gst epha2  (MedChemExpress)

Journal: Cancer Discovery

Article Title: PTEN Loss Promotes PI3Kβ Phosphorylation and EPHA2/SRC/p-PI3Kβ Y962 Complex Assembly to Drive Tumorigenesis

doi: 10.1158/2159-8290.CD-25-1126

Figure Lengend Snippet: A SRC–EPHA2–PI3Kβ tripartite complex drives oncogenic signaling in PTEN-null tumors. A, A schematic of the experiment for the targeted compound library screening to identify PI3Kβ phosphorylation inhibitors. Endogenous PI3Kβ-depleted BT549 or PC3 cells replaced with PI3Kβ-WT or PI3Kβ-Y962F mutants were treated with compounds from the library at 0.1 μmol/L. Cell viability was assessed by CCK-8 assays at 48 hours. B and C, Results of the kinase inhibitor screening in BT549 ( B ) and PC3 ( C ) cells. SRC inhibitors dasatinib and KX2-391 were among the top three significant inhibitors in both cells. D and E, Dasatinib effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 and PC3 cells. F, KX2-391 effectively inhibited p-SRC, p-PI3Kβ Y962 , p-ERK/c-Myc, and pAKT in PTEN-null BT549 cells. G, Dasatinib and KX2-391 dramatically reduced the growth of PPB cells with addback of PI3Kβ-WT but was not as effective on PPB cells with addback of PI3Kβ-Y956F. H, Knockdown of SRC or EPHA2 decreased phosphorylation of PI3Kβ-Y962 and c-MYC in BT549 cells. I, SRC coordinated with EPHA2 to phosphorylate PI3Kβ. SRC-Flag and EPHA2 plasmids were overexpressed in PTEN-WT and PTEN-KO HEK293T cells, and Flag was immunoprecipitated, followed by Western blot analysis. J, SRC and EPHA2 phosphorylated PI3Kβ-Y962 peptides in vitro . GST-SRC or GST-EPHA2 was incubated with synthetic Y962 containing PI3Kβ peptides in phosphorylation buffer, and the resulting peptides were analyzed by MS spectrum. K, In vitro phosphorylation assays showed that SRC and EPHA2 directly phosphorylated PI3Kβ-Y962, and the phosphorylation activity was blunted on PI3Kβ mutant at Y962. Particularly, SRC could strongly phosphorylate PI3Kβ-Y962. An in vitro kinase assay was performed by mixing GST-SRC or GST-EPHA2 with Flag-PI3Kβ-WT or Flag-PI3Kβ-Y962F proteins in the presence of ATP. Anti–phospho-PI3Kβ-Y962 antibody was used to detect the phosphorylated PI3Kβ-Y962. L, Knockdown of SRC (shSRC) dramatically decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; this effect can be slightly rescued by exogenous expression of EPHA2. M, Knockdown of EPHA2 (shEPHA2) decreased signaling of the p-PI3Kβ Y962 –pERK/c-Myc axis in BT549 cells; exogenous expression of SRC was unable to rescue the effect by sh EPHA2 in BT549 cells. N, SRC expression elevated p-ERK and c-Myc levels in BT549 cells, which were abolished by EPHA2 KO. O, A mechanism model to show that SRC collaborates with EPHA2 to phosphorylate PI3Kβ-Y962, whereas PTEN loss abolished PI3Kβ dephosphorylation, leading to PI3Kβ hyperphosphorylation and subsequent SRC–EPHA2–p-PI3Kβ Y962 complex formation to upregulate p-ERK/c-Myc signaling, accompanying with enhanced accessibility of PI3Kβ to phosphorylate PIP2 on the cell membrane to upregulate pAKT. Values were presented as the mean ± SEM. P values were determined by unpaired two-tailed t test (B, C). **** P < 0.0001.

Article Snippet: For peptide reactions, a 30 μL reaction system containing kinase reaction buffer added with 100 μmol/L ATP, 1 mmol/L DTT 250 ng of purified GST–SRC or GST-EPHA2 (MCE), and 0.05 mg/mL synthetic nonphosphorylated peptide were prepared.

