according to the adaption the centroid moves toward. For IKK2-EE, the Inflammatory adaption (black P value) moves away from the ARE-Down adaption. For p38i, the ARE-Down adaption (black P value) moves away from the Inflammatory adaption.

Article Snippet: Primary antibodies recognizing the following targets were used: FLAG (Sigma-Aldrich, F7425, 1:2000 dilution); HA (Roche, 11867423001, 1:1000 dilution); IKK2 (Cell Signaling Technology, 8943, 1:1000 dilution); IκBα (Cell Signaling Technology, 4814, 1:1000 dilution); MK2 (Assay Design, KAP-MA015, 1:1000 dilution); MK2 p-Thr 344 (Cell Signaling Technology, 3007, 1:1000 dilution); p38 (Santa Cruz Biotechnology, sc-535, 1:5000 dilution); p38 p-Thr 180 /Tyr 182 (Cell Signaling Technology, 4511, 1:1000 dilution); tubulin (Abcam, ab89984, 1:20,000 dilution); and vinculin (EMD Millipore, 05-386, 1:10,000 dilution).

Techniques: Expressing, Clone Assay, Dominant Negative Mutation, Plasmid Preparation, Control, RNA Sequencing

Journal: bioRxiv

Article Title: Cyld aborts hyperactivation of synovial fibroblasts in inflammatory arthritis by regulating the TAK1/IKK2 signaling axis

doi: 10.1101/2023.11.13.566552

Figure Lengend Snippet: The TAK1/ikk2 axis regulate TNF arthritogenicity in TNF-mediated arthritis (a) Detection of Tak1 ubiquitination state upon TNF stimulation in lysates from Cyld f/f and CyldM-Δ 9/Δ9 SF cultures (subjected to IP with Tab1 antibody), for the indicated time points. (n=3) (b) Detection of phosphorylation state of Tak1 upon TNF stimulation in lysates from Cyld f/f and Cyld M-Δ9/Δ9 SF cultures (subjected to IP with Tab1 antibody), for the indicated time points. (c) Histological evaluation of hTNFtg Ikk2 f/f Cyld f/f, hTNFtg Ikk2 M-Het Cyld Μ-Δ9/Δ9 and hTNFtg Ikk2/Cyld Μ-Δ9/Δ9 mice (d) Survival rates of Cyld f/f and Cyld M-Δ9/Δ9 SF SFs treated with 50ng/ml TNF, in the absence (Black bars) of absence (Grey bars) of ikk2 inhibitor ML120b, zVAD and Nec1s as indicated (T+Z: TNF 50ng/ml + zVAD, T+N1s: TNF 50ng/ml+ Nec1s)( n = 3-5)

Article Snippet: For immunoblotting, samples were collected in RIPA buffer, containing 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 10 mM Tris HCl, pH 7.4, 1 mM EDTA, protease inhibitors [Roche], and phosphatase inhibitors [Sigma-Aldrich], separated by SDS/PAGE (10-12.5%), transferred to nitrocellulose membranes [Millipore], and probed with the following antibodies: Ikk2, pJNK1/2, p-Ikk1/2, p-p38 [Cell Signaling]; ikB, JNK, p38, p-ERK1/2, ERK1/2, tubulin, actin [Santa Cruz Biotechnology].

Techniques: