RPE1 (human retinal pigmented epithelial cells stably transfected with hTERT) were cultured in DMEM/F-12 (D6421) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA). HFF-hTERT (human foreskin fibroblasts) and NIH-3T3 (mouse fibroblasts) cells were maintained in DMEM media with 2 mM l-glutamine and 10% FBS. U2OS (human osteosarcoma) cells were maintained in McCoy's media (16600082; Invitrogen, Carlsbad, CA) with 10% FBS. All cultures were grown at 37°C with 5% CO2. All culture media and supplements were purchased from Sigma-Aldrich (St. Louis, MO), and cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
The following antibodies were used in this study: Eg5, rabbit anti-Eg5 (AKIN03; Cytoskeleton, Denver, CO); actin, rabbit anti-actin (AAN01; Cytoskeleton); Eg5, mouse anti-Eg5 (627802; BioLegend, San Diego, CA); RPS5, mouse anti-RPS5 (AB58345; Abcam, Cambridge, MA); RPL10A, mouse anti-RPL10A (Ab55544; Abcam); GAPDH, rabbit anti-GAPDH (14C10) (2118; Cell Signaling, Beverly, MA); calnexin, rabbit anti-calnexin (Stressgen, Enzo Life Sciences, San Diego, CA); caspase-3, rabbit anti–caspase 3 (8G10) (9665; Cell Signaling); FLAG, mouse anti-FLAG (4049; Sigma-Aldrich); HA, mouse anti-HA (1 583 816; Roche, Indianapolis, IN); RPL4, rabbit anti-RPL4 (Proteintech Group, Chicago, IL); and phospho-H3, rabbit anti–phospho-H3 (Upstate, Millipore, Billerica, MA).
The following times and concentrations were used: 4 h, 130 μM monastrol (Tocris Bioscience, Ellisville, MO; batch 3); 2 h, 12 μM nocodazole; 2 h, 0.002 mg/ml Colcemid; 0.1 mg/ml CHX; 1 h, 0.05 mM arsenite; 1 h, 3 μM dimethylenastron (Alexis Biochemicals, San Diego, CA); and 4 h, 1.5 μM S-trityl-l-cysteine (Alexis Biochemicals). Final DMSO concentration for control experiments were as follows: experiments using monastrol and nocodazole, 0.04%; for dimethylenastron, 0.009%; for S-trityl-l-cysteine, 0.0003%. All reagents were purchased from Sigma-Aldrich, unless otherwise noted, and dissolved in DMSO, except for arsenite and Colcemid, which were purchased predissolved. For washout experiments, cells were treated for 4 h with monastrol, followed by three washes in FBS and a 4-h recovery.
Cells were reverse transfected for 24 h (unless specified) with 1.5 μg/60 mm tissue culture plate of siRNA using HiPerFect Transfection Reagent (Qiagen, Valencia, CA), following the manufacturer's protocol. Fluorescently labeled scrambled siRNA (siControl) was used (1022563) as the control. All reagents were purchased from Qiagen unless specified otherwise. Eg5 siRNA#1 (SI02653770) and Eg5 siRNA#2 (s7904) (Ambion, Austin, TX) were used.
Bicistronic plasmid transfection (plasmid 18673; Addgene, Cambridge, MA) into U2OS cells was completed using FuGene6 (1814443; Roche) transfection reagent for 24 h following the manufacturer's protocol after a 12-h knockdown of Eg5. Eg5 was knocked down for a total of 36 h. Antibodies to HA or FLAG were used for immunoblot analysis, and quantitation of immunoblots was completed using Adobe Photoshop (San Jose, CA).
35S Met/Cys incorporation assays.
Cells were grown in 60-mm plates, and the media were replaced with DMEM without methionine and cysteine (D0244; Sigma-Aldrich) plus 5% dialyzed FBS (F0392; Sigma-Aldrich), 2 mM l-glutamine, and the addition of 100 μCi/ml of 35S Met/Cys (NEG072007MC; PerkinElmer, Waltham, MA) and incubated for 30 min at 37°C. To stop reactions, 0.1 mg/ml CHX was added, and cells were trypsinized and washed in PBS prior to cell lysis in RIPA buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP-40). Protein samples were split in half; the first half was subjected to a Lowry assay and/or separated on SDS–PAGE for confirmation of equal loading or of knockdown of the specified protein. The second half was subjected to scintillation counting, in which duplicate samples of each lysate were placed on GF/C filters (28497-743; VWR, Radnor, PA), washed three times with 2 ml of 10% TCA and once with 100% ethanol, and dried before analysis.
Fractionation of cell lysates.
