Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Effect of JNK inhibition on A549 cell viability as a function of cisplatin concentration. (a, b) 7.5 μg/mL concentration of cisplatin becomes lethal in combination with JNK inhibitor SP600125. However, at higher cisplatin concentrations, the inhibitor either protects against cisplatin-induced cell death (a) or does not have any effect (b). (c) Similar effects are observed in the colon cancer cell line DLD-1. (d–g) SP600125 potentiates the appearance of cells with apoptotic morphology in 7.5 μg/mL cisplatin-treated A549 cells (d, e), in contrast to 30 μg/mL cisplatin-treated A549 cells (f, g). Relative cell viability is cell viability after 72 h treatment normalized by initial viability (measured by the MTT method). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. Fluorescent pictures were obtained with a mixture of AO/EB dyes as described in the section. SP—JNK inhibitor SP600125 (20 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Inhibition, Concentration Assay

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: JNK inhibitor SP600125 reduces JNK target transcription factor c-Jun phosphorylation in cisplatin-treated A549 cells. Different concentrations of cisplatin were used. (a) Representative Western blots from 20 and 40 h of treatments are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. (b) Repeated addition (after 24 h) of SP potentiates suppression of c-Jun phosphorylation in response to cisplatin treatment. (c) Repeated addition of SP strengthens the protective effect of JNK inhibition at 15−30 μg/mL concentrations of cisplatin in A549 cells. SP—JNK inhibitor SP600125 (20 μM). p *** < 0.0005, N = 3.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Western Blot, Staining, Inhibition

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Cisplatin concentration-dependent effect of different JNK inhibitors on A549 cell viability. Different JNK inhibitors show the same dependence on cisplatin concentration on cell viability. Statistically significant reduction in cell viability is observed at 7.5 μg/mL cisplatin in combination with 10 μM AS601245 (a), 10 μM bentamapimod (b), 2 μM JNK inhibitor IX (c), and 5 μM XG-102 (d). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. (e) Reduction in c-Jun phosphorylation upon the addition of JNK inhibitor XG-102, as determined by Western blot. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Activation of TP53 in A549 cells in response to different concentrations of cisplatin. (a, b) Expression and phosphorylation at serine-6 is induced by cisplatin and is maximal at 15 μg/mL (6 h of cisplatin treatment). (c, d) Prolonged and increasing expression and phosphorylation of TP53 in cells treated with either 7.5 μg/mL or 30 μg/mL of cisplatin. (e) TP53 activator nutlin-3a potentiates cisplatin-induced decrease in cell viability. (f) Nutlin-3a reduces viability of cisplatin + SP600125-treated cells. C—control without cisplatin; Nut—nutlin-3a (10 μM); SP—SP600125 (20 μM). Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented, p ** < 0.005, p *** < 0.0005, N = 4. (g) Nutlin induces expression of TP53. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Control, Western Blot, Staining

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Involvement of AKT signaling pathway in cisplatin-induced A549 cell death. (a) AKT protein level does not depend on cisplatin concentration. 6-h-long exposure is presented in the Western blot picture. (b) Phosphorylation/activation of AKT is cisplatin concentration-dependent. 6-h-long exposure is presented in the Western blot picture. (c) AKT protein level does not change during 40 h of cisplatin treatment. (d) Dynamics of AKT activation following cisplatin (7.5 or 30 μg/mL) treatment. (e, f) AKT inhibitor capivasertib enhances cell death at all concentrations of cisplatin used both in the absence (e) and presence (f) of JNK inhibitor SP600125. (g, h) GSK3-β inhibitor SB415286 protects A549 cells from cisplatin both in the absence (g) and presence (h) of JNK inhibition. Representative test results (all measurements were performed in quadruplicate) from more than five experiments are presented. p*<0.05, p**<0.005, p***<0.0005, N = 4. (i) Capivasertib inhibits AKT activity as shown by the inhibition of AKT molecular target GSK3-β phosphorylation at serine-9. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as loading controls. C—control without cisplatin; CAP—capivasertib (10 μM); SP—SP600125 (20 μM), SB—GSK3 inhibitor SB415286 (15 μM).

