Fig. 2. CDK11 depletion causes premature separation of condensed sister chromatids. (A,B) Partial depletion of CDK11 causes chromosome misalignment and lagging chromosomes but does not block mitotic progression. (A) Immunofluorescence analysis of HeLa cells transfected with either a CDK11 siRNA A1361 oligonucleotide or control siRNA oligonucleotide and cultured for 48 hours before immunofluorescence staining with anti--tubulin (red) and anti-CREST (green) antibodies and DAPI (blue). Bar, 5 µm. (B) Determination of the frequency of chromosome alignment and segregation. More than 100 mitotic cells from each treatment group (si-A1361, n=138; control, n=198) were analyzed for the presence of misaligned and lagging chromosomes. In this representative experiment, 29% of the CDK11 A1361 siRNA cells had misaligned and/or lagging chromosomes whereas only 1.5% of the control cells exhibited this phenotype. (C) Giemsa-stained mitotic chromosome spreads for cells treated with either control siRNA expression vector or CDK11 siRNA expression construct 2184 analyzed 48 hours after transfection. Bar, 5 µm. (D) CDK11 depletion results in premature sister chromatid separation. Data from a representative experiment in which cells were transfected with either the control or CDK11 siRNA expression vector and 100 mitotic cells were scored for chromosome appearance. Chromosome appearance was categorized as I: partially condensed with closed arms; II: condensed and separated arms with kinetochore linked; III: both condensed arms and kinetochores separated, still paired; IV: condensed and separated sister chromatids no longer paired. (Noco: nocodazole.) The mitotic index was determined from counting 1000 cells from each Giemsa-stained RNAi sample.