For fractionation of cell lysates into cytosolic and ER fractions, a digitonin fractionation protocol was used. Briefly, after the 35S Met/Cys incorporation assay cells were trypsinized and washed in PBS and the cell membrane was broken open by pipetting 25 times with a cut 200-μl pipette tip in digitonin buffer solution (10 mM 1,4-piperazinediethanesulfonic acid [PIPES; pH 6.8], 300 mM sucrose, 3 mM MgCl2, 5 mM EDTA, 0.01% digitonin, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Lysates were incubated for 8 min on ice and centrifuged at 3000 × g for 4 min, and the cytosolic fraction was removed for analysis. The pellet was washed once in PBS, centrifuged, and resuspended in RIPA buffer to retain the membrane fraction for analysis.
Polysome profiling: 10–45% sucrose gradients
Between 20 and 30 million RPE1 cells were incubated with or without 0.1 mg/ml CHX for 10 min prior to trypsinization. (Samples that were treated with CHX are labeled +CHX, whereas samples that were not treated with CHX are labeled −CHX.) Cells were lysed (20 mM Tris-HCl [pH 7.2], 130 mM KCl, 30 mM MgCl2, 2.5 mM DTT, 0.2% NP-40, 0.5% sodium deoxycholate, 0.1 mg/ml cycloheximide, 0.2 mg/ml heparin, 1 mM PMSF), incubated for 15 min on ice, the DNA pellet was removed by centrifugation, and a Lowry assay was completed to ensure equal loading onto the gradient. The lysates were placed on top of a 10–45% (wt/wt) sucrose gradient (10 mM Tris-HCl [pH 7.2], 60 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol [DTT], 0.1 mg/ml heparin), and samples were centrifuged at 27,000 rpm for 2.5 h at 4°C using a Beckman L7 Ultracentrifuge (model L7-65) in a Sorvall AH629 rotor. Gradients were fractionated by upward displacement through an ISCO UA-5 with constant UV monitoring at an absorbance of 254 nm.
In the absence of MgCl2 and in the presence of EDTA, the experiment was completed as described except that MgCl2 was omitted from the lysis buffer and the sucrose gradients and 2 mM of EDTA was added.
Immunoblot analysis of polysome profiling
For immunoblot analysis of 10–45% sucrose gradients, fractions representing each of the ribosomal subunits and/or ribosomes were pooled together. For extraction of proteins, a final concentration of 20 mM Tris, pH 7.5, was added, followed by the addition of 15–30 μl StrataClean resin (Stratagene, Santa Clara, CA). Samples were then rotated at room temperature for 30 min prior to centrifugation; pelleted beads were resuspended in 2× SDS loading dye, and samples were boiled for 10 min to elute proteins before subjection to SDS–PAGE (15% gel).
Polysomes/monosomes ratio calculations
For the calculation of P/M ratio, each polysome profile graph was photocopied and enlarged to 151%. Next, the area under each ribosomal peak (40S, 60S, 80S, and polysomes) was estimated by weighing paper cutouts of the profiles. The baseline was chosen based on the lowest point on each profile. Each peak was cut out (in triplicate) and weighed (in triplicate) on an analytical balance (Adventurer SL AS64; Ohaus, Pine Brook, NJ). Averages of the area under each ribosomal peak were calculated, and the average weight of the polysomes was divided by the average weight of the monosomes (80S ribosomes) per profile to calculate the P/M ratio. P/M ratios represent the exact polysome profile shown.
RPE1 cells were serum starved for 32 h in DMEM media without FBS. Fresh DMEM was added every 6 h, prior to CHX addition, cell lysis, and polysome profiling.
Immunoprecipitation (IP) was completed following the manufacturer's protocol, with these exceptions: 10–15 million RPE1 cells were lysed (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES; pH 7.5], 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 2.5 mM ethylene glycol tetraacetic acid [EGTA], 10% glycerol, 1 mM NaF, 0.1 mM Na3VO4, 10 mM β-glycerophosphate, 1 mM PMSF), and the DNA pellet was removed by centrifugation. Each tube received 2 μg of rabbit anti-Eg5 antibody, nonimmune serum, or 4 μl of 30% glycerol (−AB; Eg5 antibody was reconstituted in 30% glycerol) for the immunoprecipitation. For the rpS5 IP, 2.5 μg of antibody was added. Samples were subjected to SDS–PAGE (12 or 15% gel). Each IP represented 50% of the total; input represented 10% for Eg5 immunoprecipitations and 15% for the rpS5 immunoprecipitation. Nonimmune serum was used as a negative control. Protein A beads were used in the reaction (Amersham Biosciences, GE Healthcare, Piscataway, NJ).
