teton hela cell line  (InvivoGen)

Journal: The Journal of Cell Biology

Article Title: Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

doi: 10.1083/jcb.201409151

Figure Lengend Snippet: Hsp72 promotes chromosomes congression and mitotic progression. (A) HeLa:EGFP–lamin A/mCherry-H2B cells were either mock-depleted, depleted with siRNAs against Hsp72 for 72 h, or treated with Hsp70i for 4 h, as indicated, before time-lapse imaging. Stills from representative videos are shown with times (minutes) from mitotic entry indicated. (B and C) Quantification of cells from A indicating the time from NEBD to last chromosome congressed (B) and last chromosome congressed to anaphase onset (C). Data represent cumulative frequencies (±SD). (D) HeLa cells were mock depleted, depleted of Hsp72 for 72 h, or treated with Hsp70i for 4 h, as indicated, and then processed for IF with α-tubulin and CENP-A antibodies. (E) Total spindle MT intensity of cells in D was plotted relative to that of mock-depleted cells. *, P < 0.05. (F) HeLa cells treated as in D before IF with α-tubulin antibodies; enlargements show spindle pole regions. (G) Distance between spindle poles in the z axis of HeLa cells treated as in D. For box and whisker plots, boxes represent the 25th and 75th percentile, the green and white lines represent the medians and means, respectively, and whiskers show the 10th and 90th percentiles. (H) HeLa cells stably expressing mCherry-H2B were arrested in mitosis for 4 h with MG132. Time-lapse imaging was used to follow metaphase plate alignment with times (minutes) from addition of DMSO or Hsp70i as indicated. In D and F, DNA was stained with Hoechst 33258. Data are means (±SD) of 100–300 cells. Bars: (A, D, F [right], and H) 10 µm; (F, left) 2 µm.

Article Snippet: For inducible expression of Flag-TACC3, TetOn HeLa cells (Takara Bio Inc.) were used.

Techniques: Imaging, Whisker Assay, Stable Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

doi: 10.1083/jcb.201409151

Figure Lengend Snippet: Hsp72 promotes recruitment of ch-TOG–TACC3 complexes to K-fibers. (A) HeLa cells treated as indicated were placed on ice for 10 min before IF with α-tubulin and CENP-A antibodies to reveal K-fibers. (B) K-fiber intensity is plotted relative to that in mock-treated cells. (C) HeLa cells treated as indicated were incubated with 50 µM monastrol for 4 h, placed on ice for 10 min, and then processed for IF with α-tubulin and CENP-A antibodies. (D) The length of K-fibers in the monopolar spindles is plotted. For box and whisker plots, boxes represent the 25th and 75th percentile, the green and white lines represent the medians and means, respectively, and whiskers show the 10th and 90th percentiles. (E and F) HeLa cells treated as indicated were processed for IF with antibodies against α-tubulin and ch-TOG (E) or TACC3 (F). (G and H) The intensity of ch-TOG (G) and TACC3 (H) relative to tubulin from cells in E and F is indicated. (I) K-fiber intensities were measured as in B after Hsp72 or mock depletion and expression of siRNA-resistant Hsp72 constructs. siMock, mock siRNA; WT, wild type. (J) Hsp72 IPs were prepared from mitotic cells treated with or without Hsp70i and analyzed by Western blotting with the antibodies indicated. (K) The amount of proteins precipitated in J is quantified relative to that in DMSO-treated Hsp70 IPs. (L) Flag IPs prepared from mitotic cells induced to express Flag-TACC3 with doxycycline and treated with Hsp70i were analyzed by Western blotting for Flag, Hsp72, and ch-TOG. (M) The amount of proteins precipitated in L is quantified relative to DMSO-treated Flag IPs. In A, C, E, and F, DNA was stained with Hoechst 33258. Data are means (±SD) of 100–300 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 10 µm.

Article Snippet: For inducible expression of Flag-TACC3, TetOn HeLa cells (Takara Bio Inc.) were used.

