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Sino Biological epha2 expression plasmid
Epha2 Expression Plasmid, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/epha2+expression+plasmid/Human+EphA2%2FEph+Receptor+A2+Gene+ORF+cDNA+clone+expression+plasmid%2C+C-HA+tag/10__32604_slash_biocell__2025__065658-44-1-4
Average 93 stars, based on 1 article reviews

epha2 expression plasmid - by Bioz Stars, 2026-10

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Article Title: Targeting EphA2 with CRISPR/Cas9 Identifies a Novel EphA2-CDH1 Regulatory Axis in Migration of Castration-Resistant Prostate Cancer
Article Snippet: Both media were supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher, catalog number 10099141), 100 U/mL penicillin, and 100 μg/mL streptomycin (Beyotime Biotech, catalog number C0222, Shanghai, China), and cultures were incubated at 37○C in a 5% CO2 humidified atmosphere. .. The EphA2 expression plasmid (Sino Biological, catalog number: HG13926-CY, Beijing, China), control plasmid (Sino Biological, catalog number: CV011, Beijing, China) and recombinant human epidermal growth factor (EGF) (Sino Biological, catalog number: 10605-HNAE, Beijing, China) were procured from Sino Biological Inc. (Beijing, China). .. The study utilized the following antibodies: rabbit monoclonal antibody against EphA2 (CST, catalog number: 6997, Danvers, MA, USA), rabbit monoclonal antibody against CDH1 (Cell Signaling Technology, catalog number: 3195, Danvers, MA, USA), goat anti-mouse IgG-HRP secondary antibody (CST, catalog number: 7076, Danvers, MA, USA), and goat anti-rabbit IgG-HRP secondary antibody (CST, catalog number: 7074, Danvers, MA, USA).

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Fig. 2 Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 2 Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Article Snippet: To reconstitute EphA2 expression in EphA2 KO MSTO-211H cells, we used the retroviral plasmid pCLXSN-EphA2-Flag, a gift from Jin Chen (Addgene plasmid #102755, Addgene, Wartertown, MA, USA).

Techniques: Migration, Expressing, Clinical Proteomics, Injection

Fig. 4 FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A, B Parental and EphA2 KO MSTO-211H (A) and NCI-H2052 (B) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK (PTK2) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 4 FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A, B Parental and EphA2 KO MSTO-211H (A) and NCI-H2052 (B) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK (PTK2) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot, Transfection, Control

Fig. 7 Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 7 Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Incubation

Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Expressing, Western Blot

Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Techniques: Phospho-proteomics, Immunoprecipitation, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Control, Inhibition, Activity Assay

EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Techniques: Activity Assay, Expressing, Western Blot, Cytotoxicity Assay

EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy

Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Techniques: Activation Assay

