JCS007963 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Elimination of CDK11 through RNAi. (A) Schematic representation of the siRNAs used for CDK11 targeting. (B) Western blot analysis showing downregulation of the CDK11^p110 protein. HeLa cells were transfected either with CDK11 RNAi constructs pSUPER-A461, A1361 or A2184 and the control plasmid (pSUPER) by electroporation or with CDK11 siRNA oligonucleotides A1361 or control siRNA oligonucleotides by Dharmafectin1 (Dharmacon). After 48 hours, cell lysates were prepared and subjected to electrophoresis and western blot analysis using anti-CDK11 P1C antibody (Trembley et al., 2002).

• Supplemental Figure S2 -

  Fig. S2. CDK11 depletion results in mitotic arrest and cell death. HeLa cells treated with CDK11 RNAi or control RNAi for 48 hours were analyzed by time-lapse microscopy for 12 hours using a Nikon microscope equipped with an incubation chamber to maintain constant CO₂ and temperature. Top panel (control RNAi) shows that control HeLa cells form a clear metaphase plate during mitosis, and the duration of mitosis is approximately 1-1.5 hours. Lower panel (CDK11 RNAi) shows that CDK11 RNAi cells arrest at the mitotic stage without forming a clear metaphase plate and eventually undergo cell death after arresting in mitosis for more than 8 hours. Time in hours:minutes. Bar, 30 μm.

• Movie 1 -

  Movie 1. Time-lapse recording of mitotic progression of control RNAi H2B-GFP cells over a 100-minute period. Each frame represents 10 minutes.

• Movie 2 -

  Movie 2. Time-lapse recording of CDK11-depleted cells at 15-minute intervals over a 15-hour period demonstrates a prolonged mitotic arrest and eventual cell death. HeLa H2B-GFP cells were transfected with CDK11 RNAi. Recording was initiated 48 hours later using a Nikon confocal microscope equipped with a cell culture incubation chamber. Movie shows a representative CDK11 RNAi cell.