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

Fig 5. Defects in spindle dynamics upon depletion of HSPB8 correlate with impaired localization of BAG3 at G2/M.

Fig 5

(A) Representative Western blots of total cell extracts prepared from HeLa-RFP-H2B transfected with control siRNA (siCtl) or HSPB8-specific siRNAs (siHSPB8 #1 and #2), showing reduction of HSPB8 levels of ~75%; vinculin levels: loading controls. (B) Confocal time-lapse sequences from HeLa-RFP-H2B cells transfected with siHSPB8_2 and transduced with BacMam-GFP-a-tubulin, which show aberrant spindle rocking; colored asterisks designate spindle poles. Cells were synchronized by the double Thymidine block method and imaged by spinning disk confocal microscopy for 60 min at ~1.5 min intervals; Bars, 10 μm. (C) Quantification of cells from B, indicating abnormal mitosis defined as spindle rocking, stalled in mitosis +/- spindle rocking, or others (chromosome misalignment or multipolar spindle); means +/- SE from at least 281 cells from at least 3 independent experiments, as indicated (S1 Dataset). (D-E) Western blots of extracts prepared from asynchronous HeLa-RFP-H2B cells (AS), arrested in mitosis by a nocodazole-block (100 ng/ml, 16 h), or arrested at the G2/M border by a Thymidine/RO-3306-block (8 μM RO-3306, 18 h), showing hypophosphorylated BAG3 (AS, G2/M), hyperphosphorylated BAG3 (M), and cyclin B1. (E) RO-3306-treated cells that have been released in fresh medium show BAG3 supershifted band as cells exited the G2-block; AS: asynchronous. GAPDH levels: loading controls. (F, G) Deconvolved image stacks of G2-arrested HeLa cells transducted with Ad-BAG3-GFP and Ad-HSPB8-RFP, showing robust enrichment of BAG3-HSPB8 to centrosomes and the surrounding perinuclear material (red arrows). Please note that mutation of HSPB-binding domains (BAG3 IPV) like depletion of HSPB8 (siHSPB8) markedly impaired recruitment of BAG3-GFP to a defined perinuclear space at G2/M (black arrows). (H) Western blots of total extracts prepared from siRNA-treated HeLa cells arrested at prometaphase by treatment with nocodazole (100 ng/ml, 16 h), showing partial supershift of BAG3 in cells depleted of HSPB8; AS: asynchronous cells.