Journal: Cancers

Article Title: Evaluating the Therapeutic Potential of MRT68921 and Afatinib in Three-Dimensional Models of Epithelial Ovarian Cancer

doi: 10.3390/cancers18020307

Figure Lengend Snippet: Afatinib induces autophagy in EOC cell lines. ( A ) Representative immunoblots and densitometric analyses of HeyA8, OVCAR8, and ES2 cells treated with 4 μM afatinib for 24 h under adherent culture conditions. Cells were seeded and allowed to adhere for 24 h prior to treatment. Densitometric values are presented as mean fold change relative to DMSO-treated controls ± SEM (n = 4). Black, and blue outlines denote DMSO-treated and afatinib-treated respectively. Statistical significance was assessed using unpaired t -tests (* p < 0.05, *** p < 0.001, **** p < 0.0001). ( B ) Representative fluorescence images of OVCAR8- and HeyA8-autoR spheroids treated with 4 μM afatinib, starting 24 h post-seeding. Timepoints indicate the duration of treatment. Autophagic flux was assessed using the mCherry/eGFP ratio and is presented as mean ± SEM (n = 3). Black, and blue outlines denote DMSO-treated and afatinib-treated respectively. Statistical analysis performed by one-way ANOVA with Tukey’s multiple comparisons test. Letter labels above bars indicate the presence (different letters) or absence (same letter) of significance at p < 0.05. Scale bars represent 400 μm.

Article Snippet: Afatinib (5 mM in DMSO) was purchased from Tocris (#6812, Bristol, UK) and stored at −20 °C.

Techniques: Western Blot, Fluorescence

Journal: Cancers

Article Title: Evaluating the Therapeutic Potential of MRT68921 and Afatinib in Three-Dimensional Models of Epithelial Ovarian Cancer

doi: 10.3390/cancers18020307

Figure Lengend Snippet: Combined MRT68921 and afatinib treatment modulates ULK1 activity and autophagy in EOC spheroids. ( A ) Representative immunoblots and densitometric analyses of OVCAR8 (n = 4) and HeyA8 (n = 3) spheroids treated either 0.25 μM (M0.25) or 4 μM (M4) of MRT68921 and 0.125 μM afatinib (A) for 24 h. Spheroids were seeded 24 h prior to drug treatment. Densitometric values are expressed as mean fold-change relative to DMSO-treated controls ± SEM. Black, and pink outlines denote DMSO-treated and afatinib-treated spheroids respectively. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01). ( B ) Representative fluorescence images of OVCAR8-autoR and HeyA8-autoR spheroids treated with the indicated concentrations of MRT68921 and afatinib, beginning 24 h after seeding. Timepoints indicate the duration of treatment. Scale bars represent 400 μm. Autophagic flux was quantified based on the mCherry/eGFP fluorescence ratio and is presented as mean ± SEM (n = 3). Black, purple, and red outlines denote DMSO-, M0.25 + A-, and M4 +A-treated spheroids respectively. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test Tukey’s multiple comparisons test. Letter labels above bars indicate the presence (different letters) or absence (same letter) of significance at p < 0.05.

Article Snippet: Afatinib (5 mM in DMSO) was purchased from Tocris (#6812, Bristol, UK) and stored at −20 °C.

Techniques: Activity Assay, Western Blot, Fluorescence

Journal: Cancers

Article Title: Evaluating the Therapeutic Potential of MRT68921 and Afatinib in Three-Dimensional Models of Epithelial Ovarian Cancer

doi: 10.3390/cancers18020307

Figure Lengend Snippet: Combined MRT68921 and afatinib treatment impairs spheroid viability and reattachment. ( A ) Trypan Blue exclusion cell counting was performed on EOC spheroids following 72 h treatment with either 0.25 μM MRT68921 (M0.25) or 4 μM MRT68921 (M4) ± 0.125 μM afatinib ( A ). Data are presented as mean fold-change in percent viability normalized to DMSO control ± SEM (n = 3). Black, and blue outlines denote DMSO-treated and drug treated spheroids respectively. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Letter labels above bars indicate the presence (different letters) or absence (same letter) of significance at p < 0.05. ( B ) Representative images of Hema3-stained spheroids following a 72 h drug treatment and subsequent transfer to 24-well tissue culture plastic dishes for reattachment. Individual well and high-power images show qualitative differences in spheroid morphology and reattachment capacity across treatment conditions (n = 4). Scale bars represent 500 μm.

Article Snippet: Afatinib (5 mM in DMSO) was purchased from Tocris (#6812, Bristol, UK) and stored at −20 °C.

Techniques: Cell Counting, Control, Staining

Journal: Cancers

Article Title: Evaluating the Therapeutic Potential of MRT68921 and Afatinib in Three-Dimensional Models of Epithelial Ovarian Cancer

doi: 10.3390/cancers18020307

Figure Lengend Snippet: Effect of combined MRT68921 and afatinib treatment on patient ascites-derived organoids. Patient ascites-derived organoids were seeded and allowed to mature for 72 h prior to 72 h treatment with either 0.25 μM MRT68921 (M0.25) or 4 μM MRT68921 (M4) ± 0.125 μM afatinib (A). Cell viability was assessed using the alamarBlue assay and expressed as percent viability relative to DMSO-treated controls ± SEM (n = 4). Black, and purple outlines denote DMSO-treated and drug treated organoids respectively. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Letter labels above bars indicate the presence (different letters) or absence (same letter) of significance at p < 0.05.

