Journal: Cells

Article Title: Afatinib Overcomes Pemetrexed-Acquired Resistance in Non-Small Cell Lung Cancer Cells Harboring an EML4-ALK Rearrangement

doi: 10.3390/cells8121538

Figure Lengend Snippet: The PEM-acquired resistant NSCLC cell harboring EML4-ALK rearrangement model displays overexpression of the RTK signaling pathway. ( A , B ) RTK and EGFR phosphorylation arrays that were altered in NCI-H3122 R compared to the parental cell line. The densitometric ratio of duplicate spots for activated RTK and EGFR proteins to the internal loading controls on the RTK phosphorylation and EGFR phosphorylation array was calculated using Image J software (**, p < 0.01; ***, p < 0.001; N.S. not significant). ( C , D ) Western blotting analysis of A549 P, A549 R, NCI-H460 P, NCI-H460 R, NCI-H3122 P, and NCI-H3122 R cells for phospho-EGFR, EGFR, phospho-HER2, and HER2 expression was performed. GAPDH served as the loading control (SEM, serum-free media; GM; growth media). ( E ) EGFR and HER2 mRNA expression were determined in NCI-H3122 P and NCI-H3122 R cells by qRT-PCR. Fold change in EGFR, HER2, and HER3 mRNA in NCI-H3122 R cells was compared to that in parental cells (***, p < 0.001; N.S. not significant). ( F ) Western blotting analysis of NCI-H3122 P and NCI-H3122 R cells for p-AKT S473, AKT, p-MEK S217/221, p-MEK S298, and MEK. β-Actin served as the loading control (SEM, serum-free media; GM; growth media).

Article Snippet: Antibodies against protein kinase B (AKT, cs#4685), phospho-AKT S473 (cs#4058), phospho-AKT T308 (cs#9275), cleaved caspase-9 (cs#7237), cyclin B (cs#4138), epidermal growth factor receptor (EGFR, cs#2646), p-EGFR (cs#2236), erb-b2 receptor tyrosine kinase 2 (ERBB2/HER2, cs#2165), phospho-ERBB2 (cs#2243), ERBB3/HER3 (cs#4754), p-ERBB3/HER3 (cs#2842), mitogen-activated protein kinase kinase (MEK, cs#13033), phospho-MEK S217/221 (cs#9154), phospho-MEK S298 (cs#98195), extracellular-signal-regulated kinase 1/2 (ERK1/2, cs#9102), p-ERK1/2 (cs#4377s), and thymidylate synthase (TS, cs#5449) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Software, Western Blot, Expressing, Quantitative RT-PCR

Journal: Cells

Article Title: Afatinib Overcomes Pemetrexed-Acquired Resistance in Non-Small Cell Lung Cancer Cells Harboring an EML4-ALK Rearrangement

doi: 10.3390/cells8121538

Figure Lengend Snippet: Knockdown of EGFR-HER2 exerts a pro-apoptotic effect in NCI-H3122 R cells. ( A ) NCI-H3122 R cells were transfected with siEGFR and/or siHER2 and were incubated for 24 h. Cells were harvested and the expression of EGFR and HER2 was evaluated with Western blotting. β-Actin served as the loading control. ( B ) Apoptosis was evaluated using flow cytometry of Annexin V-PI double-stained NCI-H3122 R cells after transfection with siEGFR and/or siHER2 for 24 h. The Y-axis represents the PI-labeled population, whereas the X-axis represents the Annexin V positive cells. The left lower gating (Annexin V-, PI-) indicates normal cells, whereas the right lower gating (Annexin V+, PI-) and the right upper gating (Annexin V+, PI+) are the early and late apoptotic cells, respectively. ( C ) Data are presented as histograms. Means with different letters (a and b) indicate statistically significant differences ( p < 0.05; N.S., not significant). ( D ) Caspase 3/7 activity was quantified 24 h after transfection with siEGFR and/or siHER2 in NCI-H3122 R cells. Means with different letters (a and b) indicate statistically significant differences ( p < 0.05; N.S., not significant).

Article Snippet: Antibodies against protein kinase B (AKT, cs#4685), phospho-AKT S473 (cs#4058), phospho-AKT T308 (cs#9275), cleaved caspase-9 (cs#7237), cyclin B (cs#4138), epidermal growth factor receptor (EGFR, cs#2646), p-EGFR (cs#2236), erb-b2 receptor tyrosine kinase 2 (ERBB2/HER2, cs#2165), phospho-ERBB2 (cs#2243), ERBB3/HER3 (cs#4754), p-ERBB3/HER3 (cs#2842), mitogen-activated protein kinase kinase (MEK, cs#13033), phospho-MEK S217/221 (cs#9154), phospho-MEK S298 (cs#98195), extracellular-signal-regulated kinase 1/2 (ERK1/2, cs#9102), p-ERK1/2 (cs#4377s), and thymidylate synthase (TS, cs#5449) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Incubation, Expressing, Western Blot, Flow Cytometry, Staining, Labeling, Activity Assay

