etv7  (Developmental Studies Hybridoma Bank)

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: The PNT domain of ETV7 contributes to the assembly of mTORC3. ( A ). Left panel; rapamycin response curve of Karpas-299, KE7 - , and KE7 - -ETV7 M1-E144 cells treated with an escalating dose of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/mL) for three days or three population doublings. Cell densities (percent control) as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Top right; schematic representation of the flag-tagged ETV7 M1-E144 fragment with numbers indicating the number of amino acids in the different segments of the protein. Underneath the drawing is the immunoblot of the lysate of KE7 - -ETV7 M1-E144 cells immunoprecipitated with anti-mTOR or anti-FLAG antibodies and non-relevant IgG, probed for mTOR and FLAG. ( B ). Left panel: rapamycin response curves of Karpas-299, KE7 - , and KE7 - -zETV7 cells treated with an increasing dose of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/mL) for three days or three population doublings. Cell densities (percent control) as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Right panel: immunoblot of KE7 - -zETV7 cell lysate immunoprecipitated with anti-mTOR or anti-FLAG antibodies and non-relevant IgG, probed for mTOR and FLAG. ( C ). Left panel; rapamycin response curves of Karpas-299, KE7 - , and KE7 - -ETV7 Ex9 cells treated with an escalating dose of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/mL) for three days or three population doublings. Cell densities (percent control) as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Right panel: Immunoblot of KE7 - -ETV7-Ex9 cell lysate immunoprecipitated with anti-mTOR or anti-ETV7 antibodies and non-relevant IgG, probed for mTOR and ETV7. ( D ). Top, amino acid sequence of the ETV7 PNT domain with ML and EH sequences highlighted in red and yellow, respectively. Alanine mutations (green) are indicated below the sequence. Bottom left panel: rapamycin response curves of Karpas-299, KE7 - , KE7 - -M82A, KE7 - -I89A, KE7 - -V105A, and KE7 - -L109A cells treated with an increasing dose of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/mL) for three days or three population doublings. Cell densities (percent control) as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. Bottom right panel: immunoblot of lysates of KE7 - -M82A, KE7 - -I89A, KE7 - -V105A, and KE7 - -L109A cells immunoprecipitated with anti-mTOR or anti-ETV7 antibodies and nonrelevant IgG, probed for mTOR and ETV7.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: Control, Western Blot, Immunoprecipitation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: Alanine substitutions in the ETV7 ETS domain identify amino acids involved in the assembly of mTORC3. ( A ). The 3D model derived from the ETV6 ETS domain of the ETV7 ETS domain bound to DNA, showing three hydrophobic patches exposed to solvent (Patch 1, Patch 2, Patch 3) (Y239/Y242/L264; Y231/L235; W227/L289/I291/L305), which can potentially participate in ETS-mediated protein–protein interactions. ( B ). Summary of single alanine substitutions in the ETS domain of ETV7 clustering in three patches (P1, P2, P3) of solvent-exposed hydrophobic amino acids. Cells expressing the ETS mutants indicated in yellow highlighted the amino acid residues mutated, green are sensitive to rapamycin treatment, and those in red are resistant to rapamycin treatment. ( C ). Left panel: rapamycin response curves of Karpas-299, KE7 - , and KE7 - -ETV7 cells expressing the ETV7 mutants W227A, Y231A, L235A, Y239A, Y242A, L264A, L289A, I291A, L305A treated with an escalating dose of rapamycin (0.1, 0.3, 1.3, 10, 30, 100, 300, and 1000 ng/mL) for three days or three population doublings. Cell densities (percent control) as the percentage of cells treated with vehicle. Data are means ± SEM from three independent experiments. The SEM is within 5% but cannot be added to the figure due to the density of the plots. Right panel: Immunoblots of lysates of KE7 - -ETV7 - -W227A, Y231A, L235A, Y239A, Y242A, L264A, L289A, I291A, L305A cells immunoprecipitated with anti-mTOR or anti-ETV7 antibodies and non-relevant IgG, probed for mTOR and ETV7. The blots boxed in red show the rapamycin-resistant mutants.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: Derivative Assay, Solvent, Expressing, Control, Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: ETV7 binds to the FRB domain of mTOR. ( A ). Immunoblot of the coimmunoprecipitated mTOR kinase domain (amino acids 1363-2549) and ETV7 from Karpas-299 cells expressing the FLAG–mTOR kinase domain with anti-FLAG antibody, probed for FLAG and ETV7. ( B ). Immunoblot of in vitro co-IP of purified ETV7 and mTOR protein in the presence of an increasing amount of FKBP12–rapamycin (mTOR:ETV7:FKBP12/rapamycin = 1:1:0, 1:1:10, and 1:1:100), probed for mTOR and ETV7. ( C ). Immunoblot of ETV7 IP of ETV7 co-incubated with N-terminal and C-terminal deletions of the FRB-new fragment in vitro. Underneath is a schematic representation of serial N-terminal and C-terminal deletions of the FRB-new fragment. Numbers indicate the number of amino acids (aa) in the different segments of the FRB-new fragment. ( D ). FLAG/ETV7 immunoblot of ETV7 IPs of serially deleted Ndel-FRB protein fragments after association with ETV7 in vitro. The lane with the smallest fragment still binding ETV7, N27C61-Ndel-FRB, is boxed in red. The asterisks next to the FRB bands in the input blot indicate the full-length size of each fragment. Underneath is a schematic representation of serial N-terminal and C-terminal deletions of the Ndel-FRB fragment.