Journal: Antibiotics

Article Title: Two Shorter Variants of the Proline-Rich Antimicrobial Peptide B7-005 Scaffold Active Against Clinical Isolates of Pseudomonas aeruginosa and Staphylococcus aureus

doi: 10.3390/antibiotics15040412

Figure Lengend Snippet: Effects of Peptides on BEAS-2B cell line’s viability. Cell viability was measured using an MTT assay on BEAS-2B cells (4 × 10 5 cells/mL) after 24 h of exposure to the peptides. Assays were conducted in complete medium (RPMI + 10% FBS, Na-Pyruvate, Pen/Strep, L-Glutamine). Results are presented as percentages of viable cells relative to untreated control (Untr.) cells represented in grey colour (set to 100% viability). Data represent the average ± SD of three independent biological replicates, each performed in triplicate (n ≥ 9). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the untreated sample (one-way ANOVA test followed by Tukey’s multiple comparisons test).

Article Snippet: BEAS-2B cells were obtained from the American Type Culture Collection (ATCC ® CRL-3588TM, Manassas, VA, USA).

Techniques: MTT Assay, Control

Journal:

Article Title: A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species

doi: 10.1002/ijc.25587

Figure Lengend Snippet: (A) Flow cytometric analysis using 2, 7-dichlorohydrofluorescein diacetate (H2DCFDA dye) detailed the ability of DMAPT to generate ROS, which was blocked by N-acetyl cysteine (NAC) in UMUC-3 cell (upper panel) and A549 cells in (lower panel). The solid peaks are a control representing baseline ROS levels and grey peaks represent amount of ROS after exposure to drug treatment. (B) Western blotting demonstrated DMAPT treatment resulted in phosphorylation of c-Jun in both TCC (UMUC-3) and NSCLC (A549 and H522) cell lines. As observed in the left hand panel, this was substantially suppressed by pre-treatment with NAC in the UMUC-3 cell line. In the right hand panel it is noted the phosphorylation of cJUN was dose dependent in H522. (C and D) Electrophoretic mobility gel shift assays (EMSA) demonstrated constitutive NFκB DNA binding in all of the cell lines with the p65-p50 label indicating the location of this heterodimer. The presence of the most active NFκB subunit, p65, in the DNA-protein complex of both NSCLC and TCC cell lines was confirmed by a supershift with a p65 antibody. This is delineated in the last column of UMUC-3 and RT4 and the first column of A549 and BEAS2B EMSA figures. When the NFκB dimer complexes from the no DMAPT columns are compared, the location of the p65 dimer in the heterodimer complexes can be discerned. DMAPT's ability to inhibit NFκB DNA binding after 3 hours of treatment in UMUC-3 and RT4 (C) and A549 and BEAS2B (D) cell lines is shown to be dose dependent. (E) Western blotting revealed DMAPTs ability to decrease NFκB dependent proteins: TRAF-2 in UMUC-3 (left panel) as well as XIAP in RT4 and TRAF2 in A549 cells (right panel).

Article Snippet: The growth media of BEAS2B (BEGM, Lonza, Walkersville) contained Phenol red and interfered with the FACS.

Techniques: Control, Western Blot, Phospho-proteomics, Gel Shift, Binding Assay

Journal:

Article Title: A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species

doi: 10.1002/ijc.25587

Figure Lengend Snippet: (A) BrdU incorporation assay demonstrated DMAPT's ability to inhibit the in vitro proliferation of NSCLC cell lines – A549, H-222 and BEAS (left panel) and TCC cell lines - UMUC, HT1197, HT1376 and RT-4 (right panel). For each cell line 2,000 cells were placed in 100μL, drug added at concentrations of 1, 2, 5, 10 and 20 μM after 24 hours of cell growth and the percentage of cells relative to untreated control was determined after 48 hours of drug exposure. The trypan blue assay showed that after one DMAPT dose the viability for each cell line at 48 hours was 26.2% for A549 with 20 M, 20.9% for BEAS2B with 5 M; 11.8% for UMUC-3 with 10μM and 13.4% for RT4 with 10uM. (B) Using a BrdU incorporation assay NAC was observed to decrease DMAPT's in vitro anti-proliferative activity in UMUC-3, A549 and BEAS2B in a dose dependent manner. In all cell lines, 10 M DMAPT suppressed proliferation by greater than 90% and this was almost completely abrogated by 1mM NAC in UMUC-3 and A549 and DMAPT's anti-proliferative efficacy was limited to only 25% in BEAS2B cell line. (B, bottom right panel). Electrophoretic mobility gel shift assay demonstrating NAC abrogating DMAPT's ability to inhibit NFκB DNA binding in a dose dependent manner in UMUC-3.

