Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: CDCA3 protein is upregulated in EGFR mutant NSCLC. ( A ) Representative images of CDCA3 low (left panel) and CDCA3 high (right panel) staining by immunohistochemistry in NSCLC cohort. CDCA3 levels determined by stratifying on median H-score. Scale bar = 50 µm. ( B ) Stacked bar chart showing distribution of CDCA3 low (blue) and CDCA3 high (grey) levels quantified from immunohistochemistry staining of non-oncogene-driven NSCLC (EGFR-WT) cases and EGFR mutant cases (chi-square test, * p = 0.02). ( C ) Representative endogenous CDCA3 Western blot analysis from lysates of NSCLC adenocarcinoma cell lines evaluating a small panel of non-oncogene-driven (EGFR WT) and EGFR mutant (EGFR MUT) cell lines. Phosphorylated and total ERK and mTOR Western blot analysis to assess constitutive signalling in EGFR mutant cell lines. Tubulin used as a loading control. ( D ) Densitometry quantification of ( C ), with dot points representing relative CDCA3 levels from three independent experiments. Blue lines indicate median values.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Staining, Immunohistochemistry, Western Blot, Control

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulation of CDCA3 protein levels in EGFR mutant NSCLC is dependent on RTK signalling. ( A ) Violin plots showing relative CDCA3 mRNA levels in EGFR wild-type and EGFR mutant adenocarcinoma from The Cancer Genome Atlas (TCGA) RNAseq datasets. ( B ) RNAseq analysis of CDCA3 mRNA levels in cell lines examined in C comparing EGFR wild-type and EGFR mutant adenocarcinoma. ns, not significant. ( C ) Endogenous CDCA3 Western blot analysis of A549 (EGFR WT), HCC827 (EGFR exon 19 deletion) and H1975 (EGFR T790M) cells treated in the absence or presence of EGF over 24 h. Phosphorylated (S473) and total Akt Western blot analysis to assess EGF-induced signal transduction. Actin used as a loading control. ( D ) Endogenous CDCA3 Western blot analysis of HCC827 lysates assessing protein turnover by treating with cycloheximide over 6 h in the presence or absence of erlotinib. Tubulin used as a loading control. ( E ) Densitometry quantification of ( D ), showing average log 2 of relative CDCA3 protein levels relative to 0 h from three independent experiments.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Western Blot, Transduction, Control

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: CDCA3 correlates with sensitivity to EGFR TKI. ( A , B ) Scatter plots showing linear regression analysis of The Cancer Genome Atlas (TCGA) RNAseq datasets assessing the correlation between CDCA3 levels and WikiPathways of TKI resistance in ( A ) adenocarcinoma (LUAD) and ( B ) EGFR mutant LUAD. R and p values determined according to Spearman’s rank correlation. ( C ) Left panel, Dose–response curves for five EGFR mutant NSCLC cell lines treated with escalating doses of erlotinib. Cells were treated with erlotinib for 72 h before assessing cell viability. Right panel, Cell lines ranked by CDCA3 protein levels where erlotinib potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 4. ( D ) Box and whisker plot showing erlotinib potency (log IC 50 value) in CDCA3 low and CDCA3 high EGFR mutant cell lines (unpaired Student’s t test, * p = 0.0187).

