flow cytometry analysis hct116 cells  (Thermo Fisher)

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: ( A ) Expression of SMAR1 and β-catenin in various CRC cell lines. ( B ) Confocal staining of colon tissue sections co-stained with SMAR1 and β-catenin antibodies. Arrow shows the basal portion of the colon crypt. The scale bar used in the confocal experiment represents 30 μm. ( C ) Expression of SMAR1 and β-catenin levels in mouse colon tissues (polyp vs normal adjacent tissue). ( D and E ) Expression of SMAR1 and β-catenin upon stimulating HCT116 cells with Wnt3a CM or rh Wnt3a ligand (200 ng/mL). ( F ) Confocal staining of SMAR1 after Wnt3a CM stimulation in HCT116 cells. The scale bar used in the confocal experiment represents 20 μm. ( G ) SMAR1 expressions after treating HCT116 cells with both Wnt3a CM and 10 μM MG132 drug. ( H ) SMAR1 expression in FLAG-SMAR1, D1, D2 and D3 expressing cells after Wnt3a CM stimulation.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: ( A ) β-catenin expression in HCT116 cells after knockdown with sh-SMAR1. The triangle indicates increased concentration of SMAR1 plasmids of 1 and 3 μg. ( B ) Real-Time PCR experiment showing β-catenin mRNA levels in SMAR1 knockdown HCT116 cells. 18S rRNA was used for normalization. Results are shown as mean ± SD ( n = 3). The p value was determined by student's t -test. The triangle indicates increased concentration of SMAR1 plasmids of 2 and 4 μg. ( C ) β-catenin expressions in HCT116 cells after GFP-SMAR1 overexpression. The triangle indicates increased concentration of SMAR1 plasmids of 1, 2, 3 and 4 μg. ( D ) Real Time-PCR experiment showing β-catenin mRNA levels in SMAR1 overexpressed HCT116 cells. 18s rRNA was used for normalization. Results are shown as mean ± SD ( n = 3). The p value was determined by student's t -test. The triangle indicates increased concentration of SMAR1 plasmids of 1, 2.5 and 4 μg. ( E ) Luciferase promoter activities of Super 8X TOPFlash/FOPFlash (mean ± SD, n = 3) after SMAR1 overexpression and knockdown in HCT116 cells. ( F ) Expression of β-catenin in HCT116 cells after transfection with GFP-SMAR1 and simultaneous stimulation with Wnt3a CM.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Expressing, Knockdown, Concentration Assay, Real-time Polymerase Chain Reaction, Over Expression, Luciferase, Transfection

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: FACS analysis of pEGFP1-β-catenin GFP expression ( n = 3, SD) after; ( A ) Co-transfection with FLAG-vector or FLAG-SMAR1, and ( B ) Treatment with 200 ng/mL rh Wnt3a ligand. ( C ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after Wnt3a CM stimulation. ( D ) ChIP showing occupancy of SMAR1, HDAC5 and H3K9 Ac (mean ± SD, n = 3) after SMAR1 overexpression or knockdown. ( E ) ChIP showing occupancy of HDAC5 (mean ± SD, n = 3) after si-HDAC5 knockdown in HCT116 cells. ( F ) Expression of β-catenin after knockdown with 2μg si-HDAC5 plasmid. ( G ) Immunoprecipitation of SMAR1 with HDAC5. ( H ) Immunoprecipitation of HDAC5 with various truncations of SMAR1. ( I ) Immunoprecipitation of HDAC5 with SMAR1 after Wnt3a CM stimulation.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Expressing, Cotransfection, Plasmid Preparation, Over Expression, Knockdown, Immunoprecipitation

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: ( A ) Cell invasion in SW480 cells after overexpression with GFP-SMAR1 construct. ( B ) Cell migration assay in GFP-SMAR1 overexpressed SW480 cells. ( C ) Tumors generated in NOD-SCID mice ( n = 10) using various stable HCT116 cells for SMAR1. ( D and E ) Graphs showing volume and weight of tumors generated in NOD-SCID mice. ( F ) Kaplan Meier survival probability curve plotted with respect to SMAR1 expression in CRC patients. The survival curve was generated using Smith tumor colon database.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Over Expression, Construct, Cell Migration Assay, Generated, Expressing

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: ( A ) Expression of SMAR1 in HCT116 cells treated with various peptides (5 μg/mL) for 48 hrs. ( B ) Confocal staining for SMAR1 after AT-01C or AT-01D (10 μg/mL) treatment. The scale bar used in the confocal experiment represents 10 μm. β-catenin expression after treatment of HCT116 cells with increasing concentration of; ( C ) AT-01C and ( D ) AT-01D peptides. SMAR1 expression after stimulation of HCT116 cells with Wnt3a CM and simultaneously treated with ( E ) AT-01C, and ( F ) AT-01D. ( G ) Luciferase promoter activities of Super 8X TOPFlash/FOPFlash (mean ± SD, n = 3) after treatment with AT-01C or AT-01D.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Expressing, Staining, Concentration Assay, Luciferase

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: Isothermal Titration Calorimetry (ITC) showing the interaction of SMAR1 with the peptides, ( A ) AT-01C and ( B ) AT-01D. In silico AutoDock interactions of SMAR1 with: ( C ) AT-01C and ( D ) AT-01D peptide. ( E ) SMAR1 expression after treating HCT116 cells with single peptide or combination of AT-01C and AT-01D.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Isothermal Titration Calorimetry, In Silico, Expressing

Journal: Oncotarget

Article Title: SMAR1 inhibits Wnt/β-catenin signaling and prevents colorectal cancer progression

doi: 10.18632/oncotarget.25093

Figure Lengend Snippet: ( A ) Cell migration assay in SW480 cells treated with AT-01C or AT-01D for 12 hours. ( B ) Graphical representation of SW480 cells migrated (mean ± SD, distance in triplicates arbitrarily taken at three different points). ( C ) Cell invasion assays in SW480 cells after treatment with AT-01C or AT-01D (10 μg/mL) for 48 hrs. ( D ) Clonogenic assays in HCT116 cells after treatment with AT-01C or AT-01D (10 μg/mL), and ( E ) Its graphical representation ( n = 3, SD). ( F ) Tumors generated using 1 × 10 6 HCT116 cells in NOD-SCID mice after administration with AT-01C (25 mg/kg body weight). ( G and H ) Graph representing the weight and volume of the mice tumors administered with AT-01C. ( I ) Tumors generated using 1 × 10 6 HCT116 cells in NOD-SCID mice after administration with AT-01D (25 mg/kg body weight). ( J and K ) Graph representing the weight and volume of the mice tumors administered with AT-01D.

Article Snippet: For flow cytometry analysis HCT116 cells were trypsinized using Trypsin-EDTA (Gibco) and washed thrice with PBS buffer.

Techniques: Cell Migration Assay, Generated