Triton x 100

Cells cultured in 12 well plates were washed by PBS(-) and fixed in 4% paraformaldehyde (PFA) for 30 minutes, followed by permeabilization using 0.2% Triton-X 100 (Nacalai Tesque) for 15 minutes at room temperature. Background staining was blocked by Blocking One Histo (Nacalai Tesque) for 30 minutes at room temperature, followed by incubation with a primary antibody overnight at 4 °C. After washing, secondary antibodies were added and cells were incubated for 1 h at room temperature in the dark. Cells were washed and cell nuclei were stained with Hoechst 33342 (Dojindo) for 5 minutes. Cells were observed by BZ-X (Keyence). Each immunostaining-positive and -negative cells were counted by BZ-X Analyzer software (Keyence). The percentage of immunostaining-positive cells were calculated as follows: % immunostaining cells = (the number of immunostaining (+) Hoechst 33342 (+) cells)/(total number of Hoechst 33342 (+) cells) × 100.

Cells on coverglasses were treated with 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan) for 10 minutes at room temperature and permeabilized with 0.5% Triton X-100 (Nacalai Tesque, Inc.) for 7 minutes at room temperature. For staining with the anti-ucMGP antibody, cells were fixed in acetone at -20°C for 5 minutes. After washing with PBS three times, cells were treated with blocking buffer (PBS containing 10% FBS) for 30 minutes at room temperature, followed by incubation with the primary antibody diluted in blocking buffer overnight at 4°C or for 1 hour at room temperature. The primary antibodies were diluted 1:50–100, including anti-GGCX (GTX109926; GeneTex, Inc.), anti-58K Golgi (ab27043; Abcam, Cambridge, UK), anti-MGP (sc-271906; Santa Cruz), and anti-ucMGP (#ALX-804-642, mAb (BD4.4); Enzo Life Sciences, Antwerp, Belgium) [35 (link)] antibodies. Samples were reacted with secondary antibodies (1:200) (goat anti-mouse or rabbit IgG (H+L), Alexa Fluor^® 488- or 546-conjugated; Thermo Fisher Scientific Inc.) for 30 minutes at room temperature with or without Hoechst (H3570, Life Technologies Japan Ltd, Tokyo, Japan). After washing, samples were mounted in anti-fade solution (fluorescence mounting medium; Dako, Glostrup, Denmark) and images were taken with an OLYMPUS BX50 microscope (OLYMPUS), or a Keyence BZ-9000 Fluorescence Microscope (Keyence, Osaka, Japan).

HepG2 cells or PXB cells on coverslips were treated with 10M-D42AN or GFP-tagged scFv-fused peptides. For observation of nuclear actin, the cells were transfected with plasmid EYFP-NLS-Actin (kindly provided by Prof. Harada, Tohoku University) using Lipofectamine 2000 and cultured for 72 h. For early endosome staining, CellLight Early Endosomes-RFP, BacMam 2.0 (Invitrogen) was used. These cells were then fixed with 10% paraformaldehyde for 10 min followed by permeabilization with 0.5% Triton X-100 (Nacalai Tesque) for 5 min. The samples were stained with antibodies against DOCK11 (kindly provided by Prof. Matsushima, Kanazawa University), pChk1, Ack1, and γH2AX followed by Alexa488-conjugated anti-rabbit IgG (Invitrogen) and Alexa647-conjugated antimouse IgG (Invitrogen). For staining of cytoplasmic actin filaments, 100 nM rhodamine phalloidin (Acti-Stain 535, Cytoskeleton) was used. The 4′,6-diamidino-2-phenylindole solution (Thermo) was used for nuclear staining. Photobleaching was prevented using Slow Fade Gold antifade reagent (Invitrogen). The samples were observed using confocal microscopy. The cells were observed with an LSM800 confocal microscope (Zeiss) and analyzed using ZEN software (Zeiss). The fluorescence intensity of the target protein was normalized to that of 4′,6-diamidino-2-phenylindole.

Cells in chamber slides were fixed with 4% paraformaldehyde (Nacalai Tesque Inc.) in PBS for 15 min and permeabilized by three successive treatments with 0.3% Triton X-100 (Nacalai Tesque Inc.) in PBS for 2 h. Cells were treated with primary antibodies for 12 h at 4 °C, washed three times with 0.05% Tween-20 in PBS, and then treated with Alexa Fluor® 488-conjugated anti-mouse IgG (Molecular Probes) for 1 h. Fixed cells were washed three times with 0.05% Tween-20 in PBS and mounted with Fluoro-KEEPER Antifade Reagent with DAPI (Nacalai Tesque Inc.). Cells were analyzed under a confocal fluorescence microscope (OLYMPUS). The antibodies for immunocytochemistry used in this study were anti-Flag M2 (F3165, Sigma) and anti-Tuj1 (MAB1637, MECK MILLIPORE). Whole-mount in situ hybridization (n > 5) was performed as described previously [28 (link), 29 ]. We did not use randomization.

Cells were cultured on glass coverslips in 12-well plates. Cells on coverslips were washed in phosphate-buffered saline [PBS; 137 mM NaCl (Nacalai Tesque, Kyoto, Japan; 31320-34), 2.7 mM KCl (Sigma-Aldrich, St. Louis, USA; P9541), 10 mM Na2HPO4 (Wako, Osaka, Japan; 198-05955F), and 1.8 mM KH2PO4 (Nacalai Tesque, Kyoto, Japan; 28736-75)], fixed for 20 min with 4% paraformaldehyde (Nacalai Tesque, Kyoto, Japan; 26123-55) in PBS, washed again with PBS, and permeabilized with 0.3% Triton X-100 (Nacalai Tesque, Kyoto, Japan; 35501-15) in PBS for 3 min. Cells were then washed and blocked in PBS containing 4% bovine serum albumin (BSA; Wako, Osaka, Japan; 015-23295) for 30 min at room temperature. Coverslips were then incubated overnight in 4% BSA/PBS containing primary antibodies (1:200 dilution). Subsequently, cells were rinsed and incubated with secondary antibodies in 4% BSA/PBS (1:200 dilution) for 1 h at room temperature. After washing with PBS, coverslips were mounted on slides using ProLong Diamond Antifade reagent with DAPI (Invitrogen, Waltham, MA, USA; P36966), and observed on a confocal laser-scanning microscope with a 60× PlanApo/1.45NA DIC objective (Olympus, Tokyo, Japan; FV10i-LIV), as previously described [33 (link)].