Ab28144

U2OS cells were plated onto coverslips in 6-well plates and grown until 60%–70% confluency before transfection of GFP-tagged APC fragment sequences for 48 h (see Section 4.2). Cells were fixed with methanol:acetone (1:1) for 3 min, followed by three washes using PBS. Cells were blocked with 3% bovine serum albumin and stained with primary antibodies. To detect the centrosome, polyclonal PCNT (Abcam ab4448, 1:1500) and PCNT monoclonal (Abcam ab28144, 1:1500) were used. γ-tubulin (Sigma T5192, 1:800) and monoclonal γ-tubulin (Sigma T5326, 1:800) antibodies have also been used to yield a similar localization result. Cells were washed three times using PBS and subsequently incubated with the fluorescence secondary probes Alexafluor-594 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed extensively before they were mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). Cells were stained in the dark to prevent bleaching of GFP. Cells were visualized using Olympus BX-51 inverted microscope (Olympus) and optical sections were taken using an Olympus FV1000 confocal microscope (Olympus) at 60× magnification.

Cells were seeded onto coverslips, incubated for 20 h before drug treatment or viral transduction, and then fixed in PTEMF buffer containing 20 mM PIPES, 4% paraformaldehyde, 0.2% Triton-X, 10 mM EGTA and 1 mM MgCl₂ for 15 min. Primary antibodies included anti-HSP70 (GeneTex, Hsinchu, Taiwan or StressMarq, Victoria, Canada), anti-α-tubulin (GTX112141, GeneTex or T5168, Sigma, St. Louis, MO, USA), anti-γ-tubulin (T6557 or T3559, Sigma), anti-EB1 (E3406, Sigma), anti-pericentrin (ab4448 or ab28144, Abcam, Cambridge, UK), anti-CEP215 (IHC-00063, Bethyl, Cambridge, UK), anti-CEP164 (HPA037606, Sigma), anti-CEP152 (ab183911, Abcam), anti-PLK1 (No. 33-1700, Invitrogen), and anti-phospho-T210-PLK1 (PA1-126, ThermoFisher, Waltham, MA, USA). Alexa-Fluor 488-, 568-, 633-, or 647-conjugated goat anti-mouse or anti-rabbit IgG were purchased from Invitrogen, and Atto-488 goat anti-mouse IgG was from Sigma. Samples were mounted in Fluoromount-G from SouthernBiotech (Birmingham, AL, USA) for confocal or structured illumination microscopy (SIM) imaging. For ground state depletion (GSD) imaging, samples were mounted in DPBS (Sigma) containing 5% glucose, 100 mM 2-mercaptoethylamine (Sigma), 0.8 mg/ml glucose oxidase and 40 μg/ml catalase, and sealed with Twinsil (Picodent, #13001000, Wipperfürth, Germany).

The cells grown on coverslips were heat-treated and incubated at 37 °C for 72 h. Subsequently, the cells were fixed with 4% formaldehyde for 10 min, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Furthermore, the samples were blocked with 5% BSA in PBS for 60 min. After washing 3 times in PBS, the cells were incubated with primary antibodies overnight at 4 °C. Primary antibodies included rabbit anti-alpha tubulin (ab52866, Abcam, Cambridge, MA, USA) and mouse anti-pericentrin (ab28144, Abcam, Cambridge, MA, USA). Then, the cells were washed 3 times in PBS, followed by sequential incubation with secondary antibodies for 60 min at room temperature. Secondary antibodies included Goat anti-Rabbit IgG (H&L) conjugated with Alexa Fluor^® 594 (ab150080, Abcam, Cambridge, MA, USA) and Goat anti-Mouse IgG (H&L) conjugated with Alexa Fluor^® 488 (ab150113, Abcam, Cambridge, MA, USA). Finally, coverslips were mounted in PBS containing 1 μg/mL DAPI for 10 min for DNA staining. The stained cells were visualized and analyzed using a laser scanning confocal microscope (LSM780, Zeiss, Germany) with a 63X/1.4NA oil immersion objective lens and excitation wavelengths of 405 nm, 488 nm and 594 nm. All images were acquired using ZEN 2012 software.