Ribosome half-transit time assay
Thirty million cells were trypsinized, washed, and resuspended in 3 ml of BME (B1522; Sigma-Aldrich) plus 10% dialyzed FBS and 2 mM l
-glutamine for 20 min prior to the addition of 10 μCi/ml 35
S Met/Cys. At each time point, 500 μl of cells were removed, placed in an ice-cold tube with 500 μg/ml CHX, and incubated on ice. Cells were centrifuged at 4°C, washed with ice-cold PBS containing CHX, recentrifuged, lysed in 1 ml of polysome profiling buffer with the following changes (0.02 M Tris [pH 7.2], 0.130 M KCl, 0.03 M MgCl2
, 1% NP-40, 0.05% sodium deoxycholate, 0.2 mg/ml heparin, 0.25 mg/ml CHX, 1 mM DTT, 1 mM PMSF, RNasin Inhibitor [Promega]), and incubated on ice for 15 min, and the DNA was removed by centrifugation prior to splitting the lysates: 500 μl of the lysate was saved (PMS fraction) containing total proteins, whereas the other 500 μl was placed on a stepwise 20% sucrose buffer and 60% sucrose cushion. Samples were centrifuged in an S100-AT5 ultracentrifuge rotor at 55,000 rpm for 27 min, after which 500 μl of the sample was removed (PRS) containing completed proteins released from the ribosomes. The PMS and PRS fractions were then TCA precipitated on GF/C filters (as described earlier) and subjected to scintillation counting. Half-transit times were calculated by comparing the incorporation of radioactivity into total proteins and completed proteins graphed over time. This assay was completed by combining various protocols from different publications (
Ruvinsky et al., 2005
Saini et al., 2009
In vitro microtubule-binding assays
Purified tubulin (isolated from bovine brains;
Moyer et al., 1996
) was thawed on ice with the addition of an equal volume of 1× PM (10 mM PIPES [pH 7.0], 5 mM magnesium acetate, and 1 mM EGTA [pH 7.0)] buffer and 2 mM GTP, followed by a 30-min centrifugation at 10,000 × g
(4°C). The supernatant was removed, grow buffer (1× PM buffer, 0.1 mM taxol [Sigma-Aldrich], 5 mM GTP) was added to it in a 4:1 ratio (tubulin:grow buffer), followed by a 15-min incubation (34°C) with rotation. Fractions representing the 80S ribosome from polysome profiling were pooled together, inverted, and split: half received the binding reaction (5× PM buffer, 100 mM NaCl, 0.04 mM taxol, 1 mM GTP) plus 12% tubulin and was incubated with rotation for 45 min (34°C), whereas the other half received the binding reaction without tubulin, taxol, or GTP and remained at 4°C. After incubation, cells were centrifuged at 10,000 × g
for 30 min at 34 or 4°C. The supernatant was removed (containing non–microtubule-bound proteins), pellets were washed twice in PBS and recentrifuged, and the supernatant was discarded. The pellet contained microtubules and microtubule-bound proteins; 20% of the supernatant and 50% of the pellet were loaded on 12% SDS–PAGE gels.
Mitotic index analyses by DAPI
Cells were fixed in 0.2% Triton-X 100 in 4% paraformaldehyde for 15 min prior to the addition of DAPI. A minimum of 300 cells was counted per trial, and the experiment was completed in triplicate. All cells were analyzed on an Olympus (Center Valley, PA) BX60 epifluorescence microscope with a 100× oil immersion objective, unless specified. A Hamamatsu (Hamamatsu, Japan) Argus-20 charge-coupled device (CCD) camera was used to record images.
Mitotic index analyses by phospho-H3 staining
Cells were fixed in 0.2% Triton-X 100 in 4% paraformaldehyde for 30 min prior to a 30-min block in 1.5% BSA in PBST. Phospho-H3 antibody was diluted (1:500) in blocking buffer and incubated on cells for 30 min prior to three washes in PBS, the addition of secondary for 30 min, three additional washes in PBS, and DAPI staining. A minimum of 300 cells was counted per trial, and the experiment was completed in triplicate. All cells were analyzed on an Olympus BX60 epifluorescence microscope with a 100× oil immersion objective, unless specified. A Hamamatsu Argus-20 CCD camera was used to record images.
Cell cycle analysis
RPE1 cells were grown to 70% confluency in 100-mm Petri dishes before monastrol treatment. Cells were incubated for either 4 or 24 h in monastrol prior to trypsinization, two washes in PBS, and fixation in 100% ice-cold ethanol and storage overnight at 4°C. Next, the cells were pelleted by centrifugation, followed by two washes in PBS and incubation with 100 μl of 100 μg/ml RNase A for 8 h. Finally, 500 μl of a 50 μg/ml solution of propidium iodide was added to cells immediately before analysis by flow cytometry.
Caspase-3 antibody was used to determine apoptosis after a 4- to 16-h monastrol treatment; 16 h of 1 μM staurosporine was used as a positive control to demonstrate caspase-3 cleavage.
Metabolic activity assay
A CellTiter 96 AQueous One Solution Cell Proliferation Assay was performed following the manufacturer's protocol (Promega, Madison, WI) after a 24-h knockdown of Eg5 (siEg5#1 or siEg5#2) or after Eg5 inhibition or recovery by monastrol.