Techniques: Drug discovery, Phospho-proteomics, CCK-8 Assay, Knockdown, Immunoprecipitation, Western Blot, In Vitro, Incubation, Activity Assay, Mutagenesis, Kinase Assay, Expressing, De-Phosphorylation Assay, Membrane, Two Tailed Test

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

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Ligand‐independent phosphorylation of EphA2 is related to chemoresistance of SCLC. (A) mRNA expression of EPHA2 was compared in normal lung tissue ( n = 20) and SCLC ( n = 150) FFPE samples by RT‐qPCR. Data are presented in the form of mean ± SD based on independent biological replicates, **** p ≤ 0.0001. (B) Kaplan–Meier analysis of the overall survival of 150 patients with SCLC divided into high ( n = 75) and low groups ( n = 75), separated by the median level, based on EPHA2 expression levels. * p ≤ 0.05. (C) Western blot analysis shows that the expression of EphA2 (total and pS897) was enhanced in drug‐resistant sublines H69AR and H446CDDP in comparison with drug‐sensitive sublines H69 and H446, while EphA2‐pY588 showed the opposite. (D) Western blots analysis shows that the expression of ephrin‐A1 was decreased in H69AR and H446CDDP compared with H69 and H446. The expression of Akt‐pS473 showed the opposite. The expression of Akt showed no significant difference between H69 and H69AR, and between H446 and H446CDDP. (E) Western blots analysis shows that EphA2‐pS897 expression was decreased in H69AR and H446CDDP after ephrin‐A1 treatment, while EphA2‐pY588 showed the opposite. EphA2 (total) expression remained unaltered after ephrin‐A1 treatment. (F) Western blot analysis showed that Akt‐pS473 was decreased in H69AR and H446CDDP after MK2206 treatment. Akt (total) expression remained unaltered. (G) Western blot analysis shows that EphA2‐pS897 expression was decreased in H69AR and H446CDDP after MK2206 treatment. EphA2 (total) expression remained unaltered. Western blot analysis shows inhibition of Akt expression by Akt siRNA suppressed phosphorylation of EphA2‐S897 in H69AR (H) and H446CDDP (I), while EphA2 (total) expression remained unaltered. (J) Expression levels of CD44, Myc, and SOX2 were higher in drug‐resistant sublines (H69AR and H446CDDP) compared with drug‐sensitive sublines (H69 and H446). (K) Western blots show that the expression levels of N‐cadherin and vimentin were higher in drug‐resistant sublines compared with drug‐sensitive sublines, while E‐cadherin expression was the opposite. These experiments were repeated at least three times and representative images are shown.

