Journal: Pharmaceuticals

Article Title: New Genetic Bomb Trigger: Design, Synthesis, Molecular Dynamics Simulation, and Biological Evaluation of Novel BIBR1532-Related Analogs Targeting Telomerase against Non-Small Cell Lung Cancer

doi: 10.3390/ph15040481

Figure Lengend Snippet: Telomerase activity in cancer cells as a percent of control cells with target compounds 29a , 36b , and 39b .

Article Snippet: The human A549 (epithelial cell lung carcinoma, ATCC, Manassas, VA, USA), HCC44 (non-small cell lung adenocarcinoma, Leibniz Institute DSMZ-German Collection of the Microorganisms and the Cell Cultures, Braunschweig, Germany), and NCI-H23 (non-small cell lung adenocarcinoma, ATCC, Manassas, VA, USA) cell lines (compounds 29a , 36b , and 39b ) were diluted to 10 μM and incubated for 48 h before being tested using the TRAP method.

Techniques: Activity Assay, Control

Journal: iScience

Article Title: Development of human alveolar epithelial cell models to study distal lung biology and disease

doi: 10.1016/j.isci.2022.103780

Figure Lengend Snippet: Derivation of AEC lines that proliferate in culture but do not form tumors in nude mice (A) AEC line derivation scheme. Previously frozen purified human AT2 cells from Lung-FT were first cultured in Y-27632 medium. Proliferative cells (a) were then transduced with LeGO iT (tdTomato) and LeGO iG (eGFP) lentiviruses carrying hTERT (b), CDK4 R24C (c), CDK4 R24C + hTERT (d), and SV40 LgT (e). Scale bar, 100 μm. (B, C) Additional SV40 LgT-transduced cell lines were established from AT2 cells purified from (B) Lung-ON and (C) Lung-TN in Y-27632 medium (f and h), then transduced with SV40 LgT (g and i). Scale bar, 100 μm. (D) Cell proliferation assays in technical triplicates from three independent experiments (mean total number of cells ± std dev). ∗p<0.05, ∗∗p< 0.005 by independent two-sample t test relative to AEC-FT-ROCKinh. (E) Cell proliferation assays for AEC-ON and AEC-TN, under standard (1,000 cells/well) and high-density (5,000 cells/well) conditions. (F) Anchorage-independent growth assays. A549 cells, positive control. Colonies were stained with crystal violet and counted using ImageJ/Fiji after 1 month. Inset images, |×2.5×| magnified to show colonies. (G) Colony quantification of six technical replicates from at least three independent experiments (mean ± std dev). N.S., not significant; ∗p< 0.05, nonparametric Wilcoxon test. (H) Subcutaneous injection into NU/J mice to assess tumorigenicity of AEC-FT, AEC-ON, and AEC-TN cell lines over 3 months. A549 cells, positive control; AEC-hTERT line, negative control. Equal numbers of male and female mice per group. Sample labels indicate which cell line was injected into the left and right flanks (left/right). (I) Photos of excised nodules (scale bar, 5 mm) and corresponding H&E stainings. (J) Graph of nodule growth starting at 3 weeks postinjection. See also Figure S1 , and .

Article Snippet: Human A549 lung adenocarcinoma cells , ATCC , CRM-CCL-185; RRID:CVCL_0023.

Techniques: Purification, Cell Culture, Transduction, Positive Control, Staining, Injection, Negative Control

Journal: iScience

Article Title: Development of human alveolar epithelial cell models to study distal lung biology and disease

doi: 10.1016/j.isci.2022.103780

Figure Lengend Snippet:

Article Snippet: Human A549 lung adenocarcinoma cells , ATCC , CRM-CCL-185; RRID:CVCL_0023.

Techniques: Transduction, Control, Plasmid Preparation, Virus, Recombinant, Membrane, Software

Journal: Scientific Reports

Article Title: Protein serine/threonine phosphatase PPEF-1 suppresses genotoxic stress response via dephosphorylation of PDCD5

doi: 10.1038/srep39222

Figure Lengend Snippet: ( a ) PPEF-1 overexpression blocks the effect of MG132 on PDCD5 stabilization. HCT116 cells were transfected with Flag-tagged PPEF-1. Two days after transfection, cells were treated with MG132 (10 μM) for 6 h. Total lysates were immunoblotted with the indicated antibodies. ( b , c ) PPEF-1 reduces the stability of PDCD5 protein. HCT116 cells were transfected with either Flag-PPEF-1 or si-PPEF1, cells were treated with 10 μg/mL cycloheximide (CHX) for the indicated times, and then immunoblotted with the indicated antibodies. ( d ) PPEF-1 does not affect the mRNA level of PDCD5. A549 cells were transfected with either Flag-PPEF-1 or si-PPEF1, and qRT-PCR analyses were performed to measure PDCD5 mRNA expression levels. * P < 0.05 versus control. ( e ) PPEF-1 knockdown increases PDCD5 phosphorylation. Cells were harvested and immunoblotted 48 h after siRNA transfection. ( f ) Inactive PPEF-1 D172N mutant does not affect PDCD5 Ser-119 phosphorylation. Forty-eight hours after transfection, cells were treated with 50 μM etoposide (ET) for 2 h and then harvested. Cell lysates were immunoblotted with the indicated antibodies. ( g ) PPEF-1 overexpression increases PDCD5 ubiquitination. HCT116 cells were transfected with HA-tagged ubiquitin, Flag-tagged PPEF-1, Flag-tagged PPEF-1 D172N , or Myc-tagged PDCD5 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using the Myc antibody and immunoblotted with the HA antibody. Full-length blots are presented in .

