Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Mig-6 is overexpressed in gefitinib-resistant PC9/GR cell lines compared with parental PC9 cell lines. a Mig-6 expression was examined by immunofluorescence staining of endogenous Mig-6 (red) in parental PC9 cells and in PC9/GR cells (Original magnification, × 100 as displayed in the figures), lower panel is optical microscopy images of PC9 and PC9/GR cells (scale bar represents 100 μm). b Western blot experiments for proteins related to epidermal growth factor receptor (EGFR) signaling and the epithelial-mesenchymal transition (EMT) in PC9 and PC9/GR cells. The expressions Mig6, p-EGFR (Tyr1068), p-EGFR (Tyr1045), EGFR, c-MET, p-AKT, AKT, p-ERK, ERK, E-cadherin, Zo-1 and Vimentin were evaluated. β-actin was used as a loading control. c Phos-tag immunoblot analysis of Mig-6 with or without phosphatase. To check the phosphorylated status of Mig-6, the lysates we employed phos-tag SDS-PAGE. The lysate was loaded in 6% Acrylamide 100uM Mn 2+ -Phos-tag™ Acrylamide. To confirm that those slower migrating bands are really phosphorylated species of Mig-6, we treated a part of the lysate with protein phosphatase for 9 h. d RT-PCR analysis. mRNA levels of EGFR, E-cadherin, Vimentin, and c-MET was evaluated in PC9 and PC9/GR. GAPDH was used as an internal control. Uncropped blots were shown in Additional file : Fig. S1

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Mig-6 overexpression increased the viability and migration of PC9 cells. a and b Mig-6 expression was examined by western blot and immunofluorescence staining of endogenous Mig-6 in PC9 cells and Mig-6-overexpressing PC9 cells. c The cell viability of PC9 and Mig-6-overexpressing PC9 cells was measured using the Cell Counting Kit-8 (CCK-8) assay. The optical density values were detected on a microplate reader at 450 nm. d Cell migration ability was tested using wound-healing and transwell migration assays. Boyden chambers were stained with crystal violet for the transwell migration and invasion assays. The number of cells was counted under a microscope (* P < 0.05, ** P < 0.01). e The viability of cells treated with different EGFR-TKI doses was measured using the CCK-8 assay. f PC9 cells and Mig-6-overexpressing PC9 cells were incubated with medium alone (control) or various concentrations of gefitinib for 24 h, and cleaved PARP/PARP expression was assessed by western blot analysis. g Proteins related to epidermal growth factor receptor (EGFR) signaling were checked with Western blot in PC9 and Mig-6 overexpressed PC9 cells. The expressions Mig6, p-EGFR (Tyr1068), p-EGFR (Tyr1045), EGFR, C-MET, p-AKT, AKT, p-ERK, ERK, E-cadherin, Zo-1 and Vimentin were evaluated. β-actin was used as a loading control. Uncropped blots were shown in Additional file : Fig. S2

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Over Expression, Migration, Expressing, Western Blot, Immunofluorescence, Staining, Cell Counting, CCK-8 Assay, Microscopy, Incubation

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Mig-6 regulates the proliferation and migration of lung adenocarcinoma cells. a Western blot analysis of Mig-6 in PC/GR cells following transfection with Mig-6 siRNA or scrambled siRNA. b Cell viability was assessed using the CCK-8 assay after transfection of PC9 and PC9/GR cells with Mig-6 siRNA or scrambled siRNA. c Cell migration ability was tested using wound-healing and transwell migration assays. PC9 and PC9/GR cells were transfected with vector or siMig-6. Movement of cells into wounds is shown at 0 and 24 h post-scratching. Scale bar: 200 mm. Data are shown as the mean ± standard deviation (SD); * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. d Western blot analysis of E-cadherin and Vimentin in PC9 and PC9/GR cells with Mig-6 siRNA or scrambled siRNA. e Western blot analysis of AKT and p-AKT in PC9 and PC9/GR cells with Mig-6 siRNA or scrambled siRNA. f Western blot analysis of EGFR, p-EGFR (Tyr1068), p-EGFR (Tyr1045), ERK and p-ERK in PC9 and PC9/GR cells with Mig-6 siRNA or scrambled siRNA. β-actin was used as a loading control. Uncropped blots were shown in Additional file : Fig. S3