Prostate cancer cells were plated on coverslips in 12‐well plates and transfected with siRNA for 24 h, followed by synchronization using thymidine/nocodazole blockage and released. Cells were fixed at different time points in PFA 4%, permeabilized with triton 0.5%, washed, blocked in PBS (3% BSA) and incubated with the anti‐beta‐tubulin (cat. 2146s; Cell Signaling) and anti‐pericentrin (Ab‐28144; Abcam) primary antibodies. Antigens were visualized using Alexa Fluor 488/594‐conjugated antibodies. Nuclei were stained with DAPI Vectashield mounting reagent (Vector Laboratories). Images were acquired on LSM‐780 confocal microscope at 63× magnification.

Primary antibodies anti-EEA1 (ab70521), anti-58K Golgi (ab27043), antipericentrin (ab28144), and anticatalase (ab110292) were purchased from Abcam, anti-TGN46-8 (SAB4200355) from Sigma and anti-Tom20 (sc-17764) from Santa Cruz Biotechnology. All primary antibodies were monoclonal mouse antibodies. Polyclonal secondary antimouse antibodies conjugated to Atto647N (50185, Sigma Aldrich) as well as isotype-specific secondary antimouse antibodies conjugated to Atto647N (610-156-041, Rockland Immunochemicals) and Alexa Fluor 568 (A-21124, Thermo Fisher Scientific) were used for detection. The nanoboosters—single-domain alpaca antibody fragments covalently coupled to fluorescent dyes—GFP-Booster Atto488 and RFP-Booster Atto594 (Chromotek) were used to boost eYFP and mCherry fluorescence, respectively. Live cell stains SiR-tubulin, SiR-actin, and SiR-lysosome were from Spirochrome and MitoTracker Deep Red FM was from Thermo Fisher Scientific.

Polyclonal rabbit antibodies specific for Samp1 previously described in Buch et al.^{12 (link)} were used at a dilution of 1:500. Mouse antibodies against anti-phosphoERK (pERK) and rabbit antibodies against Myf5 were kind gifts from I.Faye and J. Nedergaard, respectively. The following antibody concentrations were used: mouse anti-myogenin 1:100 (F5D, DSHB); mouse anti-myosin heavy chain 1:50 (MyHC) (Mf-20, DSHB); mouse anti-MyoD 1:1000 (SC 32758, SCBT); rabbit anti-Myf6 1:1000 (ab 82842, Abcam); mouse anti-β-actin 1:5000 (A 5441, Sigma); mouse anti-Lamin A/C 1:100 (131c3, SCBT); rabbit anti-emerin 1:500 (HPA000609, Atlas antibodies) mouse anti-pericentrin 1:1000 (ab 28144, Abcam); mouse anti-BrdU (Sigma-Aldrich); horseradish-peroxidase-coupled donkey anti-mouse IgG (NA931, GE health care) or donkey anti-rabbit IgG (NA934, GE health care); Alexa Fluor 488 goat anti-rabbit IgG (A11008, Invitrogen); Alexa Fluor 568 goat anti-mouse IgG (A11001, Invitrogen); Alexa Fluor 568 goat anti-rabbit IgG (A11011, Invitrogen).

Cells were seeded on coverslips in 24-well plates and, after 24 h, they were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilised with 0.2% Triton-X-100 for 2 min at RT. Samples were blocked with 3% Bovine Serum Albumin (BSA)/PBS solution for 1 h at RT and stained with anti-N6AMT1 (1:100, CQA1550, Cohesion Biosciences, London, UK), anti-N6AMT1 (1:100, HPA059242, Atlas antibodies, Bromma, Sweden), anti-N6AMT1 (1:100, 16211-1-AP, Proteintech, Rosemont, IL, USA), anti-N6AMT1 (1:100, PA5-121076, Invitrogen, Waltham, MA, USA), anti-N6AMT1 (1:100, ARP45845_P050, Aviva Systems Biology, San Diego, CA, USA), anti-α-tubulin (1:5000, T5168, Sigma-Aldrich, St. Louis, MO, USA), and pericentrin (1:1000, ab28144, Abcam, Cambridge, UK) diluted in 3% BSA/PBS solution, followed by three washes with PBS, and incubation with secondary anti-mouse and anti-rabbit antibodies conjugated to Alexa Fluor 568 or 488 (1:1000, Invitrogen, Carlsbad, CA, USA) diluted in 3% BSA/PBS solution. The nuclei were counterstained with DAPI. The samples were analysed using a Zeiss LSM 900 confocal microscope and Zen blue 3.1 software (Carl Zeiss AG, Oberkochen, Germany).