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. 2014 Jun 4;4(6):130108. doi: 10.1098/rsob.130108

Figure 2.

Figure 2.

Rescue of TAO1 depletion-induced chromosome congression defects. (a) Schematic showing drug treatment regime for analysing kinetochore–microtubule attachment status. HeLa FRT/TO:YFP-TAO1siRes cell line was treated with siRNA for 72 h, tetracycline-induced for 24 h and exposed to MG132 for 90 min prior to immunostaining cells with antibodies against GFP (for YFP) and tubulin, CREST antisera (centromeric marker) and DAPI (to stain for DNA). (b) Immunoblot of lysates of HeLa FRT/TO:YFP-TAO1siRes cell line showing tetracycline-inducible expression of siRNA-resistant wild-type TAO1 (YFP-TAO1siRes) (marked as ##) in TAO1-St1 or Control-St siRNA-treated cells. In Control-St siRNA-treated cells, endogenous TAO1 can be visualized (marked as #). β-Tubulin (β-tub) was used as loading control. (c) Graph showing chromosome congression efficiency in TAO1-St1 or Control-St siRNA transfected and MG132-treated HeLa FRT/TO:YFP-TAO1siRes cell line with or without YFP-TAO1siRes expression. n = number of cells from three independent experiments. p-Values representing significance level were derived using proportion test. Asterisk (*) indicates significant difference. (d) Deconvolved immunoflourescence images of Control-St or TAO1-St1 siRNA transfected HeLa FRT/TO:YFP-TAO1siRes cells with and without YFP-TAO1siRes expression. Cells were transfected with siRNA as indicated and tetracycline-induced as shown in (a). Scale bar, 5 μm. Yellow arrowheads mark uncongressed chromosomes.