the negative control fopflash  (Upstate Biotechnology Inc)

Journal: Cell Death & Disease

Article Title: Alternative splicing of BAZ1A in colorectal cancer disrupts the DNA damage response and increases chemosensitization

doi: 10.1038/s41419-024-06954-6

Figure Lengend Snippet: A Immunoblotting in HCT116 cells after stable transfection with scrambled shCtrl or three different shRNAs targeting BAZ1A, with β-Actin as loading control. B RT-qPCR analysis of BAZ1A , CTNNB1 , CCAR2 , and c- MYC in shCtrl and shBAZ1A cells. C shCtrl and shBAZ1A KD cells were transiently transfected with TCF/LEF-responsive luciferase reporter construct TOPFlash or negative control FOPFlash vectors, along with Renilla pRL-TK plasmid to correct for transfection efficiency. D BAZ1A interactions on c- MYC were examined using ChIP assays in parental HCT116 CCAR2 +/+ or HCT116 CCAR2 -/- cells, with IgG serving as negative control. Statistical significance determined by Student’s t-test for n = 3 replicates, indicated by ***p < 0.001, ****p < 0.0001 vs. shCtrl.

Article Snippet: BAZ1A KD cells (see above) were transiently transfected with 1 μg of plasmid DNA for the Wnt reporter construct TOPflash, or the negative control FOPflash (Upstate Biotechnology, Lake Placid, NY, USA).

Techniques: Western Blot, Stable Transfection, Control, Quantitative RT-PCR, Transfection, Luciferase, Construct, Negative Control, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

doi: 10.1186/s13578-023-01175-4

Figure Lengend Snippet: p38α modulate β-catenin target gene expression. A Chromatin immunoprecipitation assays of Wnt target genes in HT-29 cells under serum starvation (24 h) and upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. Quantification was done using the % input method. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. untreated cells, and # P < 0.05 vs. no ralimetinib. B RTqPCR analysis of Wnt target genes in HT-29 cells upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h after p38α genetic ablation for 24 h or as a pre-treatment before p38α inhibition with ralimetinib (10 μM) for 24 h. Data are presented as mRNA fold change vs. control. The dotted line corresponds to the expression levels detected in control conditions (siRNA CTRL/DMSO). Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. siRNA CTRL/DMSO. C TOPFlash/FOPFlash assay for Wnt transcriptional activity. HT-29 cells were first transfected to overexpress p38α and β-catenin; after 24 h, cells were transfected with TOP/FOP plasmids, serum-starved for 24 h and then stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with ralimetinib (10 μM) for 24 h. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. empty vector, # P < 0.05 vs. DMSO, ▲ P < 0.05 vs. no serum. Results are representative of at least three independent experiments

Article Snippet: After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM).

Techniques: Expressing, Chromatin Immunoprecipitation, Activation Assay, Inhibition, Activity Assay, Transfection, Plasmid Preparation

Journal: BMC Cancer

Article Title: Siah1 proteins enhance radiosensitivity of human breast cancer cells

doi: 10.1186/1471-2407-10-403

Figure Lengend Snippet: Effects of Siah1 on Tcf/Lef regulated transcription activity in SKBR3 and MCF-7 cells . A: Transfected cells were cotransfected with Topflash or Fopflash plasmid, the Renilla luciferase reporter plasmid (pRL-TK) as an internal control and the indicated expression plasmids. Luciferase activity was measured at 24 h after transfection and plotted after normalizing with respect to the Renilla luciferase activity. Each experiment was performed at least three times. Columns, mean; bars, SD; *, p < 0.05. B; Functional inhibition of Siah-1 ubiquitin-ligase by siRNA increased TCF/Lef transcriptional activity in MCF-7 cells at 24 hours after transfection. Each experiment was performed three times. Columns, mean; bars, SD; *, Siah1 siRNA vs. both untreated and control siRNA p < 0.05.

Article Snippet: The Tcf/Lef-responsive luciferase reporter gene (Topflash), the negative control with mutated Tcf/Lef binding site (Fopflash), and the Renilla luciferase reporter plasmid (pRL-TK) as an internal control were obtained from Upstate Biotechnology, USA.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Functional Assay, Inhibition