Bz 700

Microscopic images of the spheroids were captured with a photomicroscope (CKX41SF, Olympus, Tokyo, Japan). Phase-contrast and fluorescence images were captured with a fluorescence microscope (BZ-700, Keyence, Okayama, Japan). For the fluorescence images, HUVECS were stained red with a fluorescent probe (Qtracker 605, Thermo Fisher Scientific) according to the manufacturer’s instructions. Stereoscopic images of the 3D structures were obtained with a stereo microscope (SZX7, Olympus, Tokyo, Japan).

For the histological analyses, hearts and embryos were collected, fixed in 4% paraformaldehyde for 1 hr at 4°C, and stored in phosphate-buffered saline or embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan). Sixteen-μm-thick frozen sections were stained with hematoxylin (Merck) and eosin (Kanto Chemical, Tokyo, Japan). Immunostaining of 16-μm-thick frozen sections was performed using primary antibodies against CD31 (553370, BD Pharmingen, RRID: AB_394816, 1:100), Flk1 (555307, BD Pharmingen, RRID:AB_395720, 1:100), Isl1 (AF1837, R&D systems, RRID:AB_2126324, 1:250), Prox1 (11-002, AngioBio, RRID: AB_10013720, 1:200; AF2727, R&D Systems, RRID: AB_2170716, 1:200), LYVE1 (11-034, AngioBio, 1:200; AF2125, R&D Systems, RPID: AB_2297188, 1:150), VEGFR3 (AF743, R&D Systems, RRID: AB_355563, 1:150), and GFP (GFP-RB-AF2020, FRL, RRID:AB_2491093, 1:500). Alexa Fluor-conjugated secondary antibodies (Abcam, RRID:AB_2636877, RRID:AB_2636997, RRID:AB_2752244, 1:400) were subsequently applied. The same protocol was followed for whole-mounted hearts and embryos, with the primary and secondary antibody incubation periods extended to two nights. Immunofluorescence imaging was conducted using a Nikon C2 confocal microscope or Keyence BZ-700. All images were processed using the ImageJ and Nikon NIS Elements software.

For immunocytochemistry, samples (neurospheres or neurons: derived from the 201B7 line) were plated onto poly-L-ornithine/fibronectin-coated chamber slide glasses (Iwaki) and fixed in 4% PFA/PBS for 30 min at room temperature. The slides were rinsed with PBS three times and permeabilized with 0.3% Triton X-100/PBS for 5 min at room temperature. After blocking with Blocking One (Nacalai Tesque, 03953-95) for 15 min at room temperature, the slides were incubated at 4°C overnight with the following antibodies: rabbit anti-α-tubulin (Cell Signaling Technology, 2144; 1:500), mouse anti-β-III tubulin (Sigma-Aldrich, T8660-2ML; 1:500), rabbit anti-tau (Dako, A0024; 1:500), rabbit anti-p38 MAPK (Cell Signaling Technology, 8690; 1:500), rabbit anti-phospho-p38 MAPK (Cell Signaling Technology, 4511; 1:500), rabbit anti-phospho-CDC25B (Thermo Fisher Scientific, PA5-104568; 1:500), and rabbit anti-acetyl-α-tubulin (Lys40) (Cell Signaling Technology, 5335; 1:500). After washing three times with PBS, the samples were incubated with secondary antibodies conjugated to Alexa 488 (Thermo Fisher Scientific, A-11034) or Alexa 555 (Thermo Fisher Scientific, A-21424; 1:500) for 60 min at room temperature and then subjected to nuclear counterstaining with Hoechst 33258 (Sigma-Aldrich, B2883; 10 μg/mL). The samples were analyzed with an all-in-one fluorescence microscope (BZ-700 or BZ-800, Keyence).

For immunocytochemistry, Caco-2 and A549 cells were grown on 35-mm glass bottom dishes (Matsunami Glass). The cultured cells were fixed with or without 4% paraformaldehyde, washed thrice with PBS, and then stained with the Alexa Fluor 488-conjugated monovalent SARS-CoV-2 spike protein at room temperature for 30 min. In addition, the cells were stained with the SARS-CoV-2 spike protein (40592-V05H; 2.5 μg/ml 2% BSA-PBS), followed by the secondary antibody anti-mouse IgG-Alexa Fluor 488 (A-11001; Thermo Fisher Scientific; 5 μg/ml 2% BSA-PBS). To confirm the expression of ACE2 in Caco-2 and A549 cells, the cultured cells were fixed with 4% paraformaldehyde, washed thrice with PBS, and then stained with an anti-ACE2 antibody (PAB886Hu01; CLOUD-CLONE; 5 μg/ml 2% BSA-PBS) at room temperature for 30 min, followed by incubation with anti-rabbit IgG-Alexa Fluor 568 (ab175471; Abcam; 10 μg/ml 2% BSA-PBS) at room temperature for 30 min. The stained samples were observed with a fluorescent microscope (BZ-700, Keyence). Raw images, including the differential interference contrast image, were captured under identical settings, in the case of same experiments, and then exported as TIFF files.