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Mutagenesis, Whisker Assay

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulating CDCA3 protein levels enhances TKI sensitivity in CDCA3 low H1975 cells. ( A ) Western blot analysis of lysates from H1975 cells transfected with empty vector or CDCA3-FLAG treated in the absence (vehicle) or presence of erlotinib or osimertinib for 24 h. CDCA3 Western blot analysis was performed to detect ectopic (arrowhead) and endogenous (asterisk) CDCA3. Phosphorylated (Y1068) and total EGFR Western blot analysis was performed to confirm TKI response. Tubulin was used as a loading control. Representative Western blot analysis from three independent experiments. ( B ) Dose–response curves for CDCA3 low H1975 cells either vector transfected or ectopically overexpressing CDCA3-FLAG treated with escalating doses of erlotinib for 72 h before assessing cell viability. ( C ) Cellular proliferation analysis of vector transfected or CDCA3-FLAG ectopically expressing H1975 cells over 96 h using the Incucyte Zoom live-cell imaging system. ( D ) Beeswarm plot showing the mitotic index determined by histone H3 pS10 staining and high throughput immunofluorescence microscopy of vector transfected or CDCA3-FLAG ectopically expressing H1975 cells. Data points represent an average percentage of mitotic nuclei per field of view from a minimum of 1100 nuclei ( n = 23 fields total). Blue lines indicate median values. ( E ) Endogenous CDCA3 Western blot analysis of H1975 cells treated in the absence or presence of CX-4945 (5 µM) over 24 h. Phosphorylated CK2 substrate probe to assess impact of CK2 inhibition with CX-4945. Tubulin was used as a loading control. ( F ) Dose–response curve for H1975 cells treated with escalating doses of CX-4945. Cells were treated with CX-4945 for 72 h before assessing cell viability. CX-4945 potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 3. ( G ) Dose–response curves showing the impact of combining IC 25 or IC 50 CX-4945 concentrations with osimertinib in H1975 cell. Potency values (IC 50 ) were calculated using GraphPad Prism. n = 3.

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Expressing, Live Cell Imaging, Staining, High Throughput Screening Assay, Immunofluorescence, Microscopy, Inhibition

Journal: Cancers

Article Title: Elevating CDCA3 Levels Enhances Tyrosine Kinase Inhibitor Sensitivity in TKI-Resistant EGFR Mutant Non-Small-Cell Lung Cancer

doi: 10.3390/cancers13184651

Figure Lengend Snippet: Upregulating CDCA3 protein levels in models of isogenic EGFR TKI resistance cells enhances TKI sensitivity. ( A , B ) Dose–response curves for vector transfected or ectopic CDCA3-FLAG overexpression in isogenic parental and resistant ( A ) HCC827 and ( B ) PC-9 cells treated with escalating doses of osimertinib for 72 h before assessing cell viability. ( C ) Plot showing erlotinib potency (log IC 50 value) comparing vector transfected and ectopic CDCA3-FLAG overexpression in isogenic parental and TKI-resistant cell lines determined in from four independent experiments. Lines connect respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.029, ns = not significant). ( D ) Plot showing osimertinib potency (log IC 50 value) comparing vector transfected and ectopic CDCA3-FLAG overexpression in isogenic parental and TKI-resistant cell lines determined in ( A , B ) from four independent experiments with lines connecting respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.0339, ns = not significant). ( E ) Dose–response curves for HCC827 and PC-9 isogenic parental and TKI-resistant cell lines treated with escalating doses of CX-4945. Cells were treated with CX-4945 for 72 h before assessing cell viability. CX-4945 potency values (IC 50 ) were calculated using GraphPad Prism and listed for each cell line. n = 3. ( F ) Plot showing potency (log IC 50 value) of osimertinib alone or in combination with CX-4945 (IC 50 ) comparing isogenic parental and TKI-resistant cell lines from three independent experiments. Lines connect respective isogenic parental and resistant cell lines (2-way ANOVA, * p = 0.0331, ns = not significant).

Article Snippet: The Ethics Committee approved tissue microarray of EGFR wild-type and mutant NSCLC tissue was purchased from Tristar Technology Group (cat. No. 69572826-2826) and handled in accordance with the guidelines and regulations approved by Queensland University of Technology (approval number 1900000269).

