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Fig. 3. Poleward transport of SAC proteins is suppressed upon dynein depletion. Cells were depleted of dynein (siDHC), spindly (siSpindly), or both (siDHC/siSpindly), subjected to ATP reduction assay, and stained for (A) Mad2, (B) BubR1 and (C) Bub3 (analyzed using the EGFP-Bub3 expressing cell line). Note that in all cases, SAC proteins accumulate on unaligned but not on aligned kinetochores. Mad1 behaves identically to Mad2 (not shown); and Bub1 behaves identically to Bub3 (not shown). Bars, 5 lm.

Journal: FEBS letters

Article Title: Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles.

doi: 10.1016/j.febslet.2014.07.011

Figure Lengend Snippet: Fig. 3. Poleward transport of SAC proteins is suppressed upon dynein depletion. Cells were depleted of dynein (siDHC), spindly (siSpindly), or both (siDHC/siSpindly), subjected to ATP reduction assay, and stained for (A) Mad2, (B) BubR1 and (C) Bub3 (analyzed using the EGFP-Bub3 expressing cell line). Note that in all cases, SAC proteins accumulate on unaligned but not on aligned kinetochores. Mad1 behaves identically to Mad2 (not shown); and Bub1 behaves identically to Bub3 (not shown). Bars, 5 lm.

Article Snippet: Dynein knockdown was performed using validated siRNA oligoduplexes against Dynein Heavy Chain [36] (Santa Cruz Biotechnology, sc-43738).

Techniques: Staining, Expressing

Fig. 4. KMN components are dynein-dependent cargoes. (A) EGFP-Bub3 expressing HeLa cells were subjected to ATP reduction assay and stained for Hec1. (Left) Upon the assay, Hec1 redistributes to spindle poles with a pool left on kinetochores, mirroring the behavior of Bub3. (Right) Dynein depleted cells (siDHC) were subjected to ATP reduction assay and stained for Hec1 and CREST. Note the absence of Hec1 at spindle poles in cells depleted of dynein. (B) HeLa cells were transiently transfected with pBABEblast YFP-Mis12 construct and subjected to ATP reduction assay. (Left) Upon the assay, Mis12 redistributed to spindle poles with a pool left on kinetochores. (Right) YFP-Mis12 expressing cells were depleted of dynein (siDHC) and subjected to ATP reduction; CREST was used as kinetochore marker. Note the absence of Mis12 at spindle poles in cells depleted of dynein PM = Prometaphase; M = Metaphase; Bars, 5 lm.

Journal: FEBS letters

Article Title: Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles.

doi: 10.1016/j.febslet.2014.07.011

Figure Lengend Snippet: Fig. 4. KMN components are dynein-dependent cargoes. (A) EGFP-Bub3 expressing HeLa cells were subjected to ATP reduction assay and stained for Hec1. (Left) Upon the assay, Hec1 redistributes to spindle poles with a pool left on kinetochores, mirroring the behavior of Bub3. (Right) Dynein depleted cells (siDHC) were subjected to ATP reduction assay and stained for Hec1 and CREST. Note the absence of Hec1 at spindle poles in cells depleted of dynein. (B) HeLa cells were transiently transfected with pBABEblast YFP-Mis12 construct and subjected to ATP reduction assay. (Left) Upon the assay, Mis12 redistributed to spindle poles with a pool left on kinetochores. (Right) YFP-Mis12 expressing cells were depleted of dynein (siDHC) and subjected to ATP reduction; CREST was used as kinetochore marker. Note the absence of Mis12 at spindle poles in cells depleted of dynein PM = Prometaphase; M = Metaphase; Bars, 5 lm.

Article Snippet: Dynein knockdown was performed using validated siRNA oligoduplexes against Dynein Heavy Chain [36] (Santa Cruz Biotechnology, sc-43738).

Techniques: Expressing, Staining, Transfection, Construct, Marker