Journal: Frontiers in Oncology
Article Title: Uncoupled nitric oxide synthase activity promotes colorectal cancer progression
doi: 10.3389/fonc.2023.1165326
Figure Lengend Snippet: GSK-3β knockdown mitigates the effects of SP in HCT-116 cells: (A) Confirmation by Western blot of GSK-3β knockdown in shGSK3.I and shGSK3.G clones. shConC (B) cells or shGSK3.I (C) cells were treated with 20 µM of SP for 1, 3, or 5 days, and protein expression of β-catenin, cdc25A, and pSer 473 -Akt were measured by Western blot. Representative blots are shown from three separate experiments. (D) LEF-TCF promotor activity was measured in shConC, shGSK3.G, and shGSK3.I clones. Results are normalized to untreated cells and reported as ± SD. Student’s t-test was used to assess for statistical significance; *p<0.05. (E) For in vitro clonogenic assays, scrambled and GSK3β knockdown HCT-116 cells were treated with 20 µM SP for 24 h before plating and growing colonies to 50 cells or more. Colony counts were normalized to untreated cells and assessed for statistical significance using the Student’s t-test. *p< 0.05.
Article Snippet: The following primary antibodies (and sources) were used: goat anti-actin (sc-1615, Santa Cruz Biotechnology, Dallas, TX) and mouse monoclonal anti-GAPDH (MAB374, Millipore, Burlington, MA); Cell Signaling Technology (Danvers, MA) provided rabbit polyclonal anti-cdc25A (cst-3652), rabbit polyclonal anti-Akt (cst-9272), rabbit polyclonal anti pS 33/37 -β-catenin (cst-2009), rabbit monoclonal anti-non-phospho (active) β-catenin (cst-8814), mouse monoclonal anti-pS 9 -GSK3β (cst-9832), and rabbit monoclonal anti-GSK3β (cst-9323) and anti-pS 473 -Akt (cst-4060).
Techniques: Western Blot, Clone Assay, Expressing, Activity Assay, In Vitro
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