(A, B) HeLa cells were pretreated with control or rhEphrin-A1 (5 μg/ml) for 1 h at 37°C and infected with Ctr (MOI-100) for 1 h at 4°C. Cells were fixed and immunostained for Ctr (Hsp60, green) and EphA2 (EphA2, red). (A) Images show EphA2 or EB or the co-localization of EB with EphA2 (yellow) on the cell surface. (B) Co-localization of EB with EphA2 was quantified from 8 different fields of view in 3 independent experiments. The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C, D) Purified EB was incubated with or without rhEphA2 (5 μg/ml) in a serum free RPMI media containing 0.1% BSA for 1 h at 37°C. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above. The mixture was centrifuged onto coverslips, washed 3 times with ice cold-PBS, stained and quantified as (B). The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05, **P<0.01. Error bars show mean ± SD. (E) Pull-down assay: Purified EB was incubated with rhEphA2 or with rhEphrin-A1 each 10 μg/ml in a serum free RPMI media containing 0.1% BSA for 45 min at 37°C in a rotary shaker. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above and the mixture was centrifuged at 5000 x g for 4 min at 4°C. The pellet was gently washed 3 times with ice cold-PBS. Ctr -infected cells act as a positive control for full length EphA2 expression. The samples were subjected to WB analysis to determine the full length endogenous EphA2 (~130 kDa) and rhEphA2 (~70 kDa) using N-terminal EphA2 specific antibody. rhEphrin-A1 was expressed at ~55–60 kDa. Hsp60 acts as a control for EB. (F) EB pre-incubated with rhPHB (PHB-His) (PHB is a mitochondrial inner membrane protein. rhPHB acts as a control for His-tagged rhEphA2) or with rhEphA2 (EphA2-His) each 5 μg/ml for 1 h at 4°C were added to HeLa cells for 1 h at 4°C. Cells were fixed and immunostained for extracellular EB and Actin filaments (Phalloidin). The number of extracellular EB was counted randomly from 40 different cells. Data are expressed as percentage of extracellular EB relative to control. Shown is the mean ± SD of three independent experiments normalized to control+EB. *P<0.05. Error bars show mean ± SD. (G) HeLa cells were incubated with antibody against N-terminal-EphA2 or-PDI or-EphB4 each 10 μg/ml for 1 h at 4°C. Cells were then infected with GFP-expressing Ctr for 1 h at 4°C. Cells were fixed and immunostained against Actin filaments. Graph was made similar to (F). Shown is the mean ± SD of two independent experiments normalized to IgG control. *P<0.05, **P<0.01, ***P<0.001. Error bars show mean ± SD. (A, C) Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A, B) HeLa cells were pretreated with control or rhEphrin-A1 (5 μg/ml) for 1 h at 37°C and infected with Ctr (MOI-100) for 1 h at 4°C. Cells were fixed and immunostained for Ctr (Hsp60, green) and EphA2 (EphA2, red). (A) Images show EphA2 or EB or the co-localization of EB with EphA2 (yellow) on the cell surface. (B) Co-localization of EB with EphA2 was quantified from 8 different fields of view in 3 independent experiments. The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C, D) Purified EB was incubated with or without rhEphA2 (5 μg/ml) in a serum free RPMI media containing 0.1% BSA for 1 h at 37°C. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above. The mixture was centrifuged onto coverslips, washed 3 times with ice cold-PBS, stained and quantified as (B). The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05, **P<0.01. Error bars show mean ± SD. (E) Pull-down assay: Purified EB was incubated with rhEphA2 or with rhEphrin-A1 each 10 μg/ml in a serum free RPMI media containing 0.1% BSA for 45 min at 37°C in a rotary shaker. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above and the mixture was centrifuged at 5000 x g for 4 min at 4°C. The pellet was gently washed 3 times with ice cold-PBS. Ctr -infected cells act as a positive control for full length EphA2 expression. The samples were subjected to WB analysis to determine the full length endogenous EphA2 (~130 kDa) and rhEphA2 (~70 kDa) using N-terminal EphA2 specific antibody. rhEphrin-A1 was expressed at ~55–60 kDa. Hsp60 acts as a control for EB. (F) EB pre-incubated with rhPHB (PHB-His) (PHB is a mitochondrial inner membrane protein. rhPHB acts as a control for His-tagged rhEphA2) or with rhEphA2 (EphA2-His) each 5 μg/ml for 1 h at 4°C were added to HeLa cells for 1 h at 4°C. Cells were fixed and immunostained for extracellular EB and Actin filaments (Phalloidin). The number of extracellular EB was counted randomly from 40 different cells. Data are expressed as percentage of extracellular EB relative to control. Shown is the mean ± SD of three independent experiments normalized to control+EB. *P<0.05. Error bars show mean ± SD. (G) HeLa cells were incubated with antibody against N-terminal-EphA2 or-PDI or-EphB4 each 10 μg/ml for 1 h at 4°C. Cells were then infected with GFP-expressing Ctr for 1 h at 4°C. Cells were fixed and immunostained against Actin filaments. Graph was made similar to (F). Shown is the mean ± SD of two independent experiments normalized to IgG control. *P<0.05, **P<0.01, ***P<0.001. Error bars show mean ± SD. (A, C) Magnification is indicated in size bar.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Control, Infection, Purification, Incubation, Staining, Pull Down Assay, Positive Control, Expressing, Membrane