Techniques: Plasmid Preparation, Transfection, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Design, Synthesis, and Antitumor Activity of Erlotinib Derivatives

doi: 10.3389/fphar.2022.849364

Figure Lengend Snippet: Surface plasmon resonance experiments of 3d and erlotinib. (A) Binding sensorgrams for 3d interaction with immobilized wild-type of EGFR. The K D (M) value between 3d and wild-type EGFR is 6.88 × 10 −6 . (B) Binding sensorgrams for 3d interaction with immobilized mutant protein of EGFR. The K D (M) value between 3d and mutant protein EGFR is 3.12 × 10 −6 . (C) Binding sensorgrams for erlotinib interaction with immobilized wild-type of EGFR. The K D (M) value between erlotinib and wild-type EGFR is 1.97 × 10 −6 .

Article Snippet: First, the EGFR wild-type (0.468 μg/μL, Active MOTIF, cat: 31165) and the EGFR mutant type (672-1210, L858R, Active MOTIF, cat: 81200) were covalently immobilized at densities of 2000 response units onto a CM5 sensor chip.

Techniques: SPR Assay, Binding Assay, Mutagenesis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Triple targeting of mutant EGFR L858R/T790M , COX-2, and 15-LOX: design and synthesis of novel quinazolinone tethered phenyl urea derivatives for anti-inflammatory and anticancer evaluation

doi: 10.1080/14756366.2023.2199166

Figure Lengend Snippet: EGFR inhibitors (first, second, and third generations), some reported compounds as dual COX-2/15-LOX inhibitors, and the rationale for the design of our multi-target directed ligands (MTDLs).

Article Snippet: We performed in vitro EGFR inhibition assay for all our newly synthesised compounds ( 6a–p ) against both wild-type EGFR and the double mutant EGFR L858R/T790M purchased from AssayQuant Technologies, Inc. (Marlboro, MA, USA) as per the manufacturer’s instructions.

Techniques:

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Triple targeting of mutant EGFR L858R/T790M , COX-2, and 15-LOX: design and synthesis of novel quinazolinone tethered phenyl urea derivatives for anti-inflammatory and anticancer evaluation

doi: 10.1080/14756366.2023.2199166

Figure Lengend Snippet: Reported 2,3‐disubstituted quinazolinones or urea-containing compounds with significant inhibitory activity against EGFR TK.

Article Snippet: We performed in vitro EGFR inhibition assay for all our newly synthesised compounds ( 6a–p ) against both wild-type EGFR and the double mutant EGFR L858R/T790M purchased from AssayQuant Technologies, Inc. (Marlboro, MA, USA) as per the manufacturer’s instructions.

Techniques: Activity Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Triple targeting of mutant EGFR L858R/T790M , COX-2, and 15-LOX: design and synthesis of novel quinazolinone tethered phenyl urea derivatives for anti-inflammatory and anticancer evaluation

doi: 10.1080/14756366.2023.2199166

Figure Lengend Snippet: In vitro inhibitory activities of the final synthesised compounds ( 6a–p ) and references against wild-type EGFR and the double mutant EGFR L858R/T790M .

Article Snippet: We performed in vitro EGFR inhibition assay for all our newly synthesised compounds ( 6a–p ) against both wild-type EGFR and the double mutant EGFR L858R/T790M purchased from AssayQuant Technologies, Inc. (Marlboro, MA, USA) as per the manufacturer’s instructions.

Techniques: In Vitro, Mutagenesis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Triple targeting of mutant EGFR L858R/T790M , COX-2, and 15-LOX: design and synthesis of novel quinazolinone tethered phenyl urea derivatives for anti-inflammatory and anticancer evaluation

doi: 10.1080/14756366.2023.2199166

Figure Lengend Snippet: (A) 3D Interaction diagram of compound 6e (thick green sticks) in the molecular surface of EGFR L858R/T790M (PDB: 5EDQ) binding site. (B) 2D Interaction diagram of compound 6e with amino acid residues EGFR L858R/T790M .

