Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: ALW-II-41-27 activated Nrf2 signaling whereas inhibited NF-κB signaling in Trichinella spiralis -infected mice. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B, and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control group ( P < 0.01); ∗∗ indicates a significant difference from PI-IBS group ( P < 0.01).

Article Snippet: To establish EphA2 gene overexpression cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were transfected with EphA2 CRISPR activation plasmid or Control CRISPR activation plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Infection, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 knock-out partially inhibited the effects of ALW-II-41-27 on LPS-treated cells. NCM460 cells were transfected to obtain clones knock-out for EphA2. Control cells and EphA2 KO cells were treated for 24 h with ALW-II-41-27 (100 ng/mL) and then treated with LPS (50 ng/mL) for 24 h. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control cells ( P < 0.01); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene overexpression cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were transfected with EphA2 CRISPR activation plasmid or Control CRISPR activation plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Knock-Out, Transfection, Clone Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 overexpression abolished the effects of ALW-II-41-27 on LPS-treated cells. NCM460 cells were transfected to obtain clones overexpressing for EphA2. Control cells and EphA2 overexpressing cells were treated for 24 h with ALW-II-41-27 and then treated with LPS (50 ng/mL) for 24 h. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control cells ( P < 0.01); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene overexpression cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were transfected with EphA2 CRISPR activation plasmid or Control CRISPR activation plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Over Expression, Transfection, Clone Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 overexpression abolished the protective effects of ALW-II-41-27 on LPS-induced oxidative stress and inflammation. Effects of EphA2 overexpression on cell viability (A) , LDH leakage (B) , cellular 4-HNE (C) , cellular protein carbonyl (D) , cellular 8-OHdG (E) , cellular HO-1 activity (F) , cellular TNF-α (G) , cellular IL-6 (H) , cellular IL-17 (I) , cellular ICAM-1 (J) , cellular IL-10 (K) and cellular NF-κB activity (L) . ## Indicates a significant difference from control cells ( P < 0.01); ∗ indicates a significant difference from LPS group ( P < 0.05); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene overexpression cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were transfected with EphA2 CRISPR activation plasmid or Control CRISPR activation plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Over Expression, Activity Assay, Control

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 knock-out partially inhibited the protective effects of ALW-II-41-27 on LPS-induced oxidative stress and inflammation. Effects of EphA2 knock-out on cell viability (A) , LDH leakage (B) , cellular 4-HNE (C) , cellular protein carbonyl (D) , cellular 8-OHdG (E) , cellular HO-1 activity (F) , cellular TNF-α (G) , cellular IL-6 (H) , cellular IL-17 (I) , cellular ICAM-1 (J) , cellular IL-10 (K) and cellular NF-κB activity (L) . ## Indicates a significant difference from control cells ( P < 0.01); ∗ indicates a significant difference from LPS group ( P < 0.05); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene overexpression cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were transfected with EphA2 CRISPR activation plasmid or Control CRISPR activation plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Knock-Out, Activity Assay, Control

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: ALW-II-41-27 activated Nrf2 signaling whereas inhibited NF-κB signaling in Trichinella spiralis -infected mice. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B, and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control group ( P < 0.01); ∗∗ indicates a significant difference from PI-IBS group ( P < 0.01).

Article Snippet: To establish EphA2 gene knockout cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were co-transfected with EphA2 CRISPR/Cas9 KO plasmid or Control CRISPR/Cas9 plasmid and EphA2 HDR Plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Infection, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 knock-out partially inhibited the effects of ALW-II-41-27 on LPS-treated cells. NCM460 cells were transfected to obtain clones knock-out for EphA2. Control cells and EphA2 KO cells were treated for 24 h with ALW-II-41-27 (100 ng/mL) and then treated with LPS (50 ng/mL) for 24 h. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control cells ( P < 0.01); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene knockout cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were co-transfected with EphA2 CRISPR/Cas9 KO plasmid or Control CRISPR/Cas9 plasmid and EphA2 HDR Plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Knock-Out, Transfection, Clone Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 overexpression abolished the effects of ALW-II-41-27 on LPS-treated cells. NCM460 cells were transfected to obtain clones overexpressing for EphA2. Control cells and EphA2 overexpressing cells were treated for 24 h with ALW-II-41-27 and then treated with LPS (50 ng/mL) for 24 h. The protein expressions of EphA2, nuclear Nrf2, cytosol Nrf2, and nuclear NF-κB/p65, cytosol NF-κB/p65, Lamin B and β-actin in colon tissue lysates were measured by western blotting and densitometry analysis. The results are expressed as the mean ± SD ( n = 10 per group); ## indicates a significant difference from control cells ( P < 0.01); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene knockout cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were co-transfected with EphA2 CRISPR/Cas9 KO plasmid or Control CRISPR/Cas9 plasmid and EphA2 HDR Plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Over Expression, Transfection, Clone Assay, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 overexpression abolished the protective effects of ALW-II-41-27 on LPS-induced oxidative stress and inflammation. Effects of EphA2 overexpression on cell viability (A) , LDH leakage (B) , cellular 4-HNE (C) , cellular protein carbonyl (D) , cellular 8-OHdG (E) , cellular HO-1 activity (F) , cellular TNF-α (G) , cellular IL-6 (H) , cellular IL-17 (I) , cellular ICAM-1 (J) , cellular IL-10 (K) and cellular NF-κB activity (L) . ## Indicates a significant difference from control cells ( P < 0.01); ∗ indicates a significant difference from LPS group ( P < 0.05); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene knockout cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were co-transfected with EphA2 CRISPR/Cas9 KO plasmid or Control CRISPR/Cas9 plasmid and EphA2 HDR Plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Over Expression, Activity Assay, Control