(A) Adherence assay: HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. The transfected cells were infected with Ctr (MOI-100) for 1 h at 4°C followed by immunostaining Ctr -EB (Hsp60, green) and pEphA2 (phospho EphA2 Ser897, red). (B) Co-localization of EB with pEphA2 was quantified. The graph was made similar to . The data are expressed as a mean percentage of pEphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C) Invasion assay: The transfected cells were infected with Ctr (MOI-20-25) for 4–5 h at 35°C followed by immunostaining as (A) including Actin filaments. (A, C) Arrows indicate the co-localization of Ctr with pEphA2 (yellow). Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) Adherence assay: HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. The transfected cells were infected with Ctr (MOI-100) for 1 h at 4°C followed by immunostaining Ctr -EB (Hsp60, green) and pEphA2 (phospho EphA2 Ser897, red). (B) Co-localization of EB with pEphA2 was quantified. The graph was made similar to . The data are expressed as a mean percentage of pEphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C) Invasion assay: The transfected cells were infected with Ctr (MOI-20-25) for 4–5 h at 35°C followed by immunostaining as (A) including Actin filaments. (A, C) Arrows indicate the co-localization of Ctr with pEphA2 (yellow). Magnification is indicated in size bar.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Transfection, Luciferase, Infection, Immunostaining, Invasion Assay

(A) HeLa cells uninfected (UN) or infected with Ctr (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with Ctr (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells uninfected (UN) or infected with Ctr (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with Ctr (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Control, Immunoprecipitation, Staining

(A) HeLa cells were infected with Ctr- EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as . (B) HeLa cells were infected with Ctr- EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or Ctr -infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course Ctr- infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with Ctr -pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells were infected with Ctr- EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as . (B) HeLa cells were infected with Ctr- EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or Ctr -infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course Ctr- infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with Ctr -pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Homogenization, Clinical Proteomics, Membrane, Isolation, Activation Assay, Control, Marker, Transfection, Staining

(A) Infectivity assay performed for B) C) D) E) F): The transfected cells infected with Ctr (MOI-1) for 24 h at 35°C was referred as primary infection. The supernatant of the primary infected cells was taken to infect the fresh cells to determine the secondary infection (see -methods). (B) HeLa cells were left untransfected (control) or transfected with empty pcDNA3 or EphA2-pcDNA3 each 1 μg/ml and then left UN or infected with Ctr for 24 h. Cells were harvested for WB analysis. (C) HeLa cells were untransfected (control) or transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C and then left UN or infected with Ctr for another 24 h. Cells were harvested for WB analysis. (D, E) Primary infected cell lysates of (B) and (C) were taken to infect the fresh cells for secondary infection (Infectivity assay). (F) Number of the inclusion per cell (%) for the infectivity assay was determined by counting the inclusion on 10 independent fields. Shown is the mean ± SD of three independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. (G) Cells were transfected with siRNA against luciferase (siLuci) or EphA2 (siEphA2) or PDGFRβ (siPDGFRβ) or both together for 40 h at 37°C and then infected with Ctr . UN: uninfected cells. Cells were harvested to determine the respective proteins by WB analysis. (H) Size of the inclusion per cell (%) for (F) was determined by ImageJ software by measuring cells out of four microscopic fields. Shown is the mean ± SD of two independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. In all of the above experiments, numbers under the blot represents fold activation for phospho specific proteins with respect to total proteins and fold change for the total proteins.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) Infectivity assay performed for B) C) D) E) F): The transfected cells infected with Ctr (MOI-1) for 24 h at 35°C was referred as primary infection. The supernatant of the primary infected cells was taken to infect the fresh cells to determine the secondary infection (see -methods). (B) HeLa cells were left untransfected (control) or transfected with empty pcDNA3 or EphA2-pcDNA3 each 1 μg/ml and then left UN or infected with Ctr for 24 h. Cells were harvested for WB analysis. (C) HeLa cells were untransfected (control) or transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C and then left UN or infected with Ctr for another 24 h. Cells were harvested for WB analysis. (D, E) Primary infected cell lysates of (B) and (C) were taken to infect the fresh cells for secondary infection (Infectivity assay). (F) Number of the inclusion per cell (%) for the infectivity assay was determined by counting the inclusion on 10 independent fields. Shown is the mean ± SD of three independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. (G) Cells were transfected with siRNA against luciferase (siLuci) or EphA2 (siEphA2) or PDGFRβ (siPDGFRβ) or both together for 40 h at 37°C and then infected with Ctr . UN: uninfected cells. Cells were harvested to determine the respective proteins by WB analysis. (H) Size of the inclusion per cell (%) for (F) was determined by ImageJ software by measuring cells out of four microscopic fields. Shown is the mean ± SD of two independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. In all of the above experiments, numbers under the blot represents fold activation for phospho specific proteins with respect to total proteins and fold change for the total proteins.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Transfection, Control, Luciferase, Software, Activation Assay