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: Western Blot, Expressing, In Vitro, Co-Immunoprecipitation Assay, Purification, Incubation, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: In vitro binding of the LBE sequence of the mTOR kinase domain to ETV7. ( A ). Top drawing: schematic representation of the N- and C-terminally extended LBE (highlighted in blue)fragment. The numbers indicate the number of amino acids in each of the LBE segments. Below is a FLAG immunoblot of an IP of the 120aa LBE fragment after overnight binding to ETV7 in vitro. Control (CTRL) shows the binding of human RUVBL2 to ETV7. ( B ). Left panel, schematic representation of the different deletions of the N- and C-terminal extended LBE fragment (120 amino acids) and underneath the amino acid sequence of the LBE domain with the amino acids essential for ETV7 binding in red; Right, FLAG immunoblot of an ETV7 IP of the different LBE fragments after overnight binding to ETV7 in vitro. Lanes containing fragments that lost binding to ETV7 are boxed in red. ( C ). FRB and LBE bind to ETV7 simultaneously. FLAG immunoblot of an ETV7 IP of LBE (120 amino acids) and Ndel-FRB fragments (ETV7:LBE:Ndel-FRB = 1:1:1, 1:1:5, 1:5:1) after overnight binding to ETV7 in vitro. ( D ). Binding of Ndel-FRB to ETV7 fragments containing the PNT domain and binding of LBE (120 amino acids) to ETV7 fragments containing the PNT and ETS domains in vitro.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: In Vitro, Binding Assay, Sequencing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: Cross-linking and mass spec identification of ETV7–FRB binding. ( A ). Optimization of cross-linking conditions. From left to right, control showing binding of ETV7 and the FRB-new fragment without cross-linker; binding of ETV7 and FRB-new after treatment with 50 mM lysine crosslinker; ETV7 and FRB-new binding after treatment with 50 mM arginine cross-linker; ETV7 and FRB-new binding after treatment with 500 mM arginine cross-linker. ( B ). Cross-linked samples stained with Coomassie Brilliant Blue. From left to right: marker; control binding of ETV7 and FRB-new without cross-linker; blank; band cut out from the lysine cross-linked material in ( A ); blank (Band 2); band cut out from the material in ( A ) (boxed in red) treated with 500 mM arginine cross-linker (Band 1); blank; marker. ( C ). Diagram of the relative location of cross-linked sites in ETV7 and FRB-new. The central pink color represents the 95 aa FRB domain, while the yellow represents the kinase domain. The FRB-new fragment includes amino acids E 1921 —L 2220 of mTOR. The blue and yellow color in ETV7 represent the PNT and ETS domains, respectively.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: Mass Spectrometry, Binding Assay, Control, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: Mapping of arginine cross-linked sites in the ETV7 PNT domain and the mTOR FRB domain.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: Ndel-FRB competes with ETV7 for mTOR binding and converts Karpas-299 cells from resistant to sensitive to rapamycin. ( A ). Ndel-FRB competes with ETV7 for in vitro binding of mTOR. ETV7 and mTOR were incubated overnight at 4 °C in the presence or absence of Ndel-FRB and/or 120 aa-LBE in vitro. ETV7 Ips were immunoblotted for mTOR, ETV7, and Flag (Ndel-FRB, and 120 aa-LBE). ( B ). Karpas-299 cells that express Ndel-FRB but not ND72C61 become sensitive to rapamycin. Proliferating KE7 - , Karpas-299, and 2 different Karpas-299 Ndel-FRB cell pools [Karpas transduced once with Ndel-FRB lentivirus (Karpas 1 × Ndel-FRB) and Karpas transduced twice with Ndel-FRB lentivirus (Karpas 2 × Ndel-FRB)] and a Karpas-299 ND72C61 cell pool (Karpas-299 transduced once with ND72C61 lentivirus), as well as 2 different KE7 - Ndel-FRB cell pools [KE7 - transduced once with Ndel-FRB lentivirus (KE7 - 1 × Ndel-FRB) and KE7 - transduced twice with Ndel-FRB lentivirus (KE7 - 2 × Ndel-FRB)] were treated with increasing amounts of rapamycin for three days or three population doublings. Data are means ± SEM from three independent experiments. The SEM is within 5% but is omitted in the figure due to the density of the plots. ( C ). Cell lysates of Karpas-299, K-E7 - , Karpas-299–Ndel-FRB, K-E7 - –Ndel-FRB, and Karpas-299–ND72C61 cells treated with increasing amounts of rapamycin (0, 0.1, 1, 3, 10, 100, 1000 ng/mL), immunoblotted for p-P70S6KThr389, total p70S6K. ( D ). Immunoblots of Co-IPs of ETV7 and mTOR of lysates of Karpas-299, K-E7 - , Karpas-299–Ndel-FRB, and Karpas-299–ND72C61 cells probed for ETV7, and mTOR. ( E ). Immunoblot showing the presence of Ndel-FRB and ND71C61 in lysates of equal numbers of KE7 - –Ndel-FRB, Karpas-299–Ndel-FRB, and Karpas-299–ND72C61 cells.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: Binding Assay, In Vitro, Incubation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Exosomal miR-361-3p promotes the viability of breast cancer cells by targeting ETV7 and BATF2 to upregulate the PAI-1/ERK pathway