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Migration, Western Blot, Transfection, CCK-8 Assay, Plasmid Preparation, Standard Deviation

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Mig-6 inhibition restores sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) in PC9/GR cells. a Cell viability was assessed using the CCK-8 assay after transfection with Mig-6 siRNA or scrambled siRNA and treatment with various doses of gefitinib. Data are shown as the mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. b PC9 and PC9/GR cells were incubated with medium alone (control) or various concentrations of gefitinib for 24 h with or without siMig-6, and cleaved PARP/PARP expression was assessed by western blot analysis. β-actin was used as a loading control. c Apoptotic cells were evaluated by TUNEL assays. The number of apoptotic cells (TUNEL-stained) and the total number of cells (DAPI-stained) were counted to determine the percentage of apoptotic cells in the tumor sections. An apparent increase in the number of apoptotic PC9/GR cells was observed when cells were simultaneously treated with the EGFR-TKI and siMig-6. Data are shown as the mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t -test. Uncropped blots were shown in Additional file : Fig. S4

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Inhibition, CCK-8 Assay, Transfection, Incubation, Expressing, Western Blot, TUNEL Assay, Staining

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: High expression of Mig-6 is correlated with poor prognosis and EMT in lung cancer. a Overall survival of lung adenocarcinoma patients was compared between Mig-6-high and Mig-6-low expression groups. Histograms of Mig-6 gene expression in lung adenocarcinoma patients. Vertical red lines designate 9.887 as the optimal cutoff of mRNA derived from the log-rank test. High mRNA expression of Mig-6 was significantly associated with a poor prognosis (hazard ratio [HR]: 1.92 [95% confidence interval (CI): 1.29–2.85]). Subgroup analysis was performed in patients with or without EGFR mutation. High mRNA expression of Mig-6 without EGFR mutation was significantly associated with poor prognosis (HR: 1.86 [95% CI: 1.19–2.9]). High mRNA expression of Mig-6 with EGFR mutation was also significantly associated with poor prognosis (HR: 2.33 [95% CI: 0.97–5.59]). b The graph displays the enrichment gene set based on the hallmark and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the top 10% vs. bottom 10% groups of Mig-6 in lung adenocarcinoma patients. Gene Set Enrichment Analysis of the EMT, G2M checkpoint, hypoxia, TGF-β, cell cycle, and ERBB signature genes in the high and low Mig-6 expression groups of lung adenocarcinoma patients

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Expressing, Derivative Assay, Mutagenesis

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Comparison of Mig-6 expression in lung adenocarcinoma at baseline and after acquiring EGFR-TKI resistance. a–d Representative pictures of Mig-6 by immunohistochemistry in cases 1 and 2. a and c are initial biopsy samples. b and d are matched re-biopsy samples of case 1 and 2. The expression of Mig-6 in re-biopsy samples were significantly higher than those of initial biopsy samples. e and f Analysis of Mig-6 expressions at baseline and after acquiring EGFR-TKI resistance ( n = 26)

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Expressing, Immunohistochemistry

Journal: BMC Cancer

Article Title: Suppression of Mig-6 overcomes the acquired EGFR-TKI resistance of lung adenocarcinoma

doi: 10.1186/s12885-020-07057-z

Figure Lengend Snippet: Graphical summary of Mig-6 function in EGFR-TKI resistant lung adenocarcinoma. Phosphorylated Mig-6 has a crucial oncogenic role in the survival of EGFR-mutant lung adenocarcinoma and targeting Mig-6 may be a promising strategy to overcome EGFR-TKI resistance in lung cancer

Article Snippet: The following antibodies were used: anti-β-actin (sc-47,778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610,182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550,513, BD Biosciences), C-MET (#3148, Cell signal) and anti-PARP (#9542, Cell Signaling Technology).

Techniques: Mutagenesis