Journal: Cancer research

Article Title: Caspase-9b interacts directly with cIAP1 to drive agonist-independent activation of NF-κB and lung tumorigenesis

doi: 10.1158/0008-5472.CAN-15-2512

Figure Lengend Snippet: (A–D) A549 cells were transfected with the indicated siRNAs,48hr. HBECs were transfected with adenovirus (Ad) control (Con) or expressing C9b,24hr. RNA was isolated and analyzed by RNAseq. (A) Network computationally generated on the basis of IPA knowledge memory indicated a high relevance to the NF-κB pathway. Red, increased expression; green, reduced expression; higher color intensity indicates higher difference in expression level. Cell localization of NF-κB-related factors is indicated (Fig. S1, larger legend). (B) Total protein was subjected to SDS-PAGE/immunoblotting. (C) The network for A549 cells comprises 77 NF-κB-related genes significantly different between control siRNAs and C9b siRNA samples. The network for HBECs comprises 133 NF-κB-related genes significantly different between AdCon and AdC9b samples. There are 15 overlapping genes; (Table S2). (D) Heatmap showing hierarchical clustering of 7 overlapping NF-κB genes that expression is contrastingly affected by C9b siRNA (A549) versus up-regulation of C9b (HBEC); red=increased expression; green=reduced expression (Table S3). Results were repeated on 2 separate occasions. (E) Total RNA from C9b-downregulated A549 cells was subjected to RT-qPCR for 7 overlapping NF-κB genes in (D). Data in E are shown as mean ± SD; n=6: one-way ANOVA followed by a Tukey’s HSD test (##p<0.01, **p<0.001); C9b-siRNA sample was compared to each control siRNA sample. (F) RT-qPCR for C9b and 5 NF-κB target genes was performed on lung tissues of 22 NSCLC patients. Values=means of triplicates; each dot represents a patient; Pearson correlation coefficient (r value) and corresponding significance level (p value) are indicated (Fig.S2). (G) LCM images of pre-LCM slide, post-LCM slide after isolating tumors and post-LCM cap view of purified tumor epithelium; tumor and normal epithelial cells are indicated in pre-LCM slides. (H) RT-qPCR for C9b and BIRC5 was performed from RNA prepared from LCM purified epithelium in (G); data are shown as means ± SD (n=3). (I) RT-qPCR for C9a, C9b or BIRC5 was performed with RNA purified from the NSCLC cell lines in Table S3.; Values=means of triplicates; each dot represents a NSCLC cell line.

Article Snippet: A549, H358, H460, H838, H1299, H1792 and H1869 cells (NSCLC cell lines) were obtained from ATCC, and Primary human bronchial epithelial primary cells (HBECs) were obtained from Cell Applications.

Techniques: Transfection, Expressing, Isolation, Generated, SDS Page, Western Blot, Quantitative RT-PCR, Purification

Journal: Oncotarget

Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

doi: 10.18632/oncotarget.24772

Figure Lengend Snippet: Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.

Article Snippet: Normal human bronchial epithelial BEAS2B cells were generously provided by Prof. Rotem Karni (The Hebrew University, Jerusalem, Israel).

Techniques: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Sequencing

Journal: Journal of Innate Immunity

Article Title: Midkine Is Part of the Antibacterial Activity Released at the Surface of Differentiated Bronchial Epithelial Cells

doi: 10.1159/000346709

Figure Lengend Snippet: Production of MK by airway epithelial cells in vitro and contribution to bactericidal activity of the air surface liquid. a Primary HBEC were cultured in an ALI for 4 weeks to allow differentiation, in the absence of antibiotics. The medium was changed, the apical side rinsed with buffer, and the cells were incubated for another 24 h, whereafter the underlying medium was collected and the apical surface was rinsed with buffer. The amount of MK was determined by ELISA and the results shown represent the mean and SD from three separate experiments. b HAEpiC, primary HBEC, the bronchial epithelial cell lines BEAS-2B and 16-HBE, and the alveolar epithelial cell line A549 were grown to near confluence in a flask. The cell medium was changed and the cells incubated for 72 h, the medium collected and the MK content determined by ELISA. No MK could be detected in the medium from the A549 cell line (data not shown). The results represent the mean and SD from three different incubations. c, d To investigate possible bactericidal activity in the air surface liquid, HBEC were grown to confluence and allowed to differentiate using the air-liquid system in the presence of RA. After removal of mucus, the apical surface was rinsed with buffer which was incubated with S. pneumoniae (strain TIGR4). One part was used for the viable count assay and one part was processed for SEM. Bacteria incubated with the rinsing fluid show blebbing and disturbed integrity (d), compared with bacteria incubated in buffer alone (c). e To determine whether MK contributes to the bactericidal activity of the rinsing fluid, MK was immunoprecipitated from one portion of the rinsing fluid while another portion was immunoprecipitated using control antibodies. Thereafter, the rinsing fluids were used to investigate bactericidal activity against S. pneumoniae (strain TIGR4), using the viable count assay. The rinsing fluid depleted of MK showed significantly lower bactericidal activity compared with the rinsing fluid that had been immunoprecipitated with control antibodies, suggesting that MK constitutes a significant part of the bactericidal activity in airway surface liquid. Statistical significance was determined using Student's t test for paired observations. IP = Immunoprecipitation.