doi: 10.1186/s12967-024-04914-4

Figure Lengend Snippet: Elevated plasma exosomal miR-361-3p promotes the malignant progression of BC in mice. A Upper panel : A schematic diagram of the animal experiments. Lower panel : A T47D cell xenograft model in female BALB/c nude mice was established. Mice exhibiting a high abundance of plasma exosomal miR-361-3p, as well as control mice, were obtained through the tail vein injection of EXO-miR-361-3p or EXO-miR-NC, respectively. B Differences in volume, weight and growth rate of murine tumors in the EXO-miR-361-3p group compared to the control group. C H&E staining of murine tumor tissues (×40). D – F Compare the expression levels of miR-361-3p, ETV7 , BATF2 , and the PAI-1/ERK pathway in excised tumors of the two groups of mice. G Proposed model of exosomal miR-361-3p targeting ETVT and BATF2 to upregulate the PAI-1/ERK pathway, leading to increased viability in BC cells. (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Human ETVT cDNA plasmid ( ETV7 plasmid), BATF2 cDNA plasmid ( BATF2 plasmid), PAI-1 cDNA plasmid ( PAI-1 plasmid) and negative control plasmid (control plasmid) were transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Clinical Proteomics, Control, Injection, Staining, Expressing

Journal: Journal of Translational Medicine

Article Title: Exosomal miR-361-3p promotes the viability of breast cancer cells by targeting ETV7 and BATF2 to upregulate the PAI-1/ERK pathway

doi: 10.1186/s12967-024-04914-4

Figure Lengend Snippet: ETV7 and BATF2 are two new targets of miR-361-3p. A The GO analysis revealed distinct pathways enriched by genes that are either downregulated or upregulated in BC cells overexpressing miR-361-3p. B Intersection of our RNA-seq results (displayed as a heatmap) and miRNA target prediction algorithms from 2 databases. C – D miR-361-3p putative targeting sites in the wild-type and mutant ETV7 3'UTR and BATF2 3'UTR, and luciferase activity assays were performed to confirm the direct binding efficiency of miR-361-3p and its targets ETV7 and BATF2. E – F Relative protein expression of ETV7 and BATF2 after coculturing BC cells with EXO-miR-361-3p/EXO-miR-NC. (* P < 0.05)

Article Snippet: Human ETVT cDNA plasmid ( ETV7 plasmid), BATF2 cDNA plasmid ( BATF2 plasmid), PAI-1 cDNA plasmid ( PAI-1 plasmid) and negative control plasmid (control plasmid) were transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: RNA Sequencing, Mutagenesis, Luciferase, Activity Assay, Binding Assay, Expressing

Journal: Journal of Translational Medicine

Article Title: Exosomal miR-361-3p promotes the viability of breast cancer cells by targeting ETV7 and BATF2 to upregulate the PAI-1/ERK pathway

doi: 10.1186/s12967-024-04914-4

Figure Lengend Snippet: ETV7 and BATF2 negatively regulate the PAI-1/ERK pathway as well as the proliferation and migration of BC cells. A Relative protein expression of PAI-1, p-ERK and T-ERK after coculturing BC cells with EXO-miR-361-3p/EXO-miR-NC. B , F Relative expression of ETV7, BATF2, PAI-1 and p-ERK/T-ERK protein after ETV7 and/or BATF2 knockdown or overexpression (* P < 0.05, ** P < 0.01, *** P < 0.001). CellTiter-Glo assays ( C , G ), wound healing assays ( D , H ) and Transwell assays ( E , I ) were performed to test the effect of ETV7 and/or BATF2 knockdown or overexpression on BC cell proliferation and metastasis. Scale bars, 100 μm

Article Snippet: Human ETVT cDNA plasmid ( ETV7 plasmid), BATF2 cDNA plasmid ( BATF2 plasmid), PAI-1 cDNA plasmid ( PAI-1 plasmid) and negative control plasmid (control plasmid) were transfected into cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Migration, Expressing, Knockdown, Over Expression