Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Schematic representation of the different TLE1 constructs used in this study. The amino terminus of TLE1 is characterized by the Q domain, which is required for dimerization; a GP domain; the CcN domain, which is involved in nuclear localization as well as phosphorylation by CK2 (at residues Ser-239 and Ser-253) and Cdc2; and an SP domain. The carboxyl terminus is characterized by WD40 repeats and is involved in binding to several proteins. TLE1(201–770)-FLAG is the deletion construct that lacks the dimerization domain, whereas TLE1(451–770) lacks the dimerization domain, the CcN domain, and the SP domain. AES, Grg1-s, and Grg6 proteins that were used in this study are also shown. AES lack the carboxy-terminal region of TLE1. Grg6 lacks the amino-terminal region of TLE1, including the Q domain.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Construct, Phospho-proteomics, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Expression of TLE1 mRNA and protein during neuronal apoptosis. A, RT-PCR analysis of RNA isolated from CGNs treated with HK or LK for 6 and 9 h. Expression of TLE1, AES, and c-Jun was evaluated. The level of TLE1 mRNA was down-regulated after 6 and 9 h of LK treatment. The level of AES was up-regulated after 9 h of LK treatment. c-Jun mRNA level was up-regulated after 6 and 9 h of LK treatment. Actin served as the loading control. B, Western blot analysis of TLE1 protein levels in CGN treated with HK or LK for 6 and 9 h, respectively. At the protein level also, TLE1 is down-regulated after 6 and 9 h of treatment with LK. Tubulin served as the loading control.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Effects of TLE1 knockdown in CGNs. A, Western blot analysis of endogenous TLE1 protein after knockdown using sh1 and sh2 shRNA in HEK 293T cells. sh1 was able to completely knock down endogenous levels of TLE1, whereas sh2 had a modest effect. Shown is densitometric analysis of endogenous TLE1 protein levels from three independent experiments after knockdown using sh1 and sh2 shRNA. B, viability of CGNs transfected with shRNAs, sh1 and sh2, against TLE1. Knockdown of endogenous TLE1 kills healthy neurons treated with HK. Viability was normalized with CGNs transfected with control shRNA treated with HK. Error bars, S.E. **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Knockdown, Western Blot, shRNA, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Elevated expression of TLE1 promotes neuronal survival. A, CGNs were transfected with GFP or TLE1-FLAG for 8 h and then treated with HK or LK for 24 h. The viability of transfected neurons was evaluated using DAPI staining. Neurons with condensed or fragmented nuclei were scored as dead. B, CGNs were transfected with GFP, TLE1, and two TLE1 deletion mutants, TLE1(451–770) and TLE1(201–770). Cell viability was normalized to GFP-transfected cells in HK. Elevated expression of TLE1, but not the two amino-terminal deletion mutants, protects neurons from LK-mediated cell death. C, cortical neurons were transfected with GFP or TLE1-FLAG for 8 h and then treated with or without HCA for 18 h. Viability was normalized to GFP-transfected cortical neurons without HCA (untreated). Elevated levels of TLE1-FLAG expression help to maintain neuronal survival in HCA-mediated proapoptotic conditions. Error bars, S.E. *, p < 0.05; **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Expressing, Transfection, Staining

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: TLE1 requires phosphorylation by CK2 and signaling via the PI3K-Akt pathway to promote neuronal survival. A, CGNs were transfected with TLE1-FLAG and switched to LK medium with no additive (NA), LK medium with 10 μm TBB, 40 μm PD98059, 10 μm U0126, 1 μm TSA (Trichostatin A), 50 μm KN62, 10 μm IC261, 20 μm LY294002, 200 nm wortmannin, and 10 μm Akt inhibitor X. Viability of transfected neurons identified by immunocytochemistry was estimated after normalizing with respect to CGNs transfected with GFP with no additive. Inhibitors against CK2 (TBB) blocked survival by TLE1 as well as inhibitors against the PI3K-Akt pathway (LY294002, wortmannin, and Akt inhibitor X (AktX)). B, immunocytochemistry of CGNS transfected with TLE1-FLAG and treated with wortmannin, LY 294002, and Akt inhibitor X. DAPI staining was performed to identify the nuclei. C, viability of neurons transfected with GFP, TLE1-FLAG, TLE1S239A-FLAG, TLE1S253A-FLAG, and TLE1S239E-FLAG for 8 h and treated with HK or LK or LK supplemented with 10 μm TBB (CK2 inhibitor) for 24 h. Viability was normalized to GFP-transfected neurons treated with HK. DAPI-stained nuclei were utilized to score for neurons that were transfected with the respective plasmid constructs. The non-phosphorylatable constructs of TLE1 were unable to maintain neuronal survival and even killed healthy neurons treated with HK. The phosphomimetic form of TLE1 was able to protect neurons in LK conditions as well as in LK conditions in the presence of CK2 inhibitor, TBB, used at a concentration of 10 μm. **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Phospho-proteomics, Transfection, Immunocytochemistry, Staining, Plasmid Preparation, Construct, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: TLE1 and FoxG1 interact. A, lysates from CGNs treated with HK or LK for 6 h were immunoprecipitated with a FoxG1 antibody, and the immunoprecipitate was subjected to Western blot analysis with TLE1 antibody. 15% of the whole cell lysate was used as the input control and probed with TLE1 antibody and FoxG1 antibody. B, cell lysates were prepared from HEK293T cells co-transfected with FoxG1-HA and either TLE1-FLAG, AES-FLAG, TLE1(201–770)-FLAG, or TLE1(451–770)-FLAG. The lysates were subjected to a co-immunoprecipitation assay using FLAG antibody for pull-down, and the immunoprecipitate was subjected to Western blotting using a HA antibody. An aliquot of the preimmunoprecipitated lysate (10%) was used as the input control. FoxG1 was able to interact with full-length TLE1 as well as the two amino-terminal deletion constructs of TLE1, but it did not interact with AES. These results demonstrate that TLE1 requires its carboxyl-terminal WD40R domain to interact with FoxG1. PD, pull-down; WB, Western blot.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Immunoprecipitation, Western Blot, Control, Transfection, Co-Immunoprecipitation Assay, Construct

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: FoxG1 and TLE1 cooperate to promote neuronal survival. A, cell lysates were prepared from HEK 293T cells co-transfected with TLE1 and either GFP or FoxG1 or co-transfected with TLE1 and either FoxG137–481-FLAG or FoxG11–275-FLAG. The lysates were immunoprecipitated using TLE1 antibody, and the immunoprecipitate was subjected to Western blotting using a FLAG antibody. Full-length FoxG1 interacted with TLE1, but the 36-amino acid-deleted FoxG1 construct at the amino terminus failed to do so. 10% of the input lysate was checked for proper expression of the constructs. PD, pull-down; WB, Western blot. B, viability of CGNs transfected with GFP, FoxG1-FLAG, TLE1-HA, FoxG1T271A-FLAG, FoxG1N219A-FLAG, and FoxG137–481-FLAG as indicated in the graph and then treated with HK or LK for 24 h. Mutants of FoxG1 that do not promote neuronal survival (17) block the ability of TLE1 to promote neuronal survival. Viability was normalized to GFP-transfected neurons treated with HK. C, viability of CGNs transfected with GFP, FoxG1-FLAG, TLE1(201–770)-FLAG, and TLE1S239A-FLAG as indicated in the graph and then treated with HK or LK for 24 h. Viability of CGNs was normalized to GFP-transfected cells treated with HK. The results show that fully functional TLE1 is required by FoxG1 to mediate neuronal survival. **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Transfection, Immunoprecipitation, Western Blot, Construct, Expressing, Blocking Assay, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Phosphorylation by Akt and CK2 regulate survival promotion by TLE1-FoxG1. A, cell lysates were prepared form HEK293T cells transfected with GFP, TLE1-FLAG, and FoxG1-FLAG and subjected to a co-immunoprecipitation assay using FLAG antibody (Ab). The immunoprecipitate was subjected to Western blot analysis using a phospho-Akt substrate-specific antibody, which will only identify proteins phosphorylated by Akt. 10% of the input lysate was checked for proper expression of the constructs. The results show that FoxG1 is being phosphorylated by Akt, whereas TLE1 is not. PD, pull-down; WB, Western blotting. B, viability of CGNs transfected with FoxG1-FLAG and treated with HK, LK, or LK supplemented with TBB at 10 μm (a CK2 inhibitor) for 24 h. Viability was normalized to CGNs transfected with FoxG1 and treated with HK. Treatment with TBB abrogates the role of FoxG1 in mediating neuronal survival. **, p <0.01.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Phospho-proteomics, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Elevated expression of AES and Grg1-s promotes neuronal death. A, viability of CGNs transfected with GFP, AES-FLAG, and Grg1-s-HA and then treated with HK or LK for 24 h. Viability was normalized to CGNs transfected with GFP and treated with HK. Overexpression of AES and Grg1-s killed neurons even in HK. B, immunocytochemical analysis of AES-transfected neurons treated with HK or LK. C, viability of cortical neurons transfected with GFP, AES-FLAG, and Grg1-s-HA for 8 h and then either left untreated or treated with 1 mm HCA. Viability was normalized to GFP-transfected cortical neurons without any treatment (Untreated). AES and Grg1-s killed healthy cortical neurons. D, cell lysate of HEK293 cells co-transfected with GFP-FLAG, TLE1-FLAG, and Grg1-s-HA, as indicated in the figure, was obtained. The lysate was subjected to co-immunoprecipitation analysis using HA antibody, and the immunoprecipitate was subjected to immunoblotting using FLAG antibody. 10% of the input lysate was checked for proper expression of the constructs. PD, pull-down; WB, Western blot. ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Expressing, Transfection, Over Expression, Immunoprecipitation, Western Blot, Construct

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: AES blocks FoxG1-TLE1-mediated neuronal survival. A, CGNs were transfected with GFP, AES-FLAG, FoxG1-FLAG, and TLE1-FLAG as indicated in the graph for 8 h and then treated with HK or LK for 24 h. Viability of neurons was normalized to CGNs transfected with GFP and treated with HK. Overexpression of AES and TLE1 was able to rescue AES-mediated cell death. B, cell lysate of HEK 293T cells co-transfected with GFP-FLAG, TLE1-FLAG, FoxG1-FLAG, and AES-FLAG, as indicated in the figure, were obtained. The lysates were subjected to co-immunoprecipitation analysis using TLE1 antibody, and the immunoprecipitate was subjected to Western blot analysis using FLAG antibody. 10% of the input lysate was checked for proper expression of the constructs. The results show that AES interferes with interaction between FoxG1 and TLE1. PD, pull-down; WB, Western blot. C, viability of CGNs transfected with GFP, FoxG1-HA, Grg6-FLAG, and TLE1-HA, as indicated in the graph, and then treated with HK or LK for 24 h. Viability was normalized to GFP-transfected neurons treated with HK. **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Transfection, Over Expression, Immunoprecipitation, Western Blot, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Model to explain promotion of neuronal survival by TLE1. A, TLE1 is phosphorylated by CK2 and functions as a dimer. Although the exact stoichiometry of FoxG1 to TLE1 is not clear, interaction with FoxG1 is necessary for the survival-promoting activity of TLE1. B, AES promotes death by heteromerizing with TLE1, thus disrupting the interaction between TLE1 and FoxG1. This occurs during LK treatment when the levels of endogenous AES are increased and the expression of TLE1 is reduced or when AES is overexpressed. C, Grg6 promotes death by disrupting the interaction of TLE1 with FoxG1 through direct interaction with FoxG1.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Activity Assay, Expressing

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 1: The proximity ligation assay demonstrates SS18-SSX/TLE1 co-localization selectively in synovial sarcoma cell lines. SS18-SSX positive cell lines (SYO-1, FUJI, MoJo) demonstrate SS18-SSX/TLE1 proximity by visualization of fluorescent nuclear foci while control cell lines (MCF7, HeLa, A673) showed little nuclear staining A. The intensity of the nuclear signal was quantified and shown to be significantly greater in SS18-SSX confirmed-positive cell lines B. The SS18-SSX/TLE1 protein-protein interaction was confirmed to be specific for synovial sarcoma cell lines by co-immunoprecipitation C. Scale bars represent 20 μm. Error bars represent standard error of mean from three independent studies.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: Proximity Ligation Assay, Control, Staining, Immunoprecipitation

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 2: Knockdown of SS18-SSX results in a loss of SS18-SSX/TLE1 co-localization and a significant decrease in PLA nuclear signals. 50 pmol of siRNA was used to knockdown SS18-SSX in SYO-1 cells, as demonstrated by a loss of protein expression. Protein levels A. and PLA signals B, C. were analyzed 48 hours post siRNA treatment. CME-1 cells were stably transfected with a doxycycline-inducible shRNA against SS18-SSX. Protein level D. and PLA signals E, F. were assessed at 24 hours post 4 μg/μL doxycycline (DX) treatment. Scale bars represent 20 μm. Statistical significance compared to RNA interference controls was determined by Student t test: * denotes p < 0.05; ** denotes p < 0.01. Error bars represent standard error of mean from three images.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: Knockdown, Expressing, Stable Transfection, Transfection, shRNA

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 3: The PLA assay can be used to detect SS18-SSX/TLE1 co-localization in FFPE human synovial sarcoma tumor samples. IHC staining of SS18 A. and TLE1 B. is strongly positive in synovial sarcoma tumor tissue from the metastasectomy specimen. The PLA nuclear signal is detected in the fixed human synovial sarcoma tumor tissue C, D. but not in the immediately adjacent normal lung tissue C, E. Scale bars represent 100 μm.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: Immunohistochemistry

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 4: HDAC inhibitors disrupt SS18-SSX/TLE1 co-localization. A significant decrease in detectable PLA signal following HDAC inhibition in SYO-1 cells A, B. is also confirmed by immunoprecipitation C. The decrease in PLA co-localization signal correlates with apoptosis induction by HDAC inhibitor FK228 in SYO-1 cells D. A panel of cytotoxic compounds and HDAC1/3 inhibitors were screened on SYO-1 cell at IC50 doses for 12 hours. While all compounds were effective in decreasing cell viability, only compounds inhibiting class I HDACs were found to decrease the PLA nuclear signal, as a result of disrupting SS18-SSX/TLE1 co-localization E. Scale bars represent 20 μm. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001. Error bars represent standard error of mean from conditions performed in triplicate. Linear regression was measured by goodness of fit calculations in Prism Graphpad.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: Inhibition, Immunoprecipitation

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 5: The PLA assay can be used for high-throughput drug screening for targeted compounds in synovial sarcoma. From a 16 000 compound small molecule drug library, a compound designated SXT1596 was recognized to significantly decrease relative co-localization of SS18-SSX/TLE1 in human synovial sarcoma cells A. SXT1596 selectively decreases cell viability in synovial sarcoma cell lines, SYO-1, FUJI, Yamato-SS, HS-SY-II, YaFuss and MoJo (white bars), when compared to negative control cell lines HEK293T, MCF7, HeLa and A673 (shaded bars) at a concentration of 5 μM B. Negative control cell lines HeLa and A673 are sensitive to SXT1596 at higher doses. SXT1596 (5-(2-Nitro-1-propenyl)-1,3-benzodioxole) (structure shown in C.) mediated disruption of the SS18-SSX/TLE1 association was confirmed by immunoprecipitation D. Nuclear SS18-SSX/TLE1 PLA signal in SYO-1 cells decreased following SXT1596 treatment to a degree similar to that obtained with the HDAC inhibitor FK228 E, F. Statistical significance compared to vehicle treatment controls was determined by Student t test: * denotes p < 0.05. Error bars represent standard error of mean from conditions performed in triplicate. Small molecule C. was drawn using iChem Labs ChemDoodle software. Scale bars represent 20 μm.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: High Throughput Screening Assay, Drug discovery, Negative Control, Concentration Assay, Disruption, Immunoprecipitation, Software

Journal: Oncotarget

Article Title: Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay.

doi: 10.18632/oncotarget.8882

Figure Lengend Snippet: Figure 6: SXT1596 decreases cell viability and reactivates EGR1 expression in synovial sarcoma. The small molecule SXT1596 induces apoptosis at a similar rate to that of HDAC inhibitor FK228 in SYO-1 cells A, B. and brings about a consistent decrease in SS18-SSX/TLE1 PLA co-localization signal in SYO-1 cells C. The induction of apoptosis correlates with the decrease in PLA co- localization signal D. Chromatin immunoprecipitation at the EGR1 promoter reveals a decrease in H3K27me3 enrichment following SXT1596 treatment E. SXT1596 reactivates EGR1 expression, at the RNA F. and protein G. level. Error bars represent standard error of mean from conditions performed in triplicate. Linear regression was measured by goodness of fit calculations in Prism Graphpad.

Article Snippet: Immunohistochemistry staining was carried out using the Ventana automated staining platform, with primary antibody at a 1/150 dilution for both SS18 (SantaCruz Biotechnologies, sc-28698) and TLE1 (Origene, TA800301).

Techniques: Expressing, Chromatin Immunoprecipitation

Journal: STAR Protocols

Article Title: Fluorogenic in vitro activity assay for the main protease M pro from SARS-CoV-2 and its adaptation to the identification of inhibitors

doi: 10.1016/j.xpro.2021.100793

Figure Lengend Snippet:

Article Snippet: Ac-Abu-Tle-Leu-Gln-AMC , Biosynth Carbosynth , FA178674.

Techniques: Recombinant