Article Snippet: The purified GST‐EphA2 WT or GST‐EphA2 P817H fusion proteins were immobilized in Glutathione Sepharose (GE Healthcare, Chicago, IL, USA) and then incubated with the purified His‐PRMT1 protein at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Wild‐type EphA2 enhances the cancer stemness and chemoresistance of SCLC, whereas the P817H mutation neutralizes intrinsic function. (A) Western blots show that the expression levels of EphA2 (total and pS897) were significantly reduced in H69AR cells, which were stably transfected with sh EPHA2 . (B) IC 50 values of epirubicin (ADM), cisplatin (CDDP), and etoposide (VP‐16) were significantly reduced in response to sh EPHA2 transfection compared with the control group in H69AR cells. The number of independent biological replicates = 3, *** p ≤ 0.001, **** p ≤ 0.0001. (C) Western blots show that the expression levels of CD44, Myc, and SOX2 were decreased in H69AR cells with silenced EPHA2 . (D) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in H69AR cells with silenced EPHA2 , while E‐cadherin expression was increased. (E) SFE of sh EPHA2 groups in H69AR cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (F) Western blots show the changes in expression levels of EphA2 (total and pS897) in H69 cells, which were stably transfected with wild‐type EPHA2 (WT), A785S, P817H, or Y930D mutant EPHA2 . (G) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with upregulated wild‐type or mutant EphA2, respectively. The number of independent biological replicates = 3, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (H) Western blots show that the expression levels of CD44, Myc, and SOX2 were increased in H69 cells overexpressing wild‐type, A785S, or Y930S mutant EphA2. (I) Western blots showed the expression levels of N‐cadherin and vimentin were increased in H69 cells overexpressing wild‐type, A785S, or Y930S mutant EphA2, while E‐cadherin expression was reduced. (J) SFE of wild‐type and mutant EphA2 groups in H69 cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (K) H69 cells stably overexpressed wild‐type EphA2, P817H mutant EphA2, or the control. (L) H69AR cells were stably transfected with sh EPHA2 or shNC. Each group of cells was injected into mice, followed by chemotherapy (CDDP + VP‐16) or empty vehicles were injected intraperitoneally as indicated ( n = 5 mice for each group). (M) Tumor weights of EphA2 WT groups and EphA2 P817H groups. Data are displayed as the mean ± SD, n = 5, * p ≤ 0.05, ** p ≤ 0.01. (N) Tumor weights of sh EPHA2 groups. Data are displayed as the mean ± SD, n = 5, *** p ≤ 0.001. Each group of cells was injected into mice, followed by chemotherapy (CDDP + VP‐16) or empty vehicles were injected intraperitoneally as indicated ( n = 5 mice for each group). These experiments were repeated at least three times and representative images are shown.

Article Snippet: The purified GST‐EphA2 WT or GST‐EphA2 P817H fusion proteins were immobilized in Glutathione Sepharose (GE Healthcare, Chicago, IL, USA) and then incubated with the purified His‐PRMT1 protein at 4°C overnight.

Techniques: Mutagenesis, Western Blot, Expressing, Stable Transfection, Transfection, Injection

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: EphA2 directly interacts with PRMT1. (A) Expression of PRMT1 was higher in chemoresistant sublines H69AR and H446CDDP compared with chemosensitive sublines H69 and H446. A significant difference in PRMT5 expression was detected between H69 and H69AR, no significant difference was detected between H446 and H446CDDP. (B) Expression of PRMT1 and H4R3me2a was decreased in sh EPHA2 groups compared with the control groups. (C) Expression of PRMT1 and H4R3me2a was higher in EphA2 WT , EphA2 A785S , and EphA2 Y930D groups in comparison with EphA2 P817H and control groups in H69. (D,E) Co‐IP was performed in H69 cells using anti‐FLAG and anti‐PRMT1 antibodies, which were transfected with plasmids of FLAG‐tagged wild‐type and mutant EPHA2 or negative control. The co‐IP products were then analyzed by western blot. (F–I) Co‐IP was performed in H69 and H69AR cells using anti‐EphA2 and anti‐PRMT1 antibodies. The co‐IP products were then analyzed by western blot. (J) GST‐pull down shows that wild‐type EphA2 directly interacted with PRMT1. (K) Immunofluorescence staining shows the subcellular localization of EphA2 (total and pS897) in H69AR and H446 cells overexpressing wild‐type or P817H mutant EphA2. Scale bars = 10 μm. (L) Immunofluorescence staining shows that wild‐type EphA2 colocalized with PRMT1 in H69AR and H446 cells overexpressing wild‐type EphA2. Scale bar = 10 μm. These experiments were repeated at least three times and representative images are shown.

Article Snippet: The purified GST‐EphA2 WT or GST‐EphA2 P817H fusion proteins were immobilized in Glutathione Sepharose (GE Healthcare, Chicago, IL, USA) and then incubated with the purified His‐PRMT1 protein at 4°C overnight.