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Concentration Assay, Western Blot, Phospho-proteomics, Activation Assay, Inhibition, Activity Assay, Staining, Control

Journal: ACS Omega

Article Title: The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin

doi: 10.1021/acsomega.4c01950

Figure Lengend Snippet: Opposite changes in TP53 and AKT phosphorylation following SP600125 treatment of A549 cells exposed to different concentrations of cisplatin. (a) Expression of TP53 is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (b) Phosphorylation of TP53 (serine-6) is increased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. (c) Phosphorylation of AKT (threonine-308) is decreased in response to the combination of SP + 7.5 μg/mL cisplatin, in contrast to the combination of SP + 30 μg/mL cisplatin. Representative Western blots are shown. Total protein Coomassie-stained polyacrylamide gels serve as a loading control. SP—JNK inhibitor SP600125 (20 μM); 20 h of treatment.

Article Snippet: Human non-small-cell lung carcinoma A549 cells were purchased from Cell Lines Service GmbH, Eppelheim, Germany.

Techniques: Phospho-proteomics, Expressing, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: Two-component vector system where Cas9 is integrated into the AAVS1 safe harbor locus to sustain stable expression. The second component is a lentiviral system containing a sgRNA targeting on and off-target sites downstream of an AcrIIA4 variant that is uniquely barcoded. B. Editing efficiency measured over time within cells expressing wild-type AcrIIA4 (circular points) versus those without wild-type AcrIIA4 (rectangular). C. Indel mutation profiles spanning the on- and off-target regions. Deletions are shown in blue and insertions are in orange. D. Experimental design in which AcrIIA4 variants were delivered to A549 cells expressing Cas9, which were cultured for 10 days prior to collection and sequencing of the AcrobaTx barcode. E. Structure of the AcrobaTx barcode and definitions of gene editing activity metrics derived from measuring editing outcomes within the on- and off-target sites. F. Editing precision of alanine scanning variants normalized to the wild-type control. D69A variant is highlighted as the variant with the highest precision within the alanine scan mutant group. G. Crystal structure of AcrIIA4 bound to the SpCas9-gRNA complex. The interaction between D69 and R1335, required for PAM recognition, is highlighted. H. Distribution of editing efficiency ( EE , left) and editing precision ( EP , right) across all AcrIIA4 variants. Variants that resulted in EP in the top 90 th percentile are highlighted in red. I. Deletion profiles of DNA repair outcomes from cells without AcrIIA4 (blue) versus cells that express a variant that promotes precise editing (red). J. Editing precision measured across AcrIIA4 variants at Day 10 versus at Day 7 (Pearson Correlation Coefficient r = 0.56).

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Plasmid Preparation, Expressing, Variant Assay, Mutagenesis, Cell Culture, Sequencing, Activity Assay, Derivative Assay, Control

Journal: bioRxiv

Article Title: Protein language model-guided engineering of an anti-CRISPR protein for precise genome editing in human cells

doi: 10.1101/2023.12.13.571376

Figure Lengend Snippet: A. Experimental design in which the top 2000 AcrIIA4 variants, rank-ordered by EP , were delivered to A549 and 293T cells for another round of screening. B. Distribution of EP across all sample groups in the second round of screening versus the first round, at left. C. Frequency of editing within the AcrobaTx barcode across seven lead candidate enAcr groups. Yellow rectangles indicate areas in which editing occurs frequently while violet depicts areas with low editing activity. D. Ratio of on- to off-target editing in cells expressing seven representative lead candidate enAcrs (purple) versus ratios observed from DNA repair outcomes within cells expressing previously described benchmark AcrIIA4 variants.

Article Snippet: A549 cells expressing the pAG001 Cas9 vector (Genecopeia) were transduced at high MOI with 8 μg/mL polybrene using a spin-infection at 1200*g for 45 minutes.

Techniques: Activity Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: TGF-β1 and MUC16 collaborate to induce the alveolar epithelial to mesenchymal transition. The A549 cell line transfected with control siRNA(−) or siRNA-MUC16 was stimulated for 48 h ( A – E ) or 72 h ( F ) with 5 ng/mL TGF-β1 to measure MUC16 ( A ), collagen type I ( B ), α-SMA ( C ), Slug ( D ), and Snail ( E ) mRNA expression by real-time PCR and collagen type I protein levels by Western blotting, quantification was performed by densitometry ( F ). Data are expressed as 2 −ΔCt for mRNA levels and relative to β-actin for protein levels. The results are expressed as means ± SE. Student t-test of three independent experiments performed in triplicate. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−).

