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. 2015 Oct 23;11(10):e1005582. doi: 10.1371/journal.pgen.1005582

Fig 4. Structural requirements on BAG3 for restoration of normal spindle dynamics in BAG3-depleted cells.

Fig 4

(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 [75]; 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 2nd 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 (S5 and S6 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 S4–S8 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 (S1 Dataset). 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.