Micro cover glass

A micro cover glass (22 mm × 22 mm, thickness 0.12–0.17 mm) (Matsunami, Osaka, Japan) was placed on the top of cellophane-overlaid PDA. A small plug of the fungal culture of the GFP reporter strain infecting each of the tested viruses (MyRV2, CHV4-C18, CHV1-EP713, or CHV1-∆p69) was placed at the edge of micro cover glass, and allowed to grow for three days. After the mycelia grew between the micro cover glass and cellophane, the clover glass with mycelia was detached and placed on top of a micro slide glass (Matsunami). GFP expression was observed using Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).

The β-CD-EP/carbendazim complexes were obtained following the kneading procedure (see Section 2.3). The coating solutions (2% w/v) were prepared by dissolving chitosan in acetic acid (1% v/v). The prepared complexes were added to the chitosan solutions and the resulting mixture was stirred vigorously for 5 min to obtain a homogeneous solution. The loading concentration of carbendazim in the solution was 100 μg/mL. Afterwards, 500 μL solutions were withdrawn and coated uniformly on a micro cover glass (Matsunami glass IND., LTD., Kishiwada, Japan). The films were dried for 48 h at room temperature before further characterization. In the case of the β-CD-EP alone, the film was obtained in the same way. The chitosan film without the complexes or β-CD-EP was used as the control.

For the immunostaining of Ptc1 in HeLa cells, we used pCI-neo-based modified expression plasmid with largely compromised promoter activity (by deleting promoter region partially) to keep the expression of Flag-tagged Ptc1 protein (Ptc1-Flag) at nearly physiological levels.³⁵ (link) This was due to preclude the possibility of inappropriate aggregation of this polytopic TMD protein in the cytosol. Transfected HeLa cells were grown on micro coverglass (Matsunami, Osaka, Japan), fixed by incubating in 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 for 3 min at room temperature. All cells after fixation and permeabilization were blocked with 3% calf serum solution in PBS for 30 min at room temperature, reacted with anti-Flag M2 primary antibody at 4°C for overnight, and were subsequently reacted with secondary antibody, Alexa Fluor^R488 goat anti-mouse IgG. To observe the nucleus, cells were treated with 2.5 μg/mL Hoechst 33258. Immunofluorescent images were obtained by BIOREVO BZ9000 fluorescence microscope (Keyence, Osaka, Japan).

For immunocytochemical observations, HeLa cells were grown on micro coverglass (Matsunami, Japan), fixed by incubating in 4% paraformaldehyde for 30 min on ice, and permeabilized with 0.1% Triton X-100 for 3 min at room temperature. Fixed cells were blocked with 3% calf serum in PBS and reacted with anti-T7 monoclonal (mouse IgG) and anti-Flag polyclonal (rabbit IgG) antibodies at room temperature for 1 h. An Alexa^TM 488-conjugated anti-mouse IgG antibody and Alexa^TM 594-conjugated anti-rabbit IgG antibody (Molecular Probe) were used as secondary antibodies at 1:1000 dilution. To observe nuclei, the cells were treated with 2.5 µg/mL Hoechst 33342. Immunofluorescent images were obtained with BIOREVO BZ9000 fluorescence microscope (Keyence, Japan).

An NPG substrate was fabricated by radio frequency (RF) sputtering and dealloying (chemical corrosion of silver by nitric acid) as follows: first, a 1000-nm thick pure gold film (>99.9 mass%) was sputtered on a Φ = 22 mm micro cover glass (Matsunami Glass Ind., Ltd., Osaka, Japan) with a RF sputtering apparatus SVC-700RF (Sanyu Electron Co., Ltd., Tokyo, Japan). Next, a 300-nm thick, gold–silver alloy (atom ratio of gold:silver = 3:7) was sputtered on the gold thin film. The alloy was then immersed in nitric acid whose concentration was 70% and washed with DPBS (Nacalai Tesque) to fabricate an NPG substrate. Two ways were employed to change the pore sizes of the NPG substrates: one was to change the conditions of dealloying and the other was to change the conditions of heat treatment after dealloying (Table S1), because it was difficult to control the pore sizes with only one way. The nanostructures of the NPG substrates were observed using a scanning electron microscope (SEM, SU-6600, Hitachi High Technologies, Tokyo, Japan). The quantitative chemical composition of the NPG substrates was analyzed with energy dispersive X-ray spectroscopy (EDXS). Five areas from three different NPG samples were randomly selected to quantify the pore size and ligament size using ImageJ software.

Cells were plated on Matsunami Micro Cover Glass (Matsunami, Osaka, Japan), fixed with 4% paraformaldehyde solution and permeabilized with 0.1% Triton X‐100 containing Tween20. Then, they were stained with anticytochrome c antibody (12963; 1 : 300, Cell Signaling Technology, Danvers, MA, USA) and visualized using anti‐mouse IgG H&L secondary antibodies (Alexa Fluor 488; Invitrogen, Waltham, MA, USA) in Blocking One reagent (Nacalai Tesque, Kyoto, Japan). The nuclei were stained with DAPI Fluoromount‐G.