Uplsapo20x objective

Fig. 5A (which is the basis for Fig. 6A) and Fig. 6B were taken with a digital camera (Power Shot G12, Canon, Tokyo, Japan). The generation of Fig. 5C is described in detail above (BX51WI microscope; UPLSAPO20X objective; Olympus), as well as the generation of Fig. 6D and all panels in Figs 2, 3 and 7 (same microscope; UPLSAPO4X objective; Olympus). Fig. 5D was taken with the same microscope using an UPLSAPO60XO objective (60×, oil, N.A.=1.35) (Olympus). The final figures were constructed using Corel Photo-Paint X7 and Corel Draw X7 (both versions 17.5.0.907; Corel, Ottawa, Canada). Only minor adjustments of contrast and brightness were made using Corel Photo-Paint, without altering the appearance of the original materials.

One mussel was killed, embedded and cut into sections (including grinding and polishing of the sections) as described above for the mussels used for investigating the formation of new mineralized tissue after exposure to ESWs or sham exposure. However, in this case, soft and hard tissue were not separated before embedding. Sections were stained with Giemsa staining. Imaging of the sections was performed with a BX51WI microscope (Olympus) operated in brightfield mode, equipped with an UPLSAPO20X objective (20×, N.A.=0.75) (Olympus) and Retiga 2000R CCD camera (Q-Imaging, Surrey, Canada).

We first established MDCK cells stably expressing EKARrEV-NLS or the prototype EKAREV-NLS. Then, 2 × 10⁴ cells were seeded in a well of a 24-well glass-bottom plate coated with 0.3 mg ml⁻¹ type I collagen. After 24 h, the medium was replaced with Medium 199 supplemented with 1% BSA, 100 unit mL⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin. Fluorescence images were acquired with an UPLSAPO 20X objective (Olympus) every 2 min. During imaging, EGF (no. E9644; Sigma-Aldrich) or trametinib (no. T-8123; LC Laboratories) was added to a concentration of 100 ng ml⁻¹ or 200 nM final, respectively. After applying autothreshold, the fluorescence intensities of each nucleus were quantified to obtain FRET/CFP values as described previously (Aoki & Matsuda, 2009 (link)).