(A) HeLa cells were left UN or infected with Ctr (MOI-2) for the indicated period of times and immunoprecipitated using α-EphA2 antibody. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%). (B) Cells were transfected with EphA2-pcDNA3 and p85-PI3K expression plasmid together for 15 h followed by Ctr infection for 24 h. Cells were stained against EphA2 (red), DNA (blue) and pPI3K (green). (C) Schematic representation of full length EphA2 plasmid, EphA2 kinase dead mutant plasmid (EphA2K645R) and EphA2 mutant plasmid missing the entire cytoplasmic domain (EphA2ΔIC). TM: transmembrane domain. (D) The plasmids having full length EphA2 or EphA2ΔIC were transfected and these cells were infected with Ctr for 20 h. Cells were fixed and stained against α-EphA2 antibody (red) and Ctr using α-Hsp60 (green). (E) Arrows were marked to illustrate the difference between the size of the inclusion of untransfected (white arrows) and transfected (red arrows) cells. (F) Size of the inclusion for (E) was determined by ImageJ software. Empty-plasmid transfected cells act as a control. Shown is the mean ± SD of two independent experiments normalized to empty-plasmid transfected cells. *P<0.05, **P<0.01. Error bars show mean ± SD. (G) Cells after transfection of the constructs (each 1.5 μg/ml) indicated above the lanes followed by infection (MOI-1) for 18 h were subjected to WB analysis to analyze the proteins as indicated. EphA2ΔIC was detected by N-terminal EphA2 antibody. Due to the high intensity of total EphA2 and phospho EphA2 from the EphA2-transfected infected cells, the upregulation of endogenous EphA2 cannot be visualized. (B, D, F) Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells were left UN or infected with Ctr (MOI-2) for the indicated period of times and immunoprecipitated using α-EphA2 antibody. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%). (B) Cells were transfected with EphA2-pcDNA3 and p85-PI3K expression plasmid together for 15 h followed by Ctr infection for 24 h. Cells were stained against EphA2 (red), DNA (blue) and pPI3K (green). (C) Schematic representation of full length EphA2 plasmid, EphA2 kinase dead mutant plasmid (EphA2K645R) and EphA2 mutant plasmid missing the entire cytoplasmic domain (EphA2ΔIC). TM: transmembrane domain. (D) The plasmids having full length EphA2 or EphA2ΔIC were transfected and these cells were infected with Ctr for 20 h. Cells were fixed and stained against α-EphA2 antibody (red) and Ctr using α-Hsp60 (green). (E) Arrows were marked to illustrate the difference between the size of the inclusion of untransfected (white arrows) and transfected (red arrows) cells. (F) Size of the inclusion for (E) was determined by ImageJ software. Empty-plasmid transfected cells act as a control. Shown is the mean ± SD of two independent experiments normalized to empty-plasmid transfected cells. *P<0.05, **P<0.01. Error bars show mean ± SD. (G) Cells after transfection of the constructs (each 1.5 μg/ml) indicated above the lanes followed by infection (MOI-1) for 18 h were subjected to WB analysis to analyze the proteins as indicated. EphA2ΔIC was detected by N-terminal EphA2 antibody. Due to the high intensity of total EphA2 and phospho EphA2 from the EphA2-transfected infected cells, the upregulation of endogenous EphA2 cannot be visualized. (B, D, F) Magnification is indicated in size bar.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Staining, Mutagenesis, Software, Control, Construct

(A) HUVEC cells were left uninfected or infected with Ctr (MOI-1) for 14 h followed by treatment with DMSO or PI3K inhibitor LY294002 (15 μM) or MAPK inhibitor UO126 (15 μM) for 10 h. After 24 h of total infection, cells were subjected to WB analysis against respective proteins. (B) EphA2 plasmid-transfected HeLa cells were left uninfected or infected with Ctr for 24 h as mentioned above the lanes. The cells were treated with DMSO or UO126 (30 μM) for 3 h or 6 h as described above the lanes and subjected to WB analysis to determine the respective proteins as indicated.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HUVEC cells were left uninfected or infected with Ctr (MOI-1) for 14 h followed by treatment with DMSO or PI3K inhibitor LY294002 (15 μM) or MAPK inhibitor UO126 (15 μM) for 10 h. After 24 h of total infection, cells were subjected to WB analysis against respective proteins. (B) EphA2 plasmid-transfected HeLa cells were left uninfected or infected with Ctr for 24 h as mentioned above the lanes. The cells were treated with DMSO or UO126 (30 μM) for 3 h or 6 h as described above the lanes and subjected to WB analysis to determine the respective proteins as indicated.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Plasmid Preparation, Transfection

