All cell lines were obtained from ATCC (Manassas, VA, U.S.A.). All cell lines were grown according to manufacturer’s conditions in the presence of fetal bovine serum (Invitrogen, Carlsbad, CA, U.S.A.).
siRNA oligos and transfection
All siRNAs were obtained from the ON-TARGET plus collection purchased from Dharmacon (Lafayette, CO, U.S.A). The sense strand sequences of the Wee1 siRNA oligos employed in the study are as follows: #5 5′-AAUAGAACAUCUCGACUUA-3′, #6 5′-AAUAUGAAGUCCCGGUAUA-3′, #7 5′-GAUCAUAUGCUUAUACAGA-3′, #8 5′-CGACAGACUCCUCAAGUGA-3′. The negative control siRNA oligos: NT1 and NT2 product numbers D-001810-01 and D-001810-02. The Ran oligo targeting sequence was 5′- AGAAGAAUCUUCAGUACUAUU-3′. Transfections of siRNA duplexes were performed using RNAiMAX reagent (Invitrogen).
Cell proliferation and caspase assays
Viable cell numbers were measured using CellTiter-Glo reagent (Promega, Madison, WI, U.S.A.) or CyQuant (Invitrogen). Caspase-Glo 3/7 assay (Promega) was used to measure cellular caspase-3/7 activity.
Antibodies and Western blot analysis
Antibodies used included mouse anti-Wee1, mouse anti-P53 (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), rabbit anti-phospho-CDK1 (Y15), rabbit anti-phospho-H2AX (S139), mouse anti-phospho-Histone H3 (S10), rabbit anti-Histone H3 (Cell Signaling Technology, Danvers, MA, U.S.A.), mouse anti-CDK1 (Millipore, Billerica, MA, U.S.A.), mouse anti-P21 (BD Biosciences, San Diego, CA, U.S.A.), and mouse anti-Actin (Sigma, St. Louis, MO, U.S.A.). Western blots were performed as previously described .
DNA cloning and Engineered cell lines
The human Wee1 coding sequence was amplified using standard PCR protocols and cloned into the pLVX-puro lentiviral construct (Clontech, Mountain View, CA, U.S.A.). Kinase-altered Wee1 (K328R) and siRNA resistant constructs were introduced using the QuikChange site directed mutagenesis kit (Stratgene, Santa Clara, CA, U.S.A.). Cell lines were generated immediately after infection by mass selection in 2 μg/ml puromycin (Clontech).
siRNA rescue experiment
Engineered NCI-H1299 cell lines were grown in DMEM containing 10% fetal bovine serum and 2 μg/ml puromycin. Cell lysate samples were collected 48 hr after transfection for Western blot analysis. Five days after siRNA transfection, microscopic cell images were taken and both floating and attached cells were then collected by trypsinization and counted for viable cell number using a Vi-CELL cell viability analyzer (Beckman Coulter, Brea, CA, U.S.A.).
Time-lapse light microscopy movies
NCI-H1299 and A549 cells were plated and transfected 24 hours later with either control siRNA (NT1) or Wee1 siRNA (#8). Plates were placed in an incubator and images were captured every 30 minutes using IncuCyte™ High Definition (HD) Imaging (Essen Bioscience, Ann Arbor, MI, U.S.A). Movies were extracted for the indicated time frames (3 days of siRNA treatment) at a rate of 10 frames per second.
Analysis of cell cycle by flow cytometry
At varied times after transfection with 5nM siRNA, adherent cells were trypsinized and combined with any floating cells present, then washed with cold PBS. Cells were then stained with 0.5 ml PBS containing 50 μg/ml propidium iodide (PI), 0.1 mg/ml RNase A, 0.1% BSA, and 0.1% Triton-X100 for 20 min and cell cycle distribution was analyzed using a BD LSR-II flow cytometer (BD Biosciences, San Jose, CA, U.S.A.).
Double thymidine block
H1299 cells were treated with 2 mM thymidine (Sigma) and blocked for 20 hrs. Cells were then released by washing with PBS and feeding with regular culture medium. Four hours after the first release, cells were transfected with 5nM siRNA and incubated for another 4.5 hrs before the second treatment of 2 mM thymidine. Cells were blocked with thymidine for another 14.5 hrs and released again into regular medium. At the second release (t = 0), cells should be synchronized at G1/S phase boundary of the cell cycle. Cell samples were collected at varied time points for cell cycle and Western blot analysis as described above.
Click-iT EdU flow cytometry assay
NCI-H1299 cells were plated in 6-well plate and transfected with 5 nM siRNA as described above. After 24 or 48 hr incubation, cells were labeled with 10 μM EdU (Invitrogen) for 30 min. Both floating and attached cells were then collected for fixation, Click-iT reaction, and cell cycle staining following manufacturer’s protocol. Cell samples were analyzed for DNA content and EdU level using BD LSR-II flow cytometer.