Cellomics beta catenin activation kits

NCI-H1703, SK-LU-1, and HLCSC were first treated with glycogen synthase kinase 3 inhibitor X (GSK3i) for 24 h upon growing overnight in order to artificially activate the Wnt/β-catenin pathway before the onset of chelerythrine chloride treatment. Immunofluorescence was performed by using Cellomics^® Beta-Catenin Activation Kits (Thermo Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol or by staining with mouse anti-beta catenin 1 (cat# 610154, BD Biosciences, Franklin Lakes, NJ, USA) and counter-stained with anti-mouse-Alexa Fluor 488 (A-11001, Thermo Fisher Scientific) and DAPI (1:1000 of 2 mg/mL, Sigma-Aldrich). Five random, non-overlapping frames were captured from each well by using Axio Vert.A1 inverted microscope equipped with HXP-120V light source and Axiocam MR R3 camera (Carl Zeiss, Oberkochen, Germany) or EVOS FL cell imaging system (Thermo Fisher Scientific). Cells harboring nuclear β-catenin were manually counted by using Image’s J cell counter plugin and the positive counts were divided by the respective total number of cells in the captured frames to obtain the percentage of cells with positive nuclear β-catenin [58 (link)]. Treated samples were compared with untreated Wnt-activated negative control.

Immunofluorescence assay was performed as described previously [23 (link)]. SK-LU-1 and HLCSC were first treated with GSK3i for 24 h upon growing overnight in order to optimally increase β-catenin localization before the onset of phytochemicals treatments. Immunofluorescence was performed by using Cellomics^® Beta-Catenin Activation Kits (Thermo Scientific, Waltham, MA, USA) in accordance with the manufacturer’s protocol. Five random, non-overlapping frames were captured from each well by using Axio Vert.A1 inverted microscope equipped with HXP-120V light source and Axiocam MR R3 camera (Carl Zeiss, Oberkochen, Germany). Cells harboring nuclear β-catenin were manually counted by using ImageJ’s cell counter plugin and the positive counts were divided by the respective total number of cells in the captured frames to obtain percentage of cells with positive nuclear β-catenin [32 (link)]. Treated samples were compared with untreated Wnt-activated negative control.