(A) The transfection efficiency of siRNA directed against EphA2 was monitored by WB analysis against total EphA2. In addition, levels of pAkt, IncA and Actin were verified. (B) siLuci or siEphA2-transfected cells were left uninfected or infected with Ctr for 16 h. Cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis. Rectangle boxes (black: before infection) or (red: after infection) denotes the difference in PARP cleavage after TNF-α induction in siLuci and siEphA2 transfected cells. (C) For quantification, TUNEL positive cells from each sample were counted from ten different fields. Shown is the mean ± SD of two independent experiments. **P<0.01, *P<0.05, ns: non-significant. Error bars show mean ± SD.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) The transfection efficiency of siRNA directed against EphA2 was monitored by WB analysis against total EphA2. In addition, levels of pAkt, IncA and Actin were verified. (B) siLuci or siEphA2-transfected cells were left uninfected or infected with Ctr for 16 h. Cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis. Rectangle boxes (black: before infection) or (red: after infection) denotes the difference in PARP cleavage after TNF-α induction in siLuci and siEphA2 transfected cells. (C) For quantification, TUNEL positive cells from each sample were counted from ten different fields. Shown is the mean ± SD of two independent experiments. **P<0.01, *P<0.05, ns: non-significant. Error bars show mean ± SD.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Transfection, Infection, TUNEL Assay

(A) HeLa cells left UN or infected with Ctr -serovar D were centrifuged at 910 x g for 30 min and allowed to infect for the indicated time points. The cells were harvested and subjected to WB analysis. (B) HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. Adherence assay: The transfected cells were infected with Ctr- serovar D for 1 h at 4°C. Cells were immunostained against EB and Actin filaments. Number of extracellular EB was counted randomly from 30 different cells. Data are expressed as percentage of extracellular EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. Invasion assay: The transfected cells were infected with Ctr- serovar D for 4–5 h at 35°C. Cells were immunostained against EB and Actin filaments. Number of invaded EB was counted randomly from 30 different cells. Data are expressed as percentage of invaded EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.001. Error bars show mean ± SD. (C) FACS analysis was performed with Ctr -serovar D infected cells as . (UN: uninfected, Iso: isotype and perm: permeabilised). (D) Results of C were shown as bar charts. Shown is the mean ± SD of two independent experiments normalized to UN. ***P<0.001, *P<0.05, ns: non-significant. Error bars show mean ± SD. (E) HeLa cells were left UN or infected with Ctr- serovar D (MOI-1) for 14, 20 and 28 h and cells were analyzed via FACS as . (F) EphA2 knockdown followed by 16 h Ctr -serovar D infected cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells left UN or infected with Ctr -serovar D were centrifuged at 910 x g for 30 min and allowed to infect for the indicated time points. The cells were harvested and subjected to WB analysis. (B) HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. Adherence assay: The transfected cells were infected with Ctr- serovar D for 1 h at 4°C. Cells were immunostained against EB and Actin filaments. Number of extracellular EB was counted randomly from 30 different cells. Data are expressed as percentage of extracellular EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. Invasion assay: The transfected cells were infected with Ctr- serovar D for 4–5 h at 35°C. Cells were immunostained against EB and Actin filaments. Number of invaded EB was counted randomly from 30 different cells. Data are expressed as percentage of invaded EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.001. Error bars show mean ± SD. (C) FACS analysis was performed with Ctr -serovar D infected cells as . (UN: uninfected, Iso: isotype and perm: permeabilised). (D) Results of C were shown as bar charts. Shown is the mean ± SD of two independent experiments normalized to UN. ***P<0.001, *P<0.05, ns: non-significant. Error bars show mean ± SD. (E) HeLa cells were left UN or infected with Ctr- serovar D (MOI-1) for 14, 20 and 28 h and cells were analyzed via FACS as . (F) EphA2 knockdown followed by 16 h Ctr -serovar D infected cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Infection, Transfection, Luciferase, Invasion Assay, Knockdown