Article Snippet: We performed in vitro EGFR inhibition assay for all our newly synthesised compounds ( 6a–p ) against both wild-type EGFR and the double mutant EGFR L858R/T790M purchased from AssayQuant Technologies, Inc. (Marlboro, MA, USA) as per the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Triple targeting of mutant EGFR L858R/T790M , COX-2, and 15-LOX: design and synthesis of novel quinazolinone tethered phenyl urea derivatives for anti-inflammatory and anticancer evaluation

doi: 10.1080/14756366.2023.2199166

Figure Lengend Snippet: (A) 3D Interaction diagram of compound 6e (thick green sticks) in the molecular surface of EGFR WT (PDB: 1M17) binding site. (B) 2D Interaction diagram of compound 6e with amino acid residues of EGFR WT .

Article Snippet: We performed in vitro EGFR inhibition assay for all our newly synthesised compounds ( 6a–p ) against both wild-type EGFR and the double mutant EGFR L858R/T790M purchased from AssayQuant Technologies, Inc. (Marlboro, MA, USA) as per the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Oncology Reviews

Article Title: HER3 signaling and targeted therapy in cancer

doi: 10.4081/oncol.2018.355

Figure Lengend Snippet: Anti-HER3 antibody as HER3 therapy.

Article Snippet: , HER3 , - EGFR wild-type patients with locally advanced or metastatic NSCLC patients (with erlotinib) [ ] , Phase III (terminated) Daiichi Sankyo , Daiichi Sankyo.

Techniques: Modification, Expressing, Mutagenesis, Amplification

Journal: Oncology Reviews

Article Title: HER3 signaling and targeted therapy in cancer

doi: 10.4081/oncol.2018.355

Figure Lengend Snippet: Summary of clinical trials, outcomes and adverse effects associated with anti-HER3 therapies.

Article Snippet: , HER3 , - EGFR wild-type patients with locally advanced or metastatic NSCLC patients (with erlotinib) [ ] , Phase III (terminated) Daiichi Sankyo , Daiichi Sankyo.

Techniques: Clinical Proteomics, Infection, Modification, Activity Assay, Biomarker Discovery, Expressing, Mutagenesis, Inhibition, Amplification

Journal: JCI Insight

Article Title: Enapotamab vedotin, an AXL-specific antibody-drug conjugate, shows preclinical antitumor activity in non-small cell lung cancer

doi: 10.1172/jci.insight.128199

Figure Lengend Snippet: (A) Tumor growth curves of the EGFR-mutant NSCLC PDX model LU1868 upon treatment with EnaV (2 mg/kg or 4 mg/kg) or erlotinib or combinations as indicated. Mean tumor sizes are displayed for each group (n = 8 per group) up to the day the first mouse of a group was sacrificed. Days of EnaV or isotype-ADC treatment indicated with red arrowheads; days of erlotinib treatment indicated with orange arrowheads. (B) Same as A for EGFR-mutant NSCLC PDX model LU0858. (C–F) Statistical analysis (Mann-Whitney U test + Bonferroni’s post hoc test; P values: *P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001) was performed on the last day that both groups were intact, but no later than day 25 after randomization, because PK studies indicated all administered drug was cleared by day 25 (data not shown), to identify significant differences in tumor sizes between groups. (C and D) Tumor sizes in the PDX model LU1868, compared on day 21 after randomization. (E and F) Tumor sizes in PDX model LU0858, compared on day 11 after randomization. (G and H) Combination therapy in EGFR-mutant, erlotinib-resistant NSCLC PDX models in vivo. Kaplan-Meier plots of NSCLC PDX model LU1868 (G) and LU0858 (H) after treatment with EnaV only (2 mg/kg or 4 mg/kg), erlotinib only, or combinations as indicated. Mice were considered tumor progression–free until a cutoff tumor size of 750 mm3 was reached. Statistically significant differences determined by Mantel-Cox analysis, *P < 0.05.

Article Snippet: The expanded mouse experiments, performed in-house or by Crown Bioscience, Oncotest, or XenTech (see ), involved EGFR –wild-type or -mutant NSCLC CDX or PDX models.