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Negative Control, Western Blot, Immunofluorescence, Staining

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: PRMT1 augments stemness and chemoresistance in SCLC. (A) Western blots show the changes in expression levels of PRMT1 in H69 cells, which were stably transfected with His‐tagged PRMT1 . (B) Western blots show that the expression levels of CD44, Myc, and SOX2 were increased in H69 cells overexpressing PRMT1. (C) Western blots show that the expression levels of N‐cadherin and vimentin were increased in H69 cells overexpressing PRMT1, while E‐cadherin expression was reduced. (D) SFE of PRMT1 groups in H69 cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (E) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with upregulated PRMT1. The number of independent biological replicates = 3, *** p ≤ 0.001. (F) Western blots show that the expression levels of PRMT1 were reduced in H69 cells with co‐transfected wild‐type or P817H mutant EPHA2 and sh PRMT1 . (G) Western blots show that the expression levels of CD44, Myc, and SOX2 were reduced in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 . (H) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 , whereas E‐cadherin expression was increased. (I) SFE of sh PRMT1 groups in H69 cells with wild‐type or mutant EPHA2 . Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (J) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 . The number of independent biological replicates = 3, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. These experiments were repeated at least three times and representative images are shown.

Article Snippet: The purified GST‐EphA2 WT or GST‐EphA2 P817H fusion proteins were immobilized in Glutathione Sepharose (GE Healthcare, Chicago, IL, USA) and then incubated with the purified His‐PRMT1 protein at 4°C overnight.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Mutagenesis

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Inhibiting ligand‐independent EphA2 suppresses the growth of SCLC in vitro and in vivo. (A) Western blots show that the expression level of EphA2‐pS897 was reduced in response to treatment with ALW‐II‐41‐27 (ALW) for 2 h in a dose‐dependent manner in H69AR cells. The total EphA2 expression remained unaltered. (B) Western blots show that the expression level of EphA2‐pS897 was reduced in response to treatment with dasatinib (Dasa) for 24 h in a dose‐dependent manner in H69AR cells. The total EphA2 expression remained unaltered. (C) IC 50 values of ADM, CDDP, and VP‐16 were reduced in H69 and H69AR cells. H69 and H69AR cells were treated with empty vehicle, or 0.75 or 1.5 μmol/L of ALW‐II‐41‐27. The number of independent biological replicates = 3, *** p ≤ 0.001, **** p ≤ 0.0001. (D) IC 50 values for ADM, CDDP, and VP‐16 were reduced in H69 and H69AR cells. H69 and H69AR cells were treated with empty vehicle, 2.5 or 5 μmol/L of dasatinib. The number of independent biological replicates = 3, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (E) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in response to treatment with ALW‐II‐41‐27, in H69AR cells, while E‐cadherin expression was increased. (F) Western blots show the changes in expression levels of CD44, Myc, SOX2, and PRMT1, in response to ALW‐II‐41‐27 treatment, in H69AR cells. (G) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in response to the treatment with AMI‐1, in H69AR cells, while E‐cadherin expression was increased. (H) Western blots show the changes in expression levels of CD44, Myc, and SOX2, in response to treatment with AMI‐1, in H69AR cells. (I) SFE of H69AR cells, which were treated with ALW‐II‐41‐27. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (J) Immunofluorescence shows the changes in subcellular localization of EphA2 and EphA2‐pS897 in response to treatment with ALW‐II‐41‐27, in H446 cells overexpressing wild‐type or P817H EphA2. Scale bars = 10 μm. (K) Effect of chemotherapy (CDDP + VP‐16), ALW, or the combination of ALW‐II‐41‐27 and chemotherapy on tumor growth using SCLC PDX models. (L) Effect of chemotherapy (CDDP + VP‐16), ALW‐II‐41‐27, or the combination of ALW‐II‐41‐27 and chemotherapy on tumor growth in vivo. Nude mice were engrafted with H69AR cells subcutaneously. These experiments were repeated at least three times and representative images are shown.

Article Snippet: The purified GST‐EphA2 WT or GST‐EphA2 P817H fusion proteins were immobilized in Glutathione Sepharose (GE Healthcare, Chicago, IL, USA) and then incubated with the purified His‐PRMT1 protein at 4°C overnight.

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Immunofluorescence