Article Snippet: Human lung adenocarcinoma cell line A549 and human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis.

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Expressing, Control, Knockdown, Phospho-proteomics, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation

Journal: Scientific Reports

Article Title: Protein serine/threonine phosphatase PPEF-1 suppresses genotoxic stress response via dephosphorylation of PDCD5

doi: 10.1038/srep39222

Figure Lengend Snippet: (a) PPEF-1 overexpression induced PDCD5 degradation, but the inactive PPEF-1 D172N mutant did not affect ET-induced stabilization. A549 cells were transfected with the indicated constructs, and then treated with ET for 6 h. Fixed cells were stained with the indicated immunofluorescent antibodies. Bar scale = 10 μm (b) Active PPEF-1, but not the inactive PPEF-1 D172N mutant, antagonizes CK2-induced PDCD5 stabilization. A549 cells were transfected with Myc-CK2α or Flag-PPEF-1 as indicated. Total cell extracts were immunoblotted with the indicated antibodies. (c) Inactive PPEF-1 D172N , but not wild-type PPEF-1, fails to antagonize CK2-induced reduction of PDCD5 ubiquitination. HCT116 cells were transfected with HA-tagged ubiquitin, Myc-tagged CK2, PPEF-1, PPEF-1 D172N , or Flag-tagged PDCD5 as indicated. Cells were treated with MG132 (10 μM) for 6 h before harvesting. Total cell extracts were immunoprecipitated using Myc antibody and immunoblotted with HA antibody. Full-length blots are presented in .

Article Snippet: Human lung adenocarcinoma cell line A549 and human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis.

Techniques: Over Expression, Mutagenesis, Transfection, Construct, Staining, Ubiquitin Proteomics, Immunoprecipitation

Journal: Scientific Reports

Article Title: Protein serine/threonine phosphatase PPEF-1 suppresses genotoxic stress response via dephosphorylation of PDCD5

doi: 10.1038/srep39222

Figure Lengend Snippet: (a) PPEF-1 inhibits ET-induced p53 activation by PDCD5 dephosphorylation. A549 cells were transfected with Flag-tagged PPEF-1 and treated with ET. Cell lysates were immunoblotted with the indicated antibodies. (b) PPEF-1 reduces ET-induced p53 target gene expression. Puma and Bax mRNA expression were measured by qRT-PCR. (c) PPEF-1 phosphatase activity was necessary to inhibit ET-induced PDCD5 phosphorylation. Cells were transfected with Flag-tagged PPEF-1 WT or inactive PPEF-1 D172N mutant, and treated with ET for 6 h. Cell lysates were immunoblotted with the indicated antibodies. (d) PPEF-1 knockdown enhanced PDCD5-p53 signaling. A549 cells were transfected with siPPEF-1, and cell lysates were immunoblotted with the indicated antibodies. (e) PPEF-1 suppresses p53 activation via negative regulation of PDCD5. PDCD5 f/f MEF cells were infected with cre-Adenovirus for 24 h, and then transfected with siPPEF-1 for 48 h. Immunoblotting was performed with the indicated antibodies. Full-length blots are presented in .

Article Snippet: Human lung adenocarcinoma cell line A549 and human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis.

Techniques: Activation Assay, De-Phosphorylation Assay, Transfection, Targeted Gene Expression, Expressing, Quantitative RT-PCR, Activity Assay, Phospho-proteomics, Mutagenesis, Knockdown, Infection, Western Blot

Journal: Scientific Reports

Article Title: Protein serine/threonine phosphatase PPEF-1 suppresses genotoxic stress response via dephosphorylation of PDCD5

doi: 10.1038/srep39222

Figure Lengend Snippet: (a) PPEF-1 overexpression confers resistance to ET-induced cell apoptosis. A549 cells were transfected with either Flag-PPEF-1 or siRNAs. Cells were treated with ET after 24 h, and cell viability was determined using MTT assays. Error bars indicate standard deviation (SD; n = 3). ** P < 0.01 versus control. (b) PPEF-1 knockdown increases ET-induced apoptosis in A549 cells. A549 cells were transfected with either Flag-PPEF-1 or siRNAs. Cells were treated with ET after 24 h and cell apoptosis was measured using TUNEL assays. Error bars indicate standard deviation (SD; n = 3). * P < 0.05, ** P < 0.01 versus control. (c) PPEF-1 overexpression enhances the chemoresistance of A549 cells. PPEF-1 or PPEF-1 D172N stable overexpression cells were injected subcutaneously into the right flank of nude mice and allowed to grow for 2 weeks. Then, mice with comparable-sized tumors (~50 mm 3 ) were selected for treatment with ET (10 mg/kg) at 2-day intervals for 6 weeks. Tumor volumes were measured every other day. * P < 0.05, ** P < 0.01 versus A549-emp+ET. Error bars indicate SD ( n = 6 per group). Full-length blots are presented in .

Article Snippet: Human lung adenocarcinoma cell line A549 and human colorectal carcinoma cell line HCT116 were purchased from and authenticated by the Korean Cell Line Bank (Seoul, Korea) using short tandem repeat analysis.

Techniques: Over Expression, Transfection, Standard Deviation, Control, Knockdown, TUNEL Assay, Injection