Techniques: Mutagenesis, MANN-WHITNEY, In Vivo

Journal: Nature chemical biology

Article Title: A drug discovery platform to identify compounds that inhibit EGFR triple mutants

doi: 10.1038/s41589-020-0484-2

Figure Lengend Snippet: (a) Schematic representation of the MaMTH-DS platform workflow. (b) Dose response curves for the top three candidate EGFR L858R/T790M/C797S inhibitors (midostaurin, AZD7762 and Chembridge 5213777) showing robust, dose-responsive and mutant specific inhibition. Results are shown as the average ± SD for three independent experiments.

Article Snippet: Kinase assays were performed using recombinant proteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R, EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).

Techniques: Mutagenesis, Inhibition

Journal: Nature chemical biology

Article Title: A drug discovery platform to identify compounds that inhibit EGFR triple mutants

doi: 10.1038/s41589-020-0484-2

Figure Lengend Snippet: Validation of midostaurin and gilteritinib as EGFR ex19del/T790M/C797S and EGFR L858R/T790M/C797S activating mutant inhibitors. (a) in vitro kinase assay of recombinant kinase domain (residues 696–1022) of indicated mutant or WT EGFR in the presence of midostaurin (left panel) or gilteritinib (right panel). Results are shown as the average ± SD for two independent experiments. (b) Effect of midostaurin and gilteritinib on caspase 3/7 activity in PC9 EGFR ex19del/T790M/C797S and A549 EGFR WT cells. Results are shown as single 36-point dose response experiments. (c) Effects of midostaurin (left panels) and gilteritinib (right panels) on EGFR activation and downstream signalling in PC9 EGFR ex19del and EGFR ex19del/T790M/C797S cells after 2 hours treatment (see Supplementary Figs 21–24 for source blot images). Results are representative of at least two independent experiments. (d) Midostaurin and gilteritinib mediated reduction of PC9 EGFR ex19del/T790M/C797S organoid viability. Osimertinib control, which does not target triple mutant EGFR activity, has no effect. Results are shown as single 36-point dose response experiments.

Article Snippet: Kinase assays were performed using recombinant proteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R, EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).

Techniques: Biomarker Discovery, Mutagenesis, In Vitro, Kinase Assay, Recombinant, Activity Assay, Activation Assay, Control

Journal: Nature chemical biology

Article Title: A drug discovery platform to identify compounds that inhibit EGFR triple mutants

doi: 10.1038/s41589-020-0484-2

Figure Lengend Snippet: Validation of EMI1 as an EGFR ex19del/T790M/C797S and EGFR L858R/T790M/C797S activating mutant inhibitor. (a) Chemical structure for EMI1. (b) in vitro kinase assay of recombinant kinase domain (residues 696–1022) of indicated mutant or WT EGFR in the presence of EMI1. Results are shown as the average ± SD for two independent experiments. (c) Effect of EMI1 on the viability of PC9 EGFR ex19del/T790M/C797S and HBE bronchial epithelial lung EGFR WT control cells. Results are shown as the average ± SD for three independent experiments (d) Effect of EMI1 on caspase 3/7 activity in PC9 EGFR ex19del/T790M/C797S and HBE EGFR WT cells. Results are shown as single 36-point dose response experiments. (e) Viability assay measuring effect of EMI1 on PC9 EGFR ex19del/T790M/C797S organoid growth. Results are shown as single 36-point dose response experiments. (f) Maximum intensity projections (stream acquisition/exposure time 500 ms/100 frames) showing the effect of EMI1 on microtubule dynamics in HEK293 MaMTH reporter cells stably expressing EGFR WT or EGFR L858R/T790M/C797S transfected with EB3-TagRFP as a microtubule plus end marker. The contrast is inverted. Graph shows quantification of microtubule plus end velocity in HEK293 reporter cells for EMI1. n = 51, 41, 36 for HEK293 EGFR WT, control, 50 and 100 nM. n = 49, 47, 41 for HEK293 EGFR C797S control, 50 and 100 nM. Significant p-values are displayed and were calculated using the Mann-Whitney test. (g) Western blot analysis showing activity of EMI1 and other microtubule targeting compounds after 2 hours treatment on EGFR ex19del/T790M/C797S activation and downstream signalling in PC9 triple mutant cells (see Supplementary Fig. 25 for source blot images). Results are representative of at least two independent experiments.

