Journal: iScience

Article Title: The rates of stem cell division determine the cell cycle lengths of its lineage

doi: 10.1016/j.isci.2021.103232

Figure Lengend Snippet:

Article Snippet: Mouse Monoclonal anti-armadillo antibody , DSHB , Cat# N2 7A1 ARMADILLO, RRID: AB_528089.

Techniques: Recombinant, Software

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Real-time PCR showing relative mRNA expression of DKK4 and β-catenin (CTNNB1) in 46 bulk human GIST tumors. Lines represent the medians. Prim/UT– untreated primary GIST (n = 22), Met/Res – resistant metastatic GIST (n = 24). Student’s t test; ***P < 0.001. (B) Representative (left) and quantitative (right) staining of β-catenin in a human GIST tissue microarray of surgical specimens from the time period 1982–2004. The percentage of relative intensity of β-catenin versus total area was calculated using Metamorph software. Medians are indicated. Student’s t test; ***P < 0.001. Prim/UT– primary, untreated GIST; Met/UT– metastatic, untreated GIST; Met/Res – metastatic, resistant GIST. (C) Representative staining of β-catenin in 46 additional human GIST specimens. Scale bar, 20 μm. (D) The corresponding β-catenin intensity was scored as negative to weak (0), moderate (1+), or high (2+). The fraction of high β-catenin intensity score is significantly higher in metastatic, resistant GIST (54.5%) than in primary, untreated GIST (12.5%). (E) Western blot analysis of nuclear active β-catenin isolated from high β-catenin-expressing human GISTs. (F) Relative mRNA expression of FDZ6, FDZ7, and LRP6 in human GIST cell lines and freshly isolated KIT+ tumor cells from 8 human specimens as detected by real-time PCR, compared to Caco-2 colorectal cancer cells. Prim/UT– untreated primary GIST, Met/Res – resistant metastatic GIST, Met/Sen – sensitive metastatic GIST.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Staining, Microarray, Software, Western Blot, Isolation

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Representative staining of β-catenin in GIST T1 xenograft and 2 imatinib-resistant PDX models. Scale bar, 20 μm. (B) Immunoblots of nuclear (NE) and cytoplasmic extracts (CE) from GIST T1 xenografts and PDXs. (C) Real-time PCR showing relative mRNA expression of indicated Wnt genes in PDXs compared to GIST T1 xenografts. Bars indicate means ± SEM. Student’s t test; **P < 0.01, ***P < 0.001. (D) Representative staining of β-catenin in tumors from KitV558Δ/+ mice and murine GIST S2 orthotopic liver tumors. Scale bar, 20 μm. (E) Immunoblots of nuclear (NE) and cytoplasmic extracts (CE) from KitV558Δ/+ tumors or S2 tumors.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Expressing

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Immunoblots of GIST T1, GIST882, and murine S2 whole cell lysates after treatment with either 150 ng/ml Wnt3a or control PBS for 4h. (B) Real-time PCR of Axin 2 and CCND1 (cyclin D1) mRNA expression after treatment with either 150 ng/ml Wnt3a or control PBS for 4h. Bars indicate means ± SEM. Student’s t test; *P < 0.05. (C) TCF/LEF reporter activity in GIST T1 cells in the presence of Wnt3a and/or β-catenin overexpression. GIST T1 cells were co-transfected with a negative reporter or TCF/LEF reporter and a mock or β-catenin expression plasmid. 48h after transfection, cells were treated with either 150 ng/ml of Wnt3a or control PBS for 4h. Relative luciferase signaling (indicating TCF/LEF activity) was determined after normalizing to renilla luciferase (indicating transfection efficiency), and expressed as relative fold changes compared to indicated control. All bars, means ± SEM. Student’s t test; *P < 0.001. (D) Immunoblots of whole-cell lysates of GIST T1 cells that had been transfected with mock or DKK4 expression plasmid and 48h later treated with either 150 ng/ml of Wnt3a or PBS for 4h. Western blot bands were quantified by Image J Software and normalized to GAPDH, and then expressed as the fold-change compared to pCMV6-mock control. (E) Cell viability assay (Dojindo) of human GIST T1 cells following 72h of transfection of control siRNA or b-catenin smartpoolsiRNA. Bars, mean ± SEM. Student’s t test; *P < 0.05.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Over Expression, Transfection, Plasmid Preparation, Luciferase, Software, Viability Assay