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: MUC16 Is Overexpressed in Idiopathic Pulmonary Fibrosis and Induces Fibrotic Responses Mediated by Transforming Growth Factor-β1 Canonical Pathway

doi: 10.3390/ijms22126502

Figure Lengend Snippet: MUC16 modulates the effect of TGF-β1 on SMAD3 phosphorylation. ( A ) A549 and ( B ) MRC5 cell lines transfected with control siRNA(−) or siRNA-MUC16 were stimulated for 40 min with TGF-β1 10 ng/mL to measure p-Smad3 and for 48 h with TGF-β1 5 ng/mL to measure β-catenin by Western blot. A549 ( C ) and MRC5 ( D ) cells were stimulated with 10 ng/mL TGF-β1 for 40 min. Total protein was extracted and immunoprecipitated using MUC16 antibody and probed against p-Smad3 and MUC16 by Western blot (representative images are shown). ( E ) A549 cells were stimulated with 10 ng/mL TGF-β1 for 1 h. MUC16 and p-Smad3 co-localisation was analysed by confocal microscopy to generate a two-dimensional cytofluorogram that selected common localised points of both antibodies (white colour). Scale bars: 10 μm. ( F ) Smad Binding Element (SBE) measure following 10 ng/mL TGF-β1 stimulation during 18 h in A549 cells transfected with siRNA(−) or siRNA-MUC16. Data are expressed relative to Smad3 or β-actin protein level (A-D). The results are expressed as means ± SE of three independent experiments performed in triplicate. Student t-test was used. * p < 0.05 vs. control; Ʇ p < 0.05 vs. siRNA(−) + TGF-β1.

Article Snippet: The SBE Reporter kit (Cat#: 60654, BPSBioscience) was used for monitoring the activity of TGFβ/SMAD signalling pathway in A549 cells.

Techniques: Transfection, Western Blot, Immunoprecipitation, Confocal Microscopy, Binding Assay

Journal: Respiratory Research

Article Title: Aggregatibacter is inversely associated with inflammatory mediators in sputa of patients with chronic airway diseases and reduces inflammation in vitro

doi: 10.1186/s12931-024-02983-z

Figure Lengend Snippet: Determination of the minimal effective MOI (mMOI) of a ) A. actinomycetemcomitans and b ) A. aphrophilus based on NF-κB pathway activation and determination of the mMOI of c ) A. actinomycetemcomitans and d ) A. aphrophilus based on IL-8 production in 3-D lung epithelial cells. On the vertical axis % NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells is shown, on the horizontal axis the tested MOI is depicted. The mMOI was defined as the lowest MOI with anti-inflammatory activity (i.e. < 50% NF-kB pathway activation / IL-8 production compared to LPS-stimulated cells as visualized by a dotted line, p < 0,05). e ) Western blot analysis of proteins (i.e. IκBα, p65 and phosphorylated (p)-IκBα) produced by 3-D A549 cells stimulated with LPS in the presence or absence of A. actinomycetemcomitans (targeted MOI 50:1) for 4 h (representative replicate is shown, image contains cropped blot – the full original blot is shown in Supplementary file_Western blot image). f ) Band intensity (normalised to β-actin) of Western blot at 4 h of 3-D A549 cells stimulated with LPS with/without A. actinomycetemcomitans . Results are expressed as a percentage of the positive control (i.e. LPS). NC: negative control (untreated 3-D A549 cells in serum-free GTSF-2 medium), A.ac. A. actinomycetemcomitans . Data represent mean ± SEM, * p < 0.05, n ≥ 3

Article Snippet: A previously developed organotypic 3-D lung cell culture model of the A549 alveolar epithelial cell line (ATCC CCL185) or the NF-κB–luciferase-transfected A549 recombinant stable cell line (BPS Bioscience, San Diego, CA, US) was used that exhibits in vivo-like phenotypic and functional properties of alveolar epithelial cells, including barrier function (localized expression of tight junctional markers), apical and basolateral polarity, and responds to infection in ways that are relevant to the infection process in vivo, including cytokine secretion [ , – ].

Techniques: Activation Assay, Activity Assay, Western Blot, Produced, Positive Control, Negative Control