Structural requirements on BAG3 for restoration of normal spindle dynamics in BAG3-depleted cells.

<p>(A) Schematic of BAG3-GFP proteins bearing mutations or deletions of functional domains and a glycine/serine-rich flexible linker peptide (GSGS) that distances the GFP moiety from the rest of the BAG3, as reported in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.ref075" target="_blank">75</a>]; WT, wild type. (B, D) BAG3-GFP and GFP IPs were prepared from HeLa cells infected with recombinant adenoviruses driving expression of BAG3-GFP or GFP proteins (Ad-BAG3-GFP or Ad-GFP) using anti-GFP, and analyzed by Western blot; levels of endogenous BAG3, HSC70 and HSPB8 in total cell extracts are shown (Input). (C) Schematic of the protocol developed to achieve knockdown-rescue experiments using siBAG3_3, Ad-BAG3-GFP and BacMam-RFP-a-tubulin. Total cell extracts were prepared 46 h after transfection during the 2^nd release and analyzed by Western blot with anti-BAG3 and anti-HSPB8 antibodies. Please note that individual BAG3-GFP proteins were introduced at near the endogenous level of BAG3 and could restore HSPB8 levels in BAG3-depleted cells, except for BAG3 (IPV) unable to bind to HSPB8. (E) Representative confocal time-lapse sequences of RFP-a-tubulin in cells from C, showing restoration of spindle dynamics upon introduction of WT BAG3-GFP, but not of BAG3-GFP (IPV) in BAG3-depleted cells; red and yellow asterisks designate the position of spindle poles (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.s011" target="_blank">S5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.s012" target="_blank">S6</a> Movies); Bars, 10 μm. Mitotic cells were imaged by spinning disk confocal microscopy for 60 to 90 min at ~1.5 min intervals. Please refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.s010" target="_blank">S4</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.s014" target="_blank">S8</a> Movies for samples of the phenotypes observed. (F, G) Quantification of cells from C, indicating percentages of cells with abnormal mitosis defined as spindle rocking, stalled in mitosis +/- spindle rocking, or chromosome misalignment; means +/- SE from 88 to 184 cells from 3 to 5 independent experiments as indicated (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005582#pgen.1005582.s023" target="_blank">S1 Dataset</a>). Statistical significance was analyzed with the Exact Fisher Test; (F) siBAG3_3 + WT versus siBAG3_3 + GFP: ****, p < 0.0001; siBAG3_3 + WT versus siBAG3_3 + IPV: **, p = 0.001; siBAG3_3 + WT versus siBAG3_3 + ΔPXXP: ***, p = 0.0003; siBAG3_3 + WT versus siBAG3_3 + ΔBAG: ***, p = 0.003; (G) siBAG3_3 + WT versus siBAG3_3 + GFP: *, p = 0.005; siBAG3_3 + WT versus siBAG3_3 + R480A: ns, p = 0.884; siBAG3_3 + WT versus siCtl + GFP: ns, p = 0.46.