Article Snippet: Human pulmonary alveolar epithelial cells (HAEpiC) comprised of type 1 and type 2 pneumocytes (3H Biomedical) were grown in poly- L -lysin (Sigma) coated flasks in alveolar epithelial cell medium with supplements (3H Biomedical).

Techniques: In Vitro, Activity Assay, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Schematic representation of workflow to identify CF correctors. Differentially expressed genes from CF patients (ΔF508-CFTR homozygous) were used for connectivity mapping to identify CF candidate therapeutics. These candidate therapeutics were further prioritized by incorporating additional layers of information from systems biology of CF. Prioritized CF candidate therapeutics were experimentally validated using intestinal organoids and bronchial epithelial cells from CF patients.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques:

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: FIS in ΔF508/ΔF508-cftr intestinal organoids. Representative images of intestinal spheres demonstrating fluid secretion in response to the test compounds (Panel A). Bar graph represents quantitation of fluid secretion in the intestinal organoids (Panel B). In this preliminary screening assay, all compounds were tested at a dose of 10 µM, while the known correctors (VX-809 and VX-661) were tested at a dose of 2 µM. The highlighted compounds (PP2 and LY294002) in the bar graph were found to show forskolin-induced swelling significantly better than that seen with the treatment of VX-809 or VX-661. The related raw, and processed data for FIS assay is made available as Additional file 4.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay, Screening Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: PP2 Dose response experiments in mice. Panel A. Representative images of ΔF508/ΔF508- cftr enterospheres depict secretion under various treatment conditions: (i) PP2 (0, 0.1, 1 and 10 µM, 24 h), (ii) PP2 (1 µM, 24 h) + CFTR inh -172 (20 µM, 30 min), (iii) VX-809 (2 µM, 24 h), and (iv) PP2 (1 µM, 24 h) + VX-809 (2 µM, 24 h). Panel B . Line graph represents quantitation of fluid secretion in enterospheres under various treatment conditions as described previously (Panel A).

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Preclinical validation of PP2. Panel A shows representative images of intestinal spheres demonstrating forskolin-induced swelling (FIS) in enteroids from normal subject and those from cystic fibrosis patients in response to DMSO, PP3, VX-809, and PP2. The bar graph represents quantitation of fluid secretion in the intestinal organoids. Panel B. Western blot data depict bands B (immature or endoplasmic reticulum form) and C (mature or membrane form) of CFTR immunoprecipitated from HEK 293 cells that overexpressed FLAG ΔF508-CFTR with and without treatment with PP2 (0.5 and 2 µM, 24 h), VX-809 (2 µM, 24 h) alone, and PP2 + VX-809 (2 µM each, 24 h). Panel C . Representative CFTR-mediated short-circuit currents ( I sc ) in human bronchial epithelial cells carrying ΔF508/ΔF50-CFTR in response to VX-809, PP2, and VX-809+PP2 treatment. Bar graphs represent data quantification of maximal increase in I sc /cm 2 from n = 5 (DMSO-treated) and n=6 (PP2-treated) independent traces. Panel D . Predicted binding pose of PP2 to ATP binding site.

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Quantitation Assay, Western Blot, Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

doi: 10.1101/288324

Figure Lengend Snippet: Functional enrichment network of differentially expressed genes following treatment with PP2 (2 µM; 48 h) in human CFBE (ΔF508/ΔF508- CFTR )

Article Snippet: RNA sequencing was performed on primary human bronchial epithelial cells homozygous for ΔF508-CFTR mutation (sample ID KKCFFT004I, Charles River, Wilmington, MA).

Techniques: Functional Assay