Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Cell viability ( A ) and cell proliferation ( B ) induced by nicotine in HBEpC and/or si-mRNA-α7-HBEpC viability. ( A ): For cell viability, 7500 cells/cm 2 are plated in T25 flask (total cell number 187,000) and treated with nicotine 1 × 10 −7 M, after 1 h cells are washed three times in PBS Ca 2+ and Mg 2+ free and then incubated in drug-free medium for additionally 48 h. Then, cells (detached or floating) are counted after staining with trypan blue dye. ( B ): For cell proliferation 7500 cells/cm 2 are plated in T25 flask and treated with nicotine every 48 h. Cells are detached and viable cells are counted every 12 h. Experiments are performed at least two times in triplicate. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Incubation, Staining, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Expression of Ki67 induced by nicotine in HBEpC and/or si-mRNA-α7-HBEpC. ( A ): ELISA experiments; ( B ): regression equation linearity, performed with Prism; ( C ): Western blotting, ( D ): densitometric analysis. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate. In the , raw data of Western blotting are reported.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: SA-β-Gal in HBEpC exposed continuously to nicotine. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques:

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Evaluation of intracellular Ca 2+ after exposure to nicotine for 48 h in HBEpC and/or si-mRNA-α7-HBEpC. ( A ): ELISA experiments; ( B ): regression equation linearity, performed with Prism. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Evaluation of intracellular ATP after exposure to nicotine for 48 h in HBEpC. ( A ): ELISA experiments; ( B ): regression equation linearity, performed with Prism. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Expression of EGF and p-EGFR after exposure to nicotine for 48 h in HBEpC. ( A ): ELISA experiments for EGFR; ( B ): regression equation linearity for EGFR, performed with Prism; ( C ): ELISA experiments for p-EGFR; ( D ): regression equation linearity for p-EGFR, performed with Prism. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Induction of p53 and phospho-p53 induced by nicotine in HBEpC. ( A ): ELISA assay. ( B ): Western blotting experiments, ( C ): densometric analysis. Experiments are performed at least two times in triplicate. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. In the , raw data of Western blotting are reported.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Induction of phospho-p38 and p38 by nicotine in HBEpC. ( A ): ELISA experiments; ( B ): regression equation linearity for phosphor-p38; ( C ): regression equation linearity for phosphor-p38performed with Prism. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: EMT induced by nicotine in HBEpC. ( A ): Western blotting; ( B ): densitometric analysis. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate. In the , raw data of Western blotting are reported.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Western Blot, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Cell migration to nicotine for 48 h in HBEpC and/or si-mRNA-α7-HBEpC. HeLa cells are positive control. ( A ) HeLa positive control. ( B ): Cell migration h in HBEpC and/or si-mRNA-α7-HBEpC. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Migration, Positive Control, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Induction of VEGF by nicotine in HBEpC and/or si-mRNA-α7-HBEpC. ( A ): ELISA experiments; ( B ) regression equation linearity for VEGFR. Statistical significance is analyzed with one-way AOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Anchorage-independent growth induced by nicotine in HBEpC. HeLa cells are positive control and NIH3T3 the negative. ( A ): Representative picture of HBEpC cloned on soft agar. ( B ): Cloned cells. Statistical significance is analyzed with one-way ANOVA with multiple-comparison and post hoc test with Bonferroni correction. Experiments are performed at least two times in triplicate.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Positive Control, Clone Assay, Comparison

Journal: Molecules

Article Title: Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

doi: 10.3390/molecules26010101

Figure Lengend Snippet: Effects induced by nicotine on different pathways in human airway epithelial cells (results obtained in this work and in literature) and comparison with effects caused by SARS-CoV-2, SARS-CoV, MERS-CoV and by non-tumorigenic virus infection on the same pathways.

Article Snippet: Human Bronchial Epithelial Cells (HBEpC) were obtained from Cell Applications Inc. ( www.cellapplications.com/product no. 502K-05a) and cultured in complete Bronchial/Tracheal Epithelial Cell Growth Medium ( www.cellapplications.com/product ) as described previously [ ]. si-mRNA-α7-HBEpC were obtained as described previously [ ].

