Figure 6. Targeted down-regulation of centromeric α-satellite transcript results in mitotic defect.

(A) Six days post-transfection of empty vector, control scrambled (shRNA^scram), or α-satellite shRNA (shRNA^sat1, shRNA^sat2), cell morphology was observed by phase contrast microscopy. Scale bar: 100 pixels. (B) Cells were treated as in (A) and stained for β-actin (green) and DAPI (cyan) to reveal alterations in cell morphology. Scale bar: 5 μm. (C) Cells were treated as in (A), synchronized in mitosis and stained for α-tubulin (green), CENP-B (red) and DAPI (cyan/blue). Three independent experiments were performed and in each, a minimum of 30 cells were analyzed per slide. Scale bar: 5 µm. IF: immunofluorescence.

Figure 6—figure supplement 1. Down-regulation of centromeric RNAs by a targeted shRNA approach.

(A) The centromeric α-satellite consensus sequence was used to design α-satellite shRNA (shRNA^sat1 and shRNA^sat2) compared to control shRNA containing a scrambled sequence (shRNA^scram). Residues of the CENP-B box are marked by a black box, and the sequences of shRNA^sat1/2 are underlined. (B) Centromeric α-satellite transcript level after transfection of shRNA^scram, shRNA^sat1, or shRNA^sat2 was measured by qtPCR. The graph represents the average of three biological replicates, with relative expression (±SD) of α-satellite transcript in shRNA^sat-transfected cells compared to shRNA^scram-transfected cells.

Figure 6—figure supplement 2. Down-regulation of centromeric transcripts does not induce senescence.

Six days post-transfection of empty vector, control scrambled (shRNA^scram) or α-satellite shRNA (shRNA^sat1 or shRNA^sat2), the potential senescent status of transfected cells was tested by a β-galactosidase assay. Senescent BJ cells were used as positive control (blue).