Ckx53

For the study of the morphological features of seeds, protocorms, and seedlings at different developmental stages, the samples were examined after co-culture^{2 (link)}. The specimens were photographed under an inverted microscope (Olympus CKX53) equipped with a digital camera (Nikon D610). Furthermore, the samples were examined via scanning electron microscopy (SEM; Hitachi SU8100). The samples were washed thrice with 0.1 M PB (pH 7.4) and then transferred into 1% OsO₄ in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. Next, the tissue blocks were washed thrice in 0.1 M PB (pH 7.4), for 15 min each time. Then, the blocks were dehydrated in a gradient series of ethanol (30%, 50%, 70%, 80%, 90% and 95%) and two changes of 100% ethanol for 15 min each and then in isoamyl acetate for 15 min. After drying the samples with Critical Point Dryer, they were attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. Finally, the samples were observed and imaged by SEM.

The samples were washed with PBS, fixed with 4 % paraformaldehyde for 30 min, according to experimental requirements, were incubated with anti-Pdpn (proteintech, 1:200), anti-Grem1 (CST, 1:50), anti-Cd200 (proteintech, 1:200), anti-Ctsk (Abcam, 1:200), anti-Ptgs2 (proteintech, 1:200), anti-vinculin (proteintech, 1:200) or anti-YAP (CST, 1:200) at 4 °C for 8–12 h. After that, samples incubated with goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:300), goat anti-rabbit IgG (Servicebio, Cy3, 1:300) or goat anti-mouse IgG (Servicebio, Alexa Fluor 488, 1:200) for 1 h at room temperature. Nuclear staining was conducted with DAPI (Sigma), and F-actin stained by rhodamine phalloidin (Beyotime). Tissue samples were fixed in 4 % paraformaldehyde. After decalcification, all samples were embedded in paraffin and sectioned at 6-μm slices. The tissue sections were immunofluorescently stained with primary antibodies targeting Sry (Santa Cruz, 1:200), CD11b (Servicebio, 1:500), TNF-α (Uscn Life Science Inc., 1:1000), IL-1β (Servicebio, 1:1200) and IL-10 (Servicebio, 1:1000), and the goat anti-rabbit IgG (Servicebio, Alexa Fluor 488, 1:400) and goat anti-rabbit IgG (Servicebio, Cy3, 1:500). DAPI was used to stain the nuclei. The images were observed by a fluorescence microscope (Olympus CKX53) or Nikon confocal microscope (Nikon).

MitoSOX probes were purchased from the University of Shanghai (Shanghai, China). Briefly, cells were treated with the MitoSOX probe and then transfected with NGFR shRNA. After 48 h, the fluorescence intensity of the cells was observed under a fluorescence microscope (CKX53; Nikon).