Techniques: Comparison, Virus, Infection, Expressing, Concentration Assay, In Vitro, Activation Assay, Activity Assay, Knockdown, Control, Migration

Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Drug screening and validation of Niclosamide using CCA cell lines and primary biliary epithelial cells. ( A ) A library of 104 off-patent drugs was screened using the CCLP CCA cell line. CCLP cells were plated at 1 × 10 4 cells per well in a 96 well plate and treated with each drug at its clinically relevant peak serum concentration for 96 h. Cell viability was then determined using an MTT assay and normalised to cells treated with the corresponding vehicle control for each drug. N = 3 biological repeats with a paired, two-tailed T-test followed by Benjamini–Hochberg multiple corrections. The inhibition of viability (%) is plotted against the reciprocal of the p value for each drug and Niclosamide is labelled. ( B – E ) Niclosamide dose–response curves for CCA cell lines (CCLP, RBE, and KKU-M055) and primary biliary epithelial cells (BECs) plated as in ( A ) and treated with Niclosamide [10 nM–100 μM] or vehicle control for 72 h. Cell viability was measured using an MTT assay and normalised to vehicle control. N = 3 biological repeats each performed in triplicate. Error bars represent SEM. When error bars cannot be seen they are smaller than the symbols. ( F ) The graph shows the relative EC50 values for each cell type calculated using GraphPad Prism. Error bars represent SEM (* = p < 0.05 unpaired t -test). The inserted Western blot shows PRH levels in the CCA cell lines prior to treatment and with β-actin as a loading control.

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: Drug discovery, Biomarker Discovery, Concentration Assay, MTT Assay, Control, Two Tailed Test, Inhibition, Western Blot

Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Palbociclib decreases the viability of CCA cells but has less effect on primary biliary epithelial cells. CCA cell lines (CCLP ( A ), RBE ( B ), KKU-M055 ( C ), or primary BECs ( D )) were plated at 1 × 10 4 cells per well in a 96-well plate and treated with increasing concentrations of Palbociclib for 96 h. Cell viability was then measured using an MTT assay and normalised to the vehicle control. Three biological repeats (five for CCLP cells) each performed in triplicate. Error bars represent SEM. When error bars cannot be seen, they are smaller than the symbols. ( E ) The graph shows the relative EC50 values calculated using GraphPad Prism. Error bars represent SEM (** = p < 0.01 *** = p < 0.001 unpaired t -test).

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: MTT Assay, Control

Journal: Cancers

Article Title: Niclosamide and Palbociclib Act Synergistically to Reduce Cholangiocarcinoma Cell Viability In Vitro and Inhibit Tumour Growth in a Mouse Model

doi: 10.3390/cancers17223721

Figure Lengend Snippet: Niclosamide and Pablociclib act synergistically to reduce CCA cell viability. ( A ) CCLP cells were plated at 1 × 10 4 cells per well in a 96-well plate before treatment with Niclosamide (NIC), Palbociclib (PAL), or both drugs in combination (COM) at the concentrations shown and for 72 h. Cell viability was then measured using an MTT assay. N = 3 biological experiments each performed in triplicate. Error bars represent SEM (* = p < 0.05, ** = p < 0.01, ns = not significant) two-tailed paired t -test. ( B ) From the data shown in ( A ) combination index (CI), values were calculated and plotted against the corresponding response (Fa) values in a CI plot. CI < 1 indicates synergism. ( C ) CCLP cells were treated with 0.5 μM Niclosamide (NIC), 1 μM Palbociclib (PAL), or both drugs in combination (COM) for 24 h, then proteins were extracted for Western blot. ( D ) CCLP cells and BECs were grown as spheroids for 5 days before treatment with 0.5 μM Niclosamide, 1 μM Palbociclib, or both drugs in combination for a further 72 h. Images were taken using a Nikon brightfield microscope, and spheroid size was calculated using ImageJ. N = 3 biological repeats. Error bars represent SEM (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001) two-tailed paired t -test. ( E ) Cell viability was measured in the spheroids from ( D ) using a PrestoBlue assay. Original western blots are presented in .

Article Snippet: Primary Biliary Epithelial Cells (BECs) obtained from healthy human liver tissue (Innoprot, Elexalde Derio, Spain) were grown in Epithelial Cell Media (Innoprot, P60106 , Elexalde Derio, Spain) supplemented with 2% FBS and 1% Epithelial Growth Supplement, as per the supplier’s protocol for a maximum of ten passages.

Techniques: MTT Assay, Two Tailed Test, Western Blot, Microscopy, Prestoblue Assay