Cul3 sirna smartpool

3 × 10 5 U2OS and 6 × 10 5 MDA-MB-231 cells were seeded in a 6-well plate and 24 h later were transfected with 25 nM CUL3 siRNA SMARTpool (L-010224-00-0005; Dharmacon™ ON-Targetplus, Cambridge, UK) according to the manufacturers' instructions. 48 h post transfection cells were treated with 50 ng/ml NCS for 6 h and collected with trypsinization in a microcentrifuge tube and fixed in 70% ethanol for 30 min on ice. Cells were pelleted and incubated with various concentrations of ethanol (50%, 30%, 10%) to remove ethanol slowly and were finally resuspended in 1 × PBS. Before FACS analysis, 1 μg/ml DAPI (Sigma-Aldrich, St. Louis, MO, USA) was added to the cell suspension and incubated for 30 min followed by cell analyses using BD FACS Aria Fusion flow cytometer.

10 6 cells from both MCF-7 and MDA-MB-231 cell lines were seeded in 100 mm dishes and 48 h later at approximately 80% confluency cells were transfected with 10 μg of plasmid DNA of either pCDNA3.1-MYC-HIS (empty vector) or pCDNA3.1-MYC-CUL3 using JetPEI or Lipofectamine3000 transfection reagents as it is described below or with siRNA transfection using 25 nM CUL3 siRNA SMARTpool (L-010224-00-0005; Dharmacon™ ON-Targetplus, Cambridge, UK) according to the manufacturers' instructions. 48 h after transfection 1 × 10 5 cells were seeded in 24-well and were let to recover for 24-48 h. A scratch was made using a pipette tip to create an incision-like gap. Immediately after the scratch 100 ng/ml of NCS was applied to the indicated wells. Images were taken using Axiocam ERc 5 s under ZEISS Primovert inverted microscope immediately after the created wounded area as well as after every 24 h for 7 days. Cell migration was quantified using ImageJ and expressed as the average percentage of closure of the scratched area using GraphPad Prism version 8.4.3. (https://www.graphpad.com/) Two-way Anova was applied for statistical analysis. The experiment was performed in three biological replicates.

10 6 cells from both MCF-7 and MDA-MB-231 cell lines were seeded in 100 mm dishes and 24 h later at approximately 80% confluency cells were transfected with 10 μg of plasmid DNA of either pCDNA3.1-MYC-HIS (referred as empty vector; Addgene, Waretown, MA, USA) or pCDNA3.1-MYC-CUL3 (Addgene, Waretown, MA, USA) using JetPEI or Lipofectamine3000 transfection reagents (Invitrogen, Carlsbad, CA, USA) as it is described below or with siRNA transfection using 25 nM CUL3 siRNA SMARTpool (L-010224-00-0005; Dharmacon™ ON-Targetplus, Cambridge, UK) according to the manufacturers' instructions. 48 h after transfection, 3000 cells were seeded in 6-well plates and were incubated for 14 days to form visible colonies of at least 50 cells. 24 h after seeding 100 ng/ml of neocarzinostatin (NCS; Sigma-Aldrich, St. Louis, MO, USA) was applied to the indicated wells. Cells were washed two times with 1 × PBS and fixed for 20 min using 4% formaldehyde with constant gentle agitation. After washing with 1 × PBS cells were stained with 0.05% crystal violet for 1 h and observed under light microscope. Images were taken using the Licor Odyssey M imaging system. Colonies were counted by ImageJ and GraphPad Prism version 8.4.3. (https://www.graphpad.com/) was used for quantification. Two-way Anova was applied for statistical analysis. The experiment was performed in three biological replicates.