Journal: Frontiers in Pharmacology

Article Title: A Novel EphA2 Inhibitor Exerts Beneficial Effects in PI-IBS in Vivo and in Vitro Models via Nrf2 and NF-κB Signaling Pathways

doi: 10.3389/fphar.2018.00272

Figure Lengend Snippet: EphA2 knock-out partially inhibited the protective effects of ALW-II-41-27 on LPS-induced oxidative stress and inflammation. Effects of EphA2 knock-out on cell viability (A) , LDH leakage (B) , cellular 4-HNE (C) , cellular protein carbonyl (D) , cellular 8-OHdG (E) , cellular HO-1 activity (F) , cellular TNF-α (G) , cellular IL-6 (H) , cellular IL-17 (I) , cellular ICAM-1 (J) , cellular IL-10 (K) and cellular NF-κB activity (L) . ## Indicates a significant difference from control cells ( P < 0.01); ∗ indicates a significant difference from LPS group ( P < 0.05); ∗∗ indicates a significant difference from LPS group ( P < 0.01); ΔΔ indicates a significant difference from control cells treated LPS; && indicates a significant difference from control cells treated with ALW-II-41-27 and LPS.

Article Snippet: To establish EphA2 gene knockout cells, NCM460 cells (1 × 10 5 cells/well) were seeded in six-well plates, when 60% confluence was reached, cells were co-transfected with EphA2 CRISPR/Cas9 KO plasmid or Control CRISPR/Cas9 plasmid and EphA2 HDR Plasmid in antibiotic-free growth medium with UltraCruz ® Transfection Reagent (Santa Cruz, CA, United States) and incubate overnight.

Techniques: Knock-Out, Activity Assay, Control

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml −1 ) for 10, 20 and 60 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. ( b ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. ( c ) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml −1 ) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. ( d ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. ( e ) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: SDS Page, Staining

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HeLa ( a ) or T98G ( b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min ( b , right). ( c ) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. ( d ) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. ( e ) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: Stable Transfection, Transfection, shRNA, Expressing, Luciferase, Negative Control

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: SDS Page

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. ( c ) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. ( d ) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: Transfection, Expressing, Negative Control, Recombinant, Incubation, Western Blot

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. ( b – f ) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin ( b ) Migrated cells were counted manually under a microscope ( c ) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. At the same time, the migration border cells were immunofluorescently stained with anti-pS-EphA2 or EphA2 antibodies ( d , e ) and cells harbouring lamellipodia were counted manually under a microscope ( f ) Scale bar, 20 μm. Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. ( g , h ) MDA-MB-231 cells were transfected with siRNA against EphA2 or negative control and EphA2 mutation-expression plasmids. The immunoblotting results from whole-cell lysates with anti-pS-EphA2, EphA2 and β-actin antibodies are shown in g and the results of scratch assay are shown in h . Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by analysis of variance followed by Tukey–Kramer HSD test.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: SDS Page, Transferring, Incubation, Microscopy, Migration, Staining, Transfection, Negative Control, Mutagenesis, Expressing, Western Blot, Wound Healing Assay

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Human melanoma cells (A2058, SK-MEL-28, A375, UACC62, UACC257 and SK-MEL-2), ( b ) DLD-1 colon cancer cells and ( c ) lung adenocarcinoma cells (PC-9, HCC827, HCC4006, NCI-H1650, H2228 and A549) were treated with vemurafenib (1 μM), BI-D1870 (10 μM), gefitinib (1 μM), crizotinib (10 μM) or U0126 (5 μM) for 30–60 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pY-EGFR, EGFR, β-actin and α-tubulin.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques:

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) A multi-cancer tissue microarray, including 1,010 cores from 13 organ cancer tissues, was adopted for immunohistochemical staining using primary antibodies against pS-EphA2 and pRSK. Typical staining images of lung cancer tissues, including adenocarcinoma (AD) and squamous cell carcinoma (SCC), at low- and high-power magnifications are shown. Scale bar, 20 μm. ( b ) Typical immunohistochemical staining of pS-EphA2 and pRSK in EGFR -mutated (exon 19 deletion) lung adenocarcinoma tissues are shown. Scale bar, 20 μm. ( c – f ) Postoperative overall Kaplan–Meier survival curves of all the lung cancer patients ( c , d ) or smoking patients ( e , f ) were compared according to pRSK negativity or positivity ( c , e ) or pS-EphA2/pRSK double positivity ( d , f ) P values were calculated by the log-rank tests.

Article Snippet: Recombinant human TNF-α, ephrin-A1-Fc chimera and EGF were obtained from R&D Systems (Minneapolis, MN, USA); recombinant human active GST-EphA2, GST-RSK1 and GST-RSK2 protein were from Carna Biosciences (Kobe, Japan); anti-EGFR monoclonal antibody (clone LA1; 05-101) was from Millipore (Billerica, MA, USA); Phos-tag ligand and TPA were from Wako Pure Chemical Industries (Osaka, Japan); LY294002, SB203580 and U0126 were from Merck Biosciences (Darmstadt, Germany); MK-2206 was from Active Biochemicals (Wan Chai, Hong Kong); BI-D1870 and crizotinib were from BioVision (Milpitas, CA, USA); gefitinib was from Cayman Chemical (Ann Arbor, MI, USA); and vemurafenib was from LC Laboratories (Woburn, MA, USA).

Techniques: Microarray, Immunohistochemical staining, Staining