Journal: Cells

Article Title: Afatinib Overcomes Pemetrexed-Acquired Resistance in Non-Small Cell Lung Cancer Cells Harboring an EML4-ALK Rearrangement

doi: 10.3390/cells8121538

Figure Lengend Snippet: RTK inhibition abrogates RTK expression in NCI-H3122 R cells. ( A ) NCI-H3122 R cells were treated with afatinib or gefitinib. The expression levels of phospho-EGFR, EGFR, phospho-AKT S473, AKT, phospho-HER2, HER2, phospho-MEK S217/221, phospho-MEK S298, and MEK were assessed by Western blotting in NCI-H3122 R cells. β-Actin served as the loading control. ( B ) Basal expression of HIF-1α was evaluated with Western blotting. GAPDH served as the loading control (SEM, serum-free media; GM; growth media). ( C ) NCI-H3122 R cells were treated with afatinib of gefitinib. The expression levels of HIF-1α were evaluated with Western blotting in NCI-H3122 R cells. GAPDH served as the loading control. ( D ) Colony-forming assays were performed in NCI-H3122 R cells. Means with different letters (a, b, and c) indicate statistically significant differences ( p < 0.05; N.S., not significant).

Article Snippet: Antibodies against protein kinase B (AKT, cs#4685), phospho-AKT S473 (cs#4058), phospho-AKT T308 (cs#9275), cleaved caspase-9 (cs#7237), cyclin B (cs#4138), epidermal growth factor receptor (EGFR, cs#2646), p-EGFR (cs#2236), erb-b2 receptor tyrosine kinase 2 (ERBB2/HER2, cs#2165), phospho-ERBB2 (cs#2243), ERBB3/HER3 (cs#4754), p-ERBB3/HER3 (cs#2842), mitogen-activated protein kinase kinase (MEK, cs#13033), phospho-MEK S217/221 (cs#9154), phospho-MEK S298 (cs#98195), extracellular-signal-regulated kinase 1/2 (ERK1/2, cs#9102), p-ERK1/2 (cs#4377s), and thymidylate synthase (TS, cs#5449) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: Systematic analysis of mRNA expression profiles in NSCLC cell lines to screen metastasis-related genes

doi: 10.3892/mmr.2016.5911

Figure Lengend Snippet: Primer sequence and basic information for the 18 differentially expressed genes are listed.

Article Snippet: The NC membranes with protein were blocked in 5% fat-free powdered milk at room temperature for 2 h. The primary antibodies used areas follows: Primary rabbit polyclonal anti-A-kinase anchoring protein 12 (AKAP12; 1:200 dilution; cat. no. 25199-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), rabbit polyclonal anti-aldehyde dehydrogenase 7 family member A1 (ALDH7A1; 1:5,000 dilution; cat. no. 2070-S; Epitomics, Burlingame, CA, USA), rabbit polyclonal anti-caveolin 1 (CAV1; cat. no. 21112-1; 1:500 dilution; Signalway Antibody LLC, College Park, MD, USA), rabbit polyclonal anti-erb-b2 receptor tyrosine kinase 2 (Erbb2; 1:1,000 dilution; cat. no. 2165s; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-growth arrest and DNA damage inducible α (GADD45α; 1:200 dilution; cat. no. sc-797; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), goat polyclonal anti-keratin 18 (Krt18; 1:200 dilution; cat. no. 35624; Signalway Antibody LLC), goat polyclonal anti-myosin heavy chain 9 (MYH9; 1:500 dilution; cat. no. c2910; Santa Cruz Biotechnology, Inc.), goat polyclonal anti-proliferating cell nuclear antigen (PCNA; 1:100 dilution; cat. no. AF0239; Affinity Bioscience, Cincinnati, OH, USA), rabbit polyclonal anti-carbamoyl-phosphate synthase 1 (CPS1; 1:200 dilution; cat. no. AP16053a; Abgent, Inc., San Diego, CA, USA), rabbit polyclonal anti-phosphoglycerate dehydrogenase (PHGDH; 1:300 dilution; cat. no. AP2936c; Abgent, Inc.) and anti-β-actin (1:5,000; cat. no. ab14128; Abcam, Cambridge, MA, USA).