(1) Ctr -EB bind and activate EphA2 and enter the cell together with the activated receptor and PI3K recruited to EphA2. (2) Ctr -mediated ERK activation induces EphA2 upregulation. (3) Upregulated EphA2 does not appear at the cell surface. (4) Intracellular EphA2 is associated with the Ctr inclusion which interacts with pPI3K. (5, 6) EphA2-induced signaling cascades (pAkt) are required to inhibit apoptosis and enhance the infection.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (1) Ctr -EB bind and activate EphA2 and enter the cell together with the activated receptor and PI3K recruited to EphA2. (2) Ctr -mediated ERK activation induces EphA2 upregulation. (3) Upregulated EphA2 does not appear at the cell surface. (4) Intracellular EphA2 is associated with the Ctr inclusion which interacts with pPI3K. (5, 6) EphA2-induced signaling cascades (pAkt) are required to inhibit apoptosis and enhance the infection.

Article Snippet: An expression plasmid for EphA2 (accession NP_004422.2) was generated by PCR amplification using cDNA of pDONR223-EphA2 (Addgene ID:23926) [ ].

Techniques: Activation Assay, Infection

Effects of EphA2 on paclitaxel sensitivity of nasopharyngeal carcinoma 5-8F cells in vitro . (A) Western blot analysis was conducted to confirm the efficiency of EphA2 over-expression. (B) Proliferation assay curves of each group. EphA2, ephrin type-A receptor 2.

Journal: Molecular Medicine Reports

Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

doi: 10.3892/mmr.2014.2799

Figure Lengend Snippet: Effects of EphA2 on paclitaxel sensitivity of nasopharyngeal carcinoma 5-8F cells in vitro . (A) Western blot analysis was conducted to confirm the efficiency of EphA2 over-expression. (B) Proliferation assay curves of each group. EphA2, ephrin type-A receptor 2.

Article Snippet: The EphA2 cDNA-pEGFP-N1 expression plasmid and pEGFP-N1 vector plasmid were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: In Vitro, Western Blot, Over Expression, Proliferation Assay

Effect of EphA2 over-expression on cell-cycle distribution and apoptosis following exposure to paclitaxel. (A) Graphs of cell cycle distribution in each group. (B) Percentages of cells in different cell cycle phases in each group. (C) Graphs showing apoptosis in each group. (D) Percentage of apoptotic cells in each group. Results are presented as the mean ± standard deviation of at least three independent experiments. * P<0.05. FITC, fluorescein isothiocyanate; EphA2, ephrin type-A receptor 2.

Journal: Molecular Medicine Reports

Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

doi: 10.3892/mmr.2014.2799

Figure Lengend Snippet: Effect of EphA2 over-expression on cell-cycle distribution and apoptosis following exposure to paclitaxel. (A) Graphs of cell cycle distribution in each group. (B) Percentages of cells in different cell cycle phases in each group. (C) Graphs showing apoptosis in each group. (D) Percentage of apoptotic cells in each group. Results are presented as the mean ± standard deviation of at least three independent experiments. * P<0.05. FITC, fluorescein isothiocyanate; EphA2, ephrin type-A receptor 2.

Article Snippet: The EphA2 cDNA-pEGFP-N1 expression plasmid and pEGFP-N1 vector plasmid were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Standard Deviation

Effect of EphA2 over-expression on cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, in NPC 5-8F cells. Western blot analysis was used to detect the expression of p21Cip1, p27Kip1 CDK2, Cyclin E and p-Rb in NPC 5-8F and CNE-2 nasopharyngeal carcinoma cells. All data were obtained by three independent experiments, which produced similar results. EphA2, ephrin type-A receptor 2; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb; NPC, nasopharyngeal carcinoma.

Journal: Molecular Medicine Reports

Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

doi: 10.3892/mmr.2014.2799

Figure Lengend Snippet: Effect of EphA2 over-expression on cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, in NPC 5-8F cells. Western blot analysis was used to detect the expression of p21Cip1, p27Kip1 CDK2, Cyclin E and p-Rb in NPC 5-8F and CNE-2 nasopharyngeal carcinoma cells. All data were obtained by three independent experiments, which produced similar results. EphA2, ephrin type-A receptor 2; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb; NPC, nasopharyngeal carcinoma.