Techniques: Incubation, Whisker Assay, Expressing, Construct, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

doi: 10.1083/jcb.201409151

Figure Lengend Snippet: Nek6 phosphorylation targets Hsp72 to the mitotic spindle. (A) Schematic representation of Hsp72 showing nucleotide (NBD) and substrate (SBD) binding domains and sequence alignment of the human Hsp70 family around threonine-66. Bold indicates conserved residues. N, N terminus; C, C terminus. (B) Purified Hsp72 wild-type (WT) or T66A proteins incubated with or without purified Nek6 were subjected to Western blotting with total Hsp70 and Hsp70-pT66 (pHsp70) antibodies (C) HEK 293 cells were either untransfected (lane 1) or transfected with Flag-Hsp72 wild type (lanes 2–4) and left asynchronous (As) or synchronized at S or M before immunoprecipitation with Flag antibodies. Lysates and Flag IPs were analyzed by Western blotting with Flag and pHsp70 antibodies. (D) Lysates and Hsp72 IPs from HEK 293 cells that were either asynchronous or synchronized in different cell cycle stages were analyzed by Western blotting as indicated (pHH3, phospho–histone H3). (E) Lysates and Hsp72 IPs from HEK 293 cells transfected with Nek6, Nek7, or GAPDH siRNAs were analyzed by Western blotting as indicated. (F) HeLa cells were processed for IF with α-tubulin and Hsp72 antibodies. (G) HEK 293 cells transfected with GFP-tagged wild-type and catalytically inactive (K71E) Hsp72 were analyzed by IF with α-tubulin (red) and GFP (green) antibodies. (H and I) HeLa cells were processed for IF with pHsp70 and α-tubulin, CenpA, or CenpE antibodies, as indicated. In I, zoomed-in views of kinetochores are shown on the right from the boxed regions in the cells on the left. (J) HeLa cells were mock- or Nek6-depleted before IF with Hsp72 and α-tubulin antibodies. (K) The intensity of Hsp72 at metaphase spindle poles was quantified relative to α-tubulin. In F–J, DNA was stained with Hoechst 33258 (blue). siMock, mock siRNA. Bars: (F–I [left images] and J) 10 µm; (I, right images) 0.5 µm.

Article Snippet: For inducible expression of Flag-TACC3, TetOn HeLa cells (Takara Bio Inc.) were used.

Techniques: Phospho-proteomics, Binding Assay, Sequencing, Purification, Incubation, Western Blot, Transfection, Immunoprecipitation, Staining

Journal: The Journal of Cell Biology

Article Title: Hsp72 is targeted to the mitotic spindle by Nek6 to promote K-fiber assembly and mitotic progression

doi: 10.1083/jcb.201409151

Figure Lengend Snippet: Hsp72-T66E rescues mitotic defects that arise upon Nek6 depletion. (A) HeLa cells were mock or Nek6 depleted for 48 h before transfection with Flag-Hsp72 constructs as indicated for 24 h and IF with Flag and phospho-H3 antibodies. The mitotic index of transfected cells was counted by microscopic analysis of chromatin. (B) HeLa cells transfected with Flag-Hsp72 constructs indicated for 24 h were stained with Flag and α-tubulin antibodies and scored by IF for the occurrence of nuclear defects. Data in A and B are means (±SD) of 100–300 cells. (C) HeLa cells were treated as in A, placed on ice for 10 min, and analyzed by IF with antibodies against α-tubulin to reveal K-fibers (green) and Flag to detect transfected cells. (D) Total K-fiber intensity from C is plotted relative to that in mock-treated samples. (E and F) HeLa cells were treated and processed as in C but with ch-TOG (E) and TACC3 (F) antibodies. (G and H) Total ch-TOG and TACC3 intensity from E and F is plotted relative to that in mock-treated samples. Error bars show means ± SD. In C, E, and F, DNA was stained with Hoechst 33258 (blue). siMock, mock siRNA; WT, wild type. *, P < 0.05. Bars, 10 µm.

Article Snippet: For inducible expression of Flag-TACC3, TetOn HeLa cells (Takara Bio Inc.) were used.

Techniques: Transfection, Construct, Staining