Article Snippet: Kinase assays were performed using recombinant proteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R, EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).

Techniques: Biomarker Discovery, Mutagenesis, In Vitro, Kinase Assay, Recombinant, Control, Activity Assay, Viability Assay, Stable Transfection, Expressing, Transfection, Marker, MANN-WHITNEY, Western Blot, Activation Assay

Journal: Nature chemical biology

Article Title: A drug discovery platform to identify compounds that inhibit EGFR triple mutants

doi: 10.1038/s41589-020-0484-2

Figure Lengend Snippet: Investigating effect of EMI1 on activated EGFR L858R/T790M/C797S endosomal trafficking. (a) Total integral intensity of EGF on EEA1-positive endosomes normalized on cytoplasm area after 30 minutes of EGF stimulation upon 1 µM compound treatment in HEK293 EGFR WT cells or EGFR L858R/T790M/C797S cells. (b) Total integral intensity of pY1068 on EEA1-positive endosomes normalized on cytoplasm area after 30 minutes of EGF stimulation with 1 µM compound treatment in HEK293 EGFR WT cells or EGFR L858R/T790M/C797S cells. Results are shown as dot plots representing the average ± SD. For EGFR WT cells, n = 63, 46, 48 and 48 images were analyzed for DMSO, EMI1, midostaurin and osimertinib treatment, respectively. For EGFR-C797S cells, n = 48, 46, 46, 48 images were analyzed for DMSO, EMI1, midostaurin and osimertinib treatment, respectively. Significant p-values are displayed and were calculated using the Dunn’s multiple comparison test. (c) Cell surface biotinylation assay assessing surface levels of EGFR L858R/T790M/C797S after treatment with 5 µM compound for 2 hours. Results are representative of at least two independent experiments. (d) Cell surface biotinylation assay assessing surface levels of EGFR WT after treatment with EMI1 for 2 hours. Results are representative of at least two independent experiments (see Supplementary Fig. 26 for source blot images). (e) Number of pEGFR and EEA1 double-positive endosomes per 1000μm2 after 30 min stimulation by EGF. (f) Mean integral intensity of pEGFR on double-positive (pEGFR and EEA1) endosomes after 30 min stimulation by EGF. Results are shown as dot plots representing the average ± SD. For EGFR WT cells, n = 63, 46, 48 and 48 images were analyzed for DMSO, EMI1, midostaurin and osimertinib treatment, respectively. For EGFR-C797S cells, n = 48, 46, 46, 48 images were analyzed for DMSO, EMI1, midostaurin and osimertinib treatment, respectively. Significant p-values are displayed and were calculated using the Dunn’s multiple comparison test.

Article Snippet: Kinase assays were performed using recombinant proteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R, EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).

Techniques: Comparison, Cell Surface Biotinylation Assay

Journal: Nature chemical biology

Article Title: A drug discovery platform to identify compounds that inhibit EGFR triple mutants

doi: 10.1038/s41589-020-0484-2

Figure Lengend Snippet: Generation and testing of EMI1 chemical analogs. (b,c,d) Western blot analysis showing effects of EMI1 (b) EMI48 (c) and EMI56 (d) on total EGFR levels, activation and downstream signalling after two hours treatment in PC9 EGFR ex19del/T790M/C797S cells (see Supplementary Figs 27 and 28 for source blot images). Results are representative of at least two independent experiments.

Article Snippet: Kinase assays were performed using recombinant proteins of the kinase domain of wild-type EGFR, EGFR-C797S/T790M/L858R, EGFR-C797S/T790M/ex19del, and EGFR-C797S (Reaction Biology Corporation).

Techniques: Western Blot, Activation Assay