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: Immunoblots of nuclear extracts of GIST T1 cells treated with (A) the proteasome inhibitor MG132 10 μM for 6h or pretreated with (B) the protein biosynthesis inhibitor cycloheximide (CHX) for 6h. (C) Immunoblots of COP1 expression in whole-cell lysates of human GIST specimens. (D) Immunoblots of nuclear extracts of GIST T1 cells transfected with control siRNA or COP1 Smartpool siRNA for 48h. (E) TCF/LEF reporter activity as detected by luciferase of GIST T1 cells transfected with a negative reporter or a TCF/LEF reporter, and control siRNA or COP1 Smartpool siRNA for 48h, Bars, mean ± SEM. Student’s t test; ***P < 0.001. (F) Representative CHX-chase assay (of 2 performed) to determine the stability (half-life) of nuclear β-catenin in GIST T1 cells 48h after transfection with control siRNA or COP1 Smartpool siRNA. Cells were collected after the addition of 200 μg/ml CHX at indicated time points (0, 1, 2, 4, 6h). Relative nuclear β-catenin levels were determined by normalizing to the loading control (lamin B1) and then normalizing to the t = 0h control siRNA. Immunoblots of nuclear extracts are shown. (G) GIST T1 cells were transfected with control siRNA or COP1 Smartpool siRNA for 48h and then treated with MG132 10 μM for 2h prior to harvest. Whole-cell lysates were immunoprecipitated by either anti-COP1 or control IgG, Western blot was performed as indicated. Whole-cell lysates were used as input. (H) Immunoblots of whole-cell lysates of GIST T1 cells transfected with the indicated constructs for 48h. Western blot bands were quantified by Image J Software and normalized to GAPDH, and then expressed as the fold-change compared to control.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Transfection, Activity Assay, Luciferase, Immunoprecipitation, Construct, Software

Journal: Molecular cancer therapeutics

Article Title: Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor

doi: 10.1158/1535-7163.MCT-17-0139

Figure Lengend Snippet: (A) Cell viability assay (Dojindo) of human GIST cell lines following 72h of PKF118-310 or vehicle (DMSO control) treatment. Bars indicate means ± SEM. Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. (B) Immunoblots of GIST T1 and HG129 cells treated with 200 nM PKF118-310 or vehicle (DMSO control) at the indicated time points. (C) GIST T1 or HG129 cells were injected into the flanks of NSG mice or (D) murine S2 cells were injected into the flanks of C5BL/6J mice and when the tumors were 100 mm3, intratumoral injections of PKF118-310 (0.8 mg/kg) or vehicle 0.01% DMSO in PBS were administered every other day for 6 doses. Graph shows Mean ± SEM. Student’s t test; **P < 0.01. (E) Representative immunostaining for β-catenin in PKF118-310 or vehicle-treated murine S2 tumors. Scale bar, 20 μm. (F) Immunoblots of whole-cell lysates from vehicle or PKF118-310 treated S2 tumors.

Article Snippet: To generate overexpressing cells, GIST T1 were transfected with empty control vector (pCMV6-mock, PS100001), human β-catenin (pCMV6-β-catenin, RC208947), DKK4 (pCMV6-DKK4, RC221217), or COP1 plasmid (pCMV6-COP1, RC210492, all from Origene) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Viability Assay, Western Blot, Injection, Immunostaining

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: β-Catenin , REA480 , 50 , 130-123-546 , FITC (APC, PE) , Miltenyi Biotec.

Techniques: Imaging