Techniques: Sequencing, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Systematic analysis of mRNA expression profiles in NSCLC cell lines to screen metastasis-related genes

doi: 10.3892/mmr.2016.5911

Figure Lengend Snippet: Validation of the expression for DEGs on the protein and mRNA levels. (A) Validation of 18 DEGs at the mRNA level. (B) Validation of 10 DEGs at the protein level. DEGs, differentially expressed genes; PRSS3, protease, serine 3; Erbb2, erb-b2 receptor tyrosine kinase 2; Aldh7a1, aldehyde dehydrogenase 7 family member A1; CAV1, caveolin 1; MT1X, metallothionein 1X; AMFR, autocrine motility factor receptor; MYH9, myosin heavy chain 9; ENTPD5, ectonucleoside triphosphate diphosphohydrolase 5; AKAP12, A-kinase anchoring protein 12; GADD45α, growth arrest and DNA damage inducible α; Krt18, keratin 18; PCNA, proliferating cell nuclear antigen; PHGDH, phosphoglycerate dehydrogenase; KYNU, kynureninase; CPS-1, carbamoyl-phosphate synthase 1; FTSJ3, FtsJ homolog 3; FKBP10, FK506 binding protein 10; FABP3, fatty acid binding protein 3.

Article Snippet: The NC membranes with protein were blocked in 5% fat-free powdered milk at room temperature for 2 h. The primary antibodies used areas follows: Primary rabbit polyclonal anti-A-kinase anchoring protein 12 (AKAP12; 1:200 dilution; cat. no. 25199-1-AP; ProteinTech Group, Inc., Chicago, IL, USA), rabbit polyclonal anti-aldehyde dehydrogenase 7 family member A1 (ALDH7A1; 1:5,000 dilution; cat. no. 2070-S; Epitomics, Burlingame, CA, USA), rabbit polyclonal anti-caveolin 1 (CAV1; cat. no. 21112-1; 1:500 dilution; Signalway Antibody LLC, College Park, MD, USA), rabbit polyclonal anti-erb-b2 receptor tyrosine kinase 2 (Erbb2; 1:1,000 dilution; cat. no. 2165s; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit polyclonal anti-growth arrest and DNA damage inducible α (GADD45α; 1:200 dilution; cat. no. sc-797; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), goat polyclonal anti-keratin 18 (Krt18; 1:200 dilution; cat. no. 35624; Signalway Antibody LLC), goat polyclonal anti-myosin heavy chain 9 (MYH9; 1:500 dilution; cat. no. c2910; Santa Cruz Biotechnology, Inc.), goat polyclonal anti-proliferating cell nuclear antigen (PCNA; 1:100 dilution; cat. no. AF0239; Affinity Bioscience, Cincinnati, OH, USA), rabbit polyclonal anti-carbamoyl-phosphate synthase 1 (CPS1; 1:200 dilution; cat. no. AP16053a; Abgent, Inc., San Diego, CA, USA), rabbit polyclonal anti-phosphoglycerate dehydrogenase (PHGDH; 1:300 dilution; cat. no. AP2936c; Abgent, Inc.) and anti-β-actin (1:5,000; cat. no. ab14128; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Binding Assay

Journal: Oncotarget

Article Title: A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

doi:

Figure Lengend Snippet: A/B) FaDu cells were treated with V, HU, VPA/HU (1.5 mM each) or PBS (C). Expression of the indicated proteins was analyzed by immunoblotting. Actin served to control equal loading. VPA/HU treatment effectively reduced the levels of total and phosphorylated EGFR, whereas ERB-B2 levels were not affected. C) FaDu cells treated with VPA/HU (1.5 mM each) were FFPE and used for IHC analysis employing EGFR- or BIM-specific Ab. Treatment resulted in reduced expression and cell surface localization of the EGFR as well as increased BIM levels. D) VPA/HU suppressed the growth of FaDu HNSCC xenograft tumors. Growth curve of tumors subjected to VPA/HU (i.p., 350 mg/kg and 750 mg/kg body weight) or PBS control. Nude mice were inoculated with FaDu tumor cells. When tumors had reached the target size of 0.1 cm 3 , mice were treated once every second day for 14 days. *p<0.001, n =4 animals per treatment group, data are mean±SD. E) Enhanced BIM and reduced EGFR levels in xenograft tumors at the end of VPA/HU treatment. BIM and EGFR expression was visualized by IHC.

Article Snippet: Ab were: α-PUMA (4976) (NEB Cell Signaling, Frankfurt, Germany); α-Survivin (Novus NB 500-201; Novus Biologicals, Littleton, CO); anti-β-Actin (A2066), α-BIM (B7929), anti-alpha-Tubulin (T5168) (Sigma Aldrich, Munich, Germany); α-BCL-XL (66461A), α-Caspase-8 (9745), -9 (9501) (Pharmingen); cleaved Caspase-3 (9664) (Cell Signaling); α-BAX (sc-20067), α-Caspase-3 (sc-7272/-7148), α-ERB-B2 (sc-284), α-EGFR (sc-81449), α-ERK1/2 (sc-135900), α-STAT3 (sc-482) (Santa Cruz Biotechnology, Heidelberg, Germany).

Techniques: Expressing, Western Blot