Article Snippet: Afatinib (5 mM in DMSO) was purchased from Tocris (#6812, Bristol, UK) and stored at −20 °C.

Techniques: Derivative Assay, Alamar Blue Assay

Journal: Molecular pharmacology

Article Title: Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

doi: 10.1124/molpharm.121.000280

Figure Lengend Snippet: Fig. 7. Inhibition of EGFR and growth factor pathway components prevented TRPV3 mRNA upregulation after injury. (A) HBEC3-KT cells were treated with 5 mM solutions of inhibitors of multiple ErbB receptor tyrosine kinase isoforms: ErbB1 (EGFR)-specific inhibitor AG-1478, ErbB pan-inhibitors AZD8931 and Afatinib, and a selective ErbB2 (HER2) inhibitor CP-724 for 2 hours in the cell passaging injury model, fol- lowed by analysis of TRPV3 mRNA. (B) Downstream targets of EGFR activation were also inhibited, and TRPV3 expression was subsequently measured 2 hours after cell passaging injury. Inhibitors included 5 mM TWS119 (GSK3b), 20 mM CCT036477 (b-catenin), 10 mM PD169316 (p38 MAPK), and 10 mM SP600125 (JNK). (C) Inhibition of NF-jB (20 mM BMS-345541) and effects on TRPV3 mRNA 2 hours after cell passaging injury. (D) Inhibitors of Wnt7a and Fzd signaling were also used: 10 ng/ml secreted Frizzled-related protein (sFRP) and IWP 2 (Porcupine inhibitor) reduced TRPV3 upregulation after injury. Furthermore, SB 431542 (TGFbRI inhibitor) and 150 mM ITD-1 (TGFbRII inhibitor) were also tested, and SB 431542 treatment prevented TRPV3 expression as well. Values were normalized to vehicle controls and are presented as mean ± S.D. from n $ 3 replicates. Statistical significance was determined using one-way ANOVA with the Dunnett post-test. ***P < 0.001, ****P < 0.0001. For Fig. 4C, an unpaired t test was used. ****P < 0.0001.

Article Snippet: Afatinib dimaleate (EGFR, ErbB2, HER4/receptor tyrosine kinase 4), IWP 2 (inhibitor of Wnt production 2, targeting porcupine), SB 431542 (TGFbRI), and ITD 1 (1,4-dihydropyridine inducer of TGFbRII degradation-1) were purchased from Tocris (Minneapolis, MN).

Techniques: Inhibition, Passaging, Activation Assay, Expressing

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non-small cell lung cancer patients treated with osimertinib.

doi: 10.1016/j.lungcan.2021.05.023

Figure Lengend Snippet: Fig. 2. Relation of Her1 dimer abundance to the sensitivity of NSCLC cell lines to EGFR-TKIs. (A) Cell viability assays for 11_18, HCC827, HCC4006, and PC9 cells exposed to the indicated concentrations of erlotinib, afatinib, or osimertinib for 72 h. The IC50 values of each drug for each cell line are also shown. Data are means of triplicates from representative experiments. (B) In situ PLA for the detection of Her1 homodimers and Her1:Her2, Her1:Her3, and Her1:Her4 heterodimers in 11_18 cells that had been incubated in the absence or presence of EGFR-TKIs at the respective IC50 values for 24 h. Images are presented as in Fig. 1. Scale bars, 50 μm. (C) Quantitative analysis of Her1 dimer signals per cell in NSCLC cell lines as determined from images similar to those in Fig. 2B and Supplementary Fig. S1. Data are means + SEM from three independent experiments. (D) Correlation analysis for the ratio of Her1 heterodimers to Her1 homodimers in 11_18, HCC827, HCC4006, and PC9 cells after EGFR-TKI treatment as in (B) and the corresponding IC50 values of erlotinib, afatinib, and osimertinib.

Article Snippet: Afatinib (LKT Laboratories, St. Paul, MN, USA), erlotinib (LKT Laboratories), and osimertinib (ChemScene, Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) and stored at − 20◦ or − 80 ◦C for up to 2 weeks.

Techniques: In Situ, Incubation

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non-small cell lung cancer patients treated with osimertinib.

doi: 10.1016/j.lungcan.2021.05.023

Figure Lengend Snippet: Fig. 3. Kaplan-Meier curves of PFS during osimertinib treatment for EGFR- mutated NSCLC patients with a high or low ratio of Her1 heterodimers to Her1 homodimers (according to the median value of 4.79) in tumor specimens collected after they had developed T790M-associated drug resistance during prior treatment with erlotinib or afatinib.

Article Snippet: Afatinib (LKT Laboratories, St. Paul, MN, USA), erlotinib (LKT Laboratories), and osimertinib (ChemScene, Monmouth Junction, NJ, USA) were dissolved in dimethyl sulfoxide (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) and stored at − 20◦ or − 80 ◦C for up to 2 weeks.

Techniques: