Journal: Science Advances

Article Title: Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

doi: 10.1126/sciadv.abe0849

Figure Lengend Snippet: ( A and B ) TopFlash reporter luciferase assays in HEK293T cells treated for 16 hours with purified Wnt3a (A) or GSK3 inhibitor CHIR99021 (CHIR) (B) and transfected with a SOX9 expression plasmid. ( C ) Same as (A) and (B) except Wnt signaling was activated by transfection of N-terminally mutated (S33Y) β-catenin (β-catenin*) and with an increasing dose of SOX9. ( D ) Same as (C) except an Axin2 enhancer reporter was used instead of TopFlash. ( E ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of SOX9 and two Wnt targets performed in pTRE-SOX9 WT cells. Dox treatment was 13.5 hours. ( F ) TopFlash reporter assays of several cell lines transfected with β-catenin* with or without SOX9. See Results and Materials and Methods for details about each cell line. Each bar represents the means of biological triplicates ± SD. * P < 0.05; ** P < 0.005; *** P < 0.001.

Article Snippet: The following antibodies were used: anti-Flag–horseradish peroxidase (HRP) (clone M2, Sigma-Aldrich, 1:5000), anti–β-catenin (BD, 1:5000), anti–α-tubulin (Cell Signaling Technology, 1:5000), anti-HIS (GE Healthcare, 1:1000), anti-MAML2 (Cell Signaling Technology, 1:1000), and anti-mouse HRP/anti-rabbit HRP (Jackson Immunochemicals, 1:2000).

Techniques: Luciferase, Purification, Transfection, Expressing, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Science Advances

Article Title: Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

doi: 10.1126/sciadv.abe0849

Figure Lengend Snippet: ( A ) Western blots of endogenous β-catenin and transfected FLAG-SOX9 from pTRE-SOX9 WT cell lysates treated with 5 μM CHIR and Dox for the indicated times (hour). ( B ) Western blots of transfected FLAG–β-catenin* and FLAG-SOX9 from HEK293T cell lysates. ( C ) Western blots of parental HEK293T cells (left half) or HEK293T cells with CRISPR-induced deletions of all four TCF genes (TCFQKO cells; right half) transfected with β-catenin* and an increasing dose of SOX9. ( D ) TopFlash reporter assay performed on HEK293T cells transfected with or without FLAG-SOX9 and treated with the proteasome inhibitors MG132 and bortezomib for 8 hours before lysis. Each bar represents the means of biological triplicates ± SD. ( E ) Western blots of HEK293T cell lysates, transfected with FLAG–β-catenin* with or without SOX9 and treated with MG132 for indicated periods of time. Images cropped from the same blot for plus/minus Sox9. ( F ) Western blots of endogenous β-catenin from pTRE-SOX9 WT cells supplemented with Dox (24 hours) and 5 μM CHIR (16 hours) and then treated with CHX for indicated period of time before harvest. ( G ) Western blots of transfected FLAG–β-catenin* from HEK293T cells transfected with β-catenin* with or without SOX9 and then treated with CHX for indicated period of time before harvest. More extract was loaded for the SOX9-expressing cells to provide a fair comparison of β-catenin decay. All images in (F) and (G) were cropped from the same blot. α-Tubulin was used as a loading control for all Western blots. In numerous experiments, Sox9 down-regulated β-catenin to levels shown in (A) to (C) and (E). In (three) separate experiments, Sox9 accelerated the turnover of β-catenin, as shown in (F) and (G). *** P < 0.001.

Article Snippet: The following antibodies were used: anti-Flag–horseradish peroxidase (HRP) (clone M2, Sigma-Aldrich, 1:5000), anti–β-catenin (BD, 1:5000), anti–α-tubulin (Cell Signaling Technology, 1:5000), anti-HIS (GE Healthcare, 1:1000), anti-MAML2 (Cell Signaling Technology, 1:1000), and anti-mouse HRP/anti-rabbit HRP (Jackson Immunochemicals, 1:2000).

Techniques: Western Blot, Transfection, CRISPR, Reporter Assay, Lysis, Expressing

Journal: Science Advances

Article Title: Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

doi: 10.1126/sciadv.abe0849

Figure Lengend Snippet: ( A ) Schematic representations of wild-type (WT) full-length and transcriptionally disabled SOX9s. The location of the DNA binding HMG domain and the three known transcriptional activation domains (TAM, PQA, and TAC) are shown . All SOX9 constructs were tagged with two Flag epitopes at their N termini. SOX9 HMG has three point mutations in the N terminus of the HMG domain (star) and contains an exogenous NLS to ensure nuclear localization. SOX9 332 lacks the last 177 amino acids (aa) of the protein, and three variants of SOX9332 containing distinct transcriptional activation domains (KIX, GAL4, and VP16) were constructed (see fig. S2B for sequences of these domains). ( B ) SoxFlash reporter luciferase assays in HEK293T cells transfected with SOX9 constructs. The differences between SOX9 and the SOX9 mutants in activation of SoxFlash were significant ( P > 0.001). ( C ) TopFlash reporter assay with SOX9 constructs. Each bar represents the means of biological triplicates ± SD. ( D ) Western blots on HEK293T cell lysates from transfections of FLAG–β-catenin* and various FLAG-SOX9 proteins. α-Tubulin was used as a loading control. ( E ) Western blots of HEK293T cell lysates transfected with different β-catenin* domain mutants (fig. S3B for details) with or without SOX9. In numerous experiments, SOX9 down-regulated β-catenin proteins to levels shown in (D) and (E). *** P < 0.001.

Article Snippet: The following antibodies were used: anti-Flag–horseradish peroxidase (HRP) (clone M2, Sigma-Aldrich, 1:5000), anti–β-catenin (BD, 1:5000), anti–α-tubulin (Cell Signaling Technology, 1:5000), anti-HIS (GE Healthcare, 1:1000), anti-MAML2 (Cell Signaling Technology, 1:1000), and anti-mouse HRP/anti-rabbit HRP (Jackson Immunochemicals, 1:2000).

Techniques: Binding Assay, Activation Assay, Construct, Luciferase, Transfection, Reporter Assay, Western Blot

Journal: Science Advances

Article Title: Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

doi: 10.1126/sciadv.abe0849

Figure Lengend Snippet: ( A ) TopFlash reporter assays in parental and MAML2 KO lines in response to CHIR (1 to 4 μM). Each point represents the means of biological triplicates ± SD. ( B and C ) Western blots of parental and MAML2 KO line (#1) of endogenous β-catenin stabilized with overnight treatment of 5 μM CHIR (B) or transfected with β-catenin* (C) and treated with CHX for the indicated times. α-Tubulin was used as a loading control. Images cropped from the same blots. Blots shown were representative of three independent experiments. ( D ) Schematic representation of the N-terminal (MAML2-N) and C-terminal (MAML2-C) fragments used in the β-catenin binding assays shown in (E). The locations of the CSL/N binding domain (gray), acidic domains (stripped), and polyQ regions (black) are indicated. MAML2-N and MAML2-C are HIS-tagged at their C termini. ( E ) Western blots of in vitro pull downs using GST or GST–β-catenin with HIS-tagged MAML2-N (left) and MAML2-C (right). Input lanes contain 10% of the total binding reaction. Data are representative of three independent pull-down assays. ( F and G ) Cartoon depicting a SOX9-MAML2 axis that restricts the Wnt pathway independently of the β-catenin destruction complex, proteasome, and lysosomal degradation pathways. * P < 0.05.

Article Snippet: The following antibodies were used: anti-Flag–horseradish peroxidase (HRP) (clone M2, Sigma-Aldrich, 1:5000), anti–β-catenin (BD, 1:5000), anti–α-tubulin (Cell Signaling Technology, 1:5000), anti-HIS (GE Healthcare, 1:1000), anti-MAML2 (Cell Signaling Technology, 1:1000), and anti-mouse HRP/anti-rabbit HRP (Jackson Immunochemicals, 1:2000).

