Laser confocal microscopy

Harvested T. vaginalis trophozoite cells were washed three times with PBS (pH 7.2), and smeared on a poly-L-lysine treated glass slide for 15 min. Then, the trophozoites were fixed using PBS containing 4% paraformaldehyde for 10 min at room temperature, permeabilized using PBS containing 1% TritonX-100 for 10 min, washed three times with PBS and blocked using PBST containing 4% (w/v) BSA for 1 h at 37°C. After the slides were washed three times with PBS, rat antiserum against TvAP33 (dilution ratio 1:100) and control rat serum were added to the slides overnight at 4°C. The slides were washed three times with PBS, treated with goat anti-rat IgG antibody (Beyotime, Shanghai, China) labeled with Cy3 (dilution ratio 1:1,000) and incubated in the dark for 40 min. After three washes in PBS, DAPI (Beyotime) was used to stain the nuclei for 15 min in darkness. After washing with PBS, fluorescent mounting medium (Beyotime) was added, and the cells were examined by laser confocal microscopy (Nikon, Beijing, China).

We sought to characterize the cross-species impact of mammalian cell membrane coating on plant-derived photosynthetic organelle interactions with macrophages and mature tissue cells (chondrocytes). RAW 264.7 mouse macrophages and mouse chondrocytes were used for the cellular uptake experiments. Cells were incubated in 12-well plates (1 × 10⁵ cells per well) and cultured for 1 day. The NTUs were labelled with DiI before coating with LNPs or CM. Then, DiI-labelled NTUs, LNP-NTUs and CM-NTUs were used at a concentration of 2 × 10⁵ NTUs per cell to study the cellular internalization efficiency. RAW 264.7 mouse macrophages were incubated with the NTUs for 6 h. Chondrocytes were incubated with NTUs for 1, 3 and 6 h. The cell samples were washed three times with PBS for 5 min and fixed with 4% polyformaldehyde (PFA) for 20 min. Then, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 20 min at 25 °C to label nuclei. Finally, the cells were observed by laser confocal microscopy (LCM; Nikon) or structured illumination microscopy (Nikon). The DiI fluorescence signal was measured using a Synergy H4 hybrid microplate reader (Bio Tek). A fluorescence-based assay was performed to estimate the numbers of NTUs delivered to each cell.

CA06g13580 was cloned from Huangdenglong pepper using CcWRKY25-F/R primers (Table S1). The coding sequence (CDS) without stop codon of CcWRKY25 was inserted into the cloning vector TA before subcloning into the pCAMBIA1300: GFP vector. The pCAMBIA1300: GFP: CcWRKY25 fusion protein and pCAMBIA1300: GFP were separately and transiently synthesized in tobacco leaves after Agrobacterium infection [45 (link)]. The infective tobacco was cultured in a light-proof environment at 24 °C for 2 days. Then, fluorescence was detected by laser confocal microscopy (Nikon, Japan).

According to the instructions of the tetramethylrhodamine (TRITC)‑conjugated phalloidin (Merck, Germany, Cat. no. FAK100), drop the cell suspension on the sterilized glass slide, and fix the slide in 4% formaldehyde after climbing the slide in the incubator for 24 h. 0.1% Triton X-100 was permeabilized, TRITC-labeled phalloidin was added dropwise. After incubation with DAPI staining solution, the results were observed by laser confocal microscopy (Nikon, Japan). Images were acquired under a microscope at 1000x magnification.

CircRNF111 probes (RiboBio, Guangzhou, China) targeting the back-splicing junction region were designed to visualize circRNF111 fluorescence in situ. The differentiated preadipocytes were first fixed with in situ hybridization fixative. After prehybridization, cells were incubated with the labelled circRNF111 probes in hybridization buffer at 55°C overnight. Cell nuclei were counterstained by 4’,6-diamidino-2-phenylindole (DAPI; Sigma, USA). Laser confocal microscopy was used to observe the localization of circRNF111 (Nikon, Tokyo, Japan).

Protein subcellular localization of BjuAOP2 was performed by transient expression in tobacco leaf epidermal cells. Agrobacterium tumefaciens GV3101 containing the ProCAMV35S::GFP::BjuAOP2 vector plasmid was activated and enlarge-cultivated. Subsequently, the organisms were collected, resuspended in a resuspension solution (OD600 almost 0.5), left for 2-3 h, and introduced into the lower epidermis of 3-4 weeks old tobacco leaves with a syringe. Observations were made after 3 days of transformation. The leaves transformed with empty pTF101-GFP were used as control. In addition, laser confocal microscopy (Nikon, Tokyo, Japan) was utilized for the observation of GFP fluorescence with excitation light at 488 nm and emission light at 510 nm. Furthermore, chloroplast autofluorescence showed excitation light at 640 nm and emission light at 675 nm.