Article Snippet: The growth media of BEAS2B (BEGM, Lonza, Walkersville) contained Phenol red and interfered with the FACS.

Techniques: BrdU Incorporation Assay, In Vitro, Control, Activity Assay, Gel Shift, Binding Assay

Journal:

Article Title: A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species

doi: 10.1002/ijc.25587

Figure Lengend Snippet: (A) Flow cytometry with propidium iodine and Annexin V staining showed the ability of DMAPT to induce early and late apoptosis in UMUC-3 with 5 and 10 M. In this assay, the upper left quadrant with predominant PI staining represents necrosis, the upper right, combined PI and annexin staining represents late/atypical apoptosis and bottom right with predominant annexin staining represents early/typical apoptosis. (B) Western blotting of proteins in UMUC-3 detailed DMAPT induced cleavage of procaspase 8, procaspase 3, and PARP. Flow cytometry demonstrated DMAPT induced early and late apoptosis in RT4 (C), BEAS-2B (D) and A549 (E). In BEAS-2B, DMAPT also induced substantial necrosis. All of these types of cell death were blocked by NAC in RT4, BEAS2B and A549.

Article Snippet: The growth media of BEAS2B (BEGM, Lonza, Walkersville) contained Phenol red and interfered with the FACS.

Techniques: Flow Cytometry, Staining, Western Blot

Journal:

Article Title: A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species

doi: 10.1002/ijc.25587

Figure Lengend Snippet: (A) EMSA detailed NNK increased NFκB DNA binding in a dose dependent manner in BEAS2B (left panel) and that DMAPT was able to block NNK-induced NFκB DNA binding in BEAS2B (right panel). (B) Western blotting detailed NNK increased the levels of cyclin D1 protein and that this was blocked by DMAPT (left panel). EMSA revealed NNK did not increase NFκB DNA binding in RT4 nor did it block the ability of DMAPT to inhibit NFκB DNA binding (right panel). In all EMSA figures the first panel is p65 supershift demonstrating location of p65 in the p65-50 heterodimer in the other lanes. (C) Flow cytometry demonstrated DMAPT induced S phase and G2 phase accumulation in RT4 and BEAS2B, respectively. These changes were present at 12 and 24 hours in both cell lines (data not shown for 12 hour time point) and were blocked by NAC. (D) NNK did not alter cell cycle distribution in RT4 but did increase the number of BEAS2B cells in G1 phase at 24 hours (37% in control versus 55% with NNK treatment). The ability of DMAPT to induce S phase accumulation in RT4 was not abrogated by exposing the cells to NNK after DMAPT (top panel). In BEAS2B, whereas DMAPT alone induced G2 phase accumulation (C, lower panel), DMAPT treatment followed by NNK exposure caused cells to accumulate in G1 and S phases.

Article Snippet: The growth media of BEAS2B (BEGM, Lonza, Walkersville) contained Phenol red and interfered with the FACS.

Techniques: Binding Assay, Blocking Assay, Western Blot, Flow Cytometry, Control

Journal: Frontiers in Toxicology

Article Title: Diesel exhaust particles alter mitochondrial bioenergetics and cAMP producing capacity in human bronchial epithelial cells

doi: 10.3389/ftox.2024.1412864

Figure Lengend Snippet: Exposure of BEAS-2B cells to DEP induced oxidative stress markers and differentially affected expression of key players of the antioxidant response system.

Article Snippet: BEAS-2B cells were exposed to 100 or 300 μg/mL DEP (SRM 2975, National Institute of Standards and Technology) for 24 h. Culture medium was collected to measure IL-8 and IL-6 protein concentrations using specific enzyme-linked immunosorbent assays (ELISAs) ( ).

Techniques: Expressing, Control