Journal: Frontiers in Oncology

Article Title: The Atypical Protein Kinase C Small Molecule Inhibitor ζ-Stat, and Its Effects on Invasion Through Decreases in PKC-ζ Protein Expression

doi: 10.3389/fonc.2020.00209

Figure Lengend Snippet: PKC-ζ regulates invasion and migration of TOV21G ovarian cancer. (A) TOV21G cells were grown, serum starved for 48 h, and placed in the upper chamber of transwell plate coated with 0.1x BME and serum (10%) containing media was placed in the lower chamber as a chemo attractant. Following treatment with 10μM ζ-stat for 24 h, cells that invaded through BME and migrated into the lower chamber were stained with crystal violet and observed under microscope. Original magnification is 10x and scale bar represents 1 mm. (B) Invasion ( N = 4) and (C) migration ( N = 4) fixed and stained cells on the lower chamber were quantified using ImageJ FIJI software, average and plotted. Standard deviation error bars are represented. (** p < 0.01, *** p < 0.001). (D) Activated Rac1 (GTP bound) was pulled down using GST tagged PAK-PBD and analyzed by Western Blot. Densitometry of activated Rac1 bands were plotted ( N = 4). Standard deviation error bars are represented.

Article Snippet: The sources of cell lines, reagents and antibodies were: TOV21G and ES-2 CCOC cell lines (American Type Culture Collection, USA); SHT290 normal endometrial stromal cell line (Kerafast, USA); MCDB 121, Media 199, F12K, penicillin and streptomycin, trypsin, Dulbecco's phosphate buffered saline (DPBS) and Mito + (Corning, USA); McCoy's media (HyClone, USA); Opti-MEM I (Gibco, USA); Fetal bovine serum (FBS, Atlanta Biologicals, USA); human insulin (MP Biomedicals, LLC, France); dimethyl sulfoxide (DMSO, Sigma Aldrich, USA); Water-Soluble Tetrazolium (WST-1, Roche, USA); Halt protease and phosphatase inhibitors cocktail and Protein A/G magnetic beads (Thermo Scientific, USA); anti- PKC-ζ (9372s, 1:1000, Cell Signaling, USA); anti- PKC-ι (610178 1:1000, BD, USA); anti-β-actin (A3854, 1:40000, Sigma Aldrich, USA); anti-RhoA (ab54835, 1:4000, Abcam, USA); anti-Ect2 (07-1364, 1:1000, Millipore, USA); anti-β-tubulin (5346t, 1:1000, Cell Signaling, USA); anti-PARP (9532s, 1:1000, Cell Signaling, USA); Donkey anti-rabbit IgG Alexa-488 (A21206, 1:500, Invitrogen, USA); Goat anti-rabbit (170-6515, 1:2000, Bio-Rad Laboratories, USA); Goat anti-mouse (170-6516, 1:2000, Bio-Rad Laboratories, USA); Activated Rac1 pulldown kit (BK035, Cytoskeleton, USA); 96-well transwell insert and basement membrane extract (BME; both Corning Inc., Corning, NY, USA); RNA bee (Amsbio, United Kingdom); Qiagen RT Kit (205113, Qiagen, Germany); RhoA PCR primers (HP100025, Sino Biological, USA); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) PCR primers (Eurofins, USA); NE-PER Nuclear and Cytoplasmic Extraction Reagents (78835, Thermo Fisher Scientific, USA).

Techniques: Migration, Staining, Microscopy, Software, Standard Deviation, Western Blot