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, Protein-Protein interactions, 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: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: ETV7 was highly expressed in CRC. A CRC tissues ( n = 20) and paracancerous ( n = 20) tissues were subjected to immunochemistry staining of ETV7. B ETV7 expression in CRC ( n = 275) and control samples ( n = 349) based on the information from TCGA database. C Methylation level of the ETV7 promoter in CRC ( n = 313) and control samples ( n = 37) based on the information from TCGA database. D The protein expression of ETV7 was checked in two types of CRC cells (RKO and HCT116) and normal colorectal cells (NCM460)

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Staining, Expressing, Control, Methylation

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: Correlation of ETV7 expression and clinical features in patients with CRC

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Expressing

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: ETV7 promoted the amplification and migration of CRC cells. A The protein expression of ETV7 was checked in RKO cells transfected with pcDNA empty vector (vector) or pcDNA-ETV7 (ETV7). B Cell viability was measured in RKO cells transfected with pcDNA empty vector (vector) or pcDNA-ETV7 (ETV7). n = 3. C, D Cell colony formation and migration ability were examined in RKO cells transfected with pcDNA empty vector (vector) or pcDNA-ETV7 (ETV7). n = 3. E The protein expression of ETV7 was checked in siCtrl, siETV7#1, and siETV7#2 HCT116 cells. F Cell viability was measured in siCtrl, siETV7#1, and siETV7#2 HCT116 cells. n = 3. G, H Cell colony formation and migration ability were examined n siCtrl, siETV7#1, and siETV7#2 HCT116 cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Amplification, Migration, Expressing, Transfection, Plasmid Preparation

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: ETV7 reduced apoptosis and cell cycle arrest in colorectal cancer cells. A Rate of apoptosis in RKO cells transfected with pcDNA empty vector (vector) or pcDNA-ETV7 (ETV7). n = 3. B Rate of apoptosis in siCtrl, siETV7#1, and siETV7#2 HCT116 cells. n = 3. C Cell cycle distribution of RKO cells transfected with pcDNA empty vector (vector) or pcDNA-ETV7 (ETV7). n = 3. D Cell cycle distribution of siCtrl, siETV7#1, and siETV7#2 HCT116 cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Transfection, Plasmid Preparation

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: ETV7 positively regulated the expression of IFIT3. A–C The qPCR ( n = 3) and WB analyses detected IFIT3 expression in RKO cells transfected with pcDNA-ETV7 and HCT16 cells transfected with siETV7. D, E IFIT3 promoter luciferase activity was detected in RKO and HCT16 cells 24 h following transfection with pcDNA-ETV7 or siRNAs targeting ETV7. n = 3. F Pearson’s correlation of ETV7 and IFIT3 expression using data from TCGA database. *** p < 0.001

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Expressing, Transfection, Luciferase, Activity Assay

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: IFIT3 knockdown inhibited the cellular function in ETV7-overexpressing cells. A The protein expression of IFIT3 in RKO cells transfected with pcDNA-ETV7 and pcDNA-ETV7 + siIFIT3. B Viability of RKO cells transfected with pcDNA-ETV7 and pcDNA-ETV7 + siIFIT3. n = 3. C Colony numbers of RKO cells transfected with pcDNA-ETV7 and pcDNA-ETV7 + siIFIT3. n = 3. D Numbers of migrated RKO cells transfected with pcDNA-ETV7 and pcDNA-ETV7 + siIFIT3. n = 3. ** p < 0.01; *** p < 0.001

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Knockdown, Cell Function Assay, Expressing, Transfection

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: IFIT3 overexpression rescued the cellular function in ETV7-silenced colorectal cancer cells. A IFIT3 expression in HCT116 cells transfected with siETV7 and siETV7 + pcDNA-IFIT3 was detected using WB. B Viability of HCT116 cells transfected with siETV7 and siETV7 + pcDNA-IFIT3. n = 3. C, D Colony numbers and numbers of migrated HCT116 cells transfected with siETV7 and siETV7 + pcDNA-IFIT3. n = 3. ** p < 0.01; *** p < 0.001

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Over Expression, Cell Function Assay, Expressing, Transfection

Journal: Functional & Integrative Genomics

Article Title: ETV7 promotes colorectal cancer progression through upregulation of IFIT3

doi: 10.1007/s10142-023-01282-y

Figure Lengend Snippet: The expression of ETV7 in normal tissue and CRC tissue by IHC

Article Snippet: Of note, elevated expression of ETV7 has been linked to B lymphomagenesis (Cardone et al. ), hematopoietic malignancy (Carella et al. ; Kawagoe et al. ), hepatocellular carcinoma (Matos et al. ), and bladder cancer (Li et al. ).

Techniques: Expressing