Figure 3.

Non-phosphorylatable BAG3-GFP^DMB expression in BAG3-depleted cells, as phospho-mimetic BAG3-GFP^T285D, cannot rescue spindle dynamics and mitotic rounding defects. (A) Depletion-rescue experiments were performed in HeLa-RFP-H2B transfected with siBAG3 (3’UTR_1) or control siRNA and transduced with recombinant adenoviruses driving expression of BAG3-GFP proteins. Cells were synchronized in mitosis by a double thymidine block. (B) Total cell extracts were analyzed by Western blots using anti-BAG3 and anti-GAPDH; levels of endogenous BAG3 depletion (>75% reduction) and exogenous BAG3-GFP levels were estimated by loading increasing amounts of control cell extract (siCtl, 1/8, 1/4, 1/2, 1), or from an extract of BAG3-depleted cells transduced with Ad-BAG3-GFP, respectively. (C) Representative spinning disk confocal time-lapse sequences of cells from (B) transduced with BacMam-RFP-α-tubulin, showing normalization of spindle dynamics in BAG3-depleted cells upon reintroduction of BAG3-GFP^WT, but not by non-phosphorylatable BAG3-GFP^DMB expression or phosphomimetic BAG3-GFP^T285D. The green asterisks designate a spindle pole showing normal dynamic in a BAG3-depleted cells expressing wild-type BAG3-GFP, while magenta asterisks designate a spindle pole showing abnormal motility in a BAG3-depleted cell expressing BAG3-GFP^DMB; Bar: 10 μm. The graph shows quantification of percentages of rounded cells with spindle dynamic defects defined as spindle rocking or stalled in mitosis ± spindle rocking; means ± SE of 92 to 525 cells from at least 3 independent experiments. Statistical significance was analyzed with the Fisher Exact Test ****: p < 0.0001. See also Videos S1–S5 for representative phenotypes. (D) Epifluorescence images showing the representative phenotype of a rounded mitotic cell at metaphase (siCtl) compared to a flat mitotic cell at metaphase (siBAG3 [3′UTR_2]); F-actin, tubulin, and DNA were stained using phalloidin (green), α-tubulin antibody (magenta) and Hoechst (white), Bar: 10 μm. The graph depicts percentages of cells with mitotic cell rounding defects; means ± SE of 600 to 622 cells from 3 independent experiments. Statistical significance was analyzed by the Fisher Exact Test ****: p < 0.0001; ns, not significant. See also Figure S2A for Western blots of BAG3 depletion levels and Figure S3 for impacts of BAG3-GFP constructs on the aggresomal targeting of ubiquitinated proteins.