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

Journal: Science Advances

Article Title: Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

doi: 10.1126/sciadv.abe0849

Figure Lengend Snippet: ( A ) TopFlash reporter assays in parental and MAML2 KO lines in response to CHIR (1 to 4 μM). Each point represents the means of biological triplicates ± SD. ( B and C ) Western blots of parental and MAML2 KO line (#1) of endogenous β-catenin stabilized with overnight treatment of 5 μM CHIR (B) or transfected with β-catenin* (C) and treated with CHX for the indicated times. α-Tubulin was used as a loading control. Images cropped from the same blots. Blots shown were representative of three independent experiments. ( D ) Schematic representation of the N-terminal (MAML2-N) and C-terminal (MAML2-C) fragments used in the β-catenin binding assays shown in (E). The locations of the CSL/N binding domain (gray), acidic domains (stripped), and polyQ regions (black) are indicated. MAML2-N and MAML2-C are HIS-tagged at their C termini. ( E ) Western blots of in vitro pull downs using GST or GST–β-catenin with HIS-tagged MAML2-N (left) and MAML2-C (right). Input lanes contain 10% of the total binding reaction. Data are representative of three independent pull-down assays. ( F and G ) Cartoon depicting a SOX9-MAML2 axis that restricts the Wnt pathway independently of the β-catenin destruction complex, proteasome, and lysosomal degradation pathways. * P < 0.05.

Article Snippet: The following antibodies were used: anti-Flag–horseradish peroxidase (HRP) (clone M2, Sigma-Aldrich, 1:5000), anti–β-catenin (BD, 1:5000), anti–α-tubulin (Cell Signaling Technology, 1:5000), anti-HIS (GE Healthcare, 1:1000), anti-MAML2 (Cell Signaling Technology, 1:1000), and anti-mouse HRP/anti-rabbit HRP (Jackson Immunochemicals, 1:2000).

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

Journal: Oncogene

Article Title: Aberrantly activated AREG-EGFR signaling is required for the growth and survival of CRTC1-MAML2 fusion-positive mucoepidermoid carcinoma cells.

doi: 10.1038/onc.2013.348

Figure Lengend Snippet: Figure 4. Active EGFR signaling is dependent on expression of CRTC1–MAML2 fusion or AREG in fusion-positive MEC cells. (a) MAML2 knockdown in fusion-negative cells did not affect the level of active EGFR signaling, as assessed by western blotting for P-EGFR (Tyr1068) and the downstream P-p42/p44 MAPK and their total proteins. Cells were harvested 72 h after two infections with lentiviruses on two consecutive days. (b, c) Knockdown of CRTC1–MAML2 and MAML2 in fusion-positive H3118 and H292 MEC cells reduced the level of P-EGFR and P-p42/ p44 MAPK. (d) Two forms of lentiviral-based AREG shRNAs reduced AREG transcript levels in H3118 MEC cells by real-time RT–PCR n=3. Po0.05. (e) AREG knockdown led to reduced EGFR signaling in H3118 MEC cells shown by reduced P-EGFR and P-p42/44 MAPK levels. (f, g) Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining analyses showed that fusion-positive human H3118 and H292 MEC xenografts from nude mice displayed MEC histology and positive signals for AREG and P-p42/44 MAPK.

Article Snippet: Antibodies The following antibodies were purchased from commercial sources including anti-MAML2 (Cell Signaling Technology, Danvers, MA, USA; CST-4618), anti-MAML2 (Bethyl Laboratories, Montgomery, TX, USA; A300-681A); anti-phospho-EGF receptor (Tyr1068; CST-3777); anti-EGFR (CST-2232); anti-p44/42 MAPK (CST-9102); anti-phospho-p42/44 MAPK (CST-4370); anti-CREB (Upstate, Billerica, MA, USA; 06-863); anti-a-Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-5286); anti-b-actin (sc-47778); and anti-AREG antibody (R&D Systems, Minneapolis, MN, USA; AF262).

Techniques: Expressing, Knockdown, Western Blot, Quantitative RT-PCR, Immunohistochemical staining, Immunohistochemistry