Article Snippet: The EphA2 cDNA-pEGFP-N1 expression plasmid and pEGFP-N1 vector plasmid were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Western Blot, Expressing, Produced

Effect of paclitaxel stimulation and EphA2 overexpression on activation of the PI3K/Akt signalling pathway in NPC 5-8F cells. (A) NPC 5-8F cells were treated with varying concentrations of paclitaxel and PI3K/Akt pathway signalling molecules (total Akt, p-Akt) were measured by western blot analysis. (B) EphA2 over-expression stimulates the PI3K/Akt pathway. The total quantity Akt, p-Akt, GSK-3β and p-GSK-3β was determined by western blot analysis. EphA2, ephrin type-A receptor 2; PI3K, phosphoinositide 3-kinase; p-Akt, phosphorylated Akt; GSK-3β, glycogen synthase kinase-3β; p-GSK-3β, phosphorylated-GSK-3β.

Journal: Molecular Medicine Reports

Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

doi: 10.3892/mmr.2014.2799

Figure Lengend Snippet: Effect of paclitaxel stimulation and EphA2 overexpression on activation of the PI3K/Akt signalling pathway in NPC 5-8F cells. (A) NPC 5-8F cells were treated with varying concentrations of paclitaxel and PI3K/Akt pathway signalling molecules (total Akt, p-Akt) were measured by western blot analysis. (B) EphA2 over-expression stimulates the PI3K/Akt pathway. The total quantity Akt, p-Akt, GSK-3β and p-GSK-3β was determined by western blot analysis. EphA2, ephrin type-A receptor 2; PI3K, phosphoinositide 3-kinase; p-Akt, phosphorylated Akt; GSK-3β, glycogen synthase kinase-3β; p-GSK-3β, phosphorylated-GSK-3β.

Article Snippet: The EphA2 cDNA-pEGFP-N1 expression plasmid and pEGFP-N1 vector plasmid were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Activation Assay, Western Blot

EphA2 mediated paclitaxel sensitivity in NPC 5–8 cells via modulation of the PI3K/Akt signalling pathway. (A) PI3K/Akt signalling pathway inhibitor, LY294002, reverses paclitaxel resistance caused by EphA2 over-expression. (B) Effect of LY294002 on the cell-cycle distribution in EphA2 over-expressing NPC cells pre-treated with paclitaxel. (C) Effect of LY294002 on the apoptotic rate in NPC cells over-expressing EphA2, pretreated with paclitaxel. (D) LY294002 restores the changes in expression of cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, caused by EphA2 over-expression. * P<0.05. EphA2, ephrin type-A receptor 2; NPC, nasopharyngeal carcinoma; PI3K, phosphoinositide 3-kinase; p-Akt, phsophorylated Akt; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb.

Journal: Molecular Medicine Reports

Article Title: Ephrin type-A receptor 2 regulates sensitivity to paclitaxel in nasopharyngeal carcinoma via the phosphoinositide 3-kinase/Akt signalling pathway

doi: 10.3892/mmr.2014.2799

Figure Lengend Snippet: EphA2 mediated paclitaxel sensitivity in NPC 5–8 cells via modulation of the PI3K/Akt signalling pathway. (A) PI3K/Akt signalling pathway inhibitor, LY294002, reverses paclitaxel resistance caused by EphA2 over-expression. (B) Effect of LY294002 on the cell-cycle distribution in EphA2 over-expressing NPC cells pre-treated with paclitaxel. (C) Effect of LY294002 on the apoptotic rate in NPC cells over-expressing EphA2, pretreated with paclitaxel. (D) LY294002 restores the changes in expression of cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, caused by EphA2 over-expression. * P<0.05. EphA2, ephrin type-A receptor 2; NPC, nasopharyngeal carcinoma; PI3K, phosphoinositide 3-kinase; p-Akt, phsophorylated Akt; Rb, retinoblastoma protein; p-Rb, phosphorylated Rb.

Article Snippet: The EphA2 cDNA-pEGFP-N1 expression plasmid and pEGFP-N1 vector plasmid were obtained from GeneChem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Expressing

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