Tcs sp8 system

The subcellular localization analysis was carried out as described in previous work [25 (link)]. In brief, the full sequence of LcASC ORF was amplified with specific primers for sASC-F/R, as shown in Table S1. The whole CDS region of LcASC was inserted into the pEGFP-N1 plasmid to construct a eukaryotic expression vector. pEGFP-N1-LcASC and pEGFP-N1 were extracted with a small amount of the plasmid extraction kit (TIANMO, China). Large yellow croaker kidney cell lines were inoculated onto a 24-well cell culture plate and cultured until cells adhered to 80–90%. The pEGFP-N1-LcASC and pEGFP-N1 were transfected into the kidney cells for 24 h by the electron-transfected method, and the cells were then fixed and stained. Finally, the cells were observed with laser scanning confocal observation (Leica TCS SP8 system, Berlin, Germany) under two channels of DAPI excitation light and GFP excitation light.
For overexpression, the whole CDS region of LcASC was inserted into the pcDNA3.1 vector and transfected into HEK 293T cells using the Lipo8000 TM transfection reagent. After 24 h post-transfection, the cells were harvested for Western blot to examine the expression level of the LcASC protein.

Confocal images were recorded on an (inverted) confocal Leica TCS SP8 system equipped with a 63 × 1.20 NA water immersion objective. Chloroplasts were freshly isolated, and Chls were excited at 633 nm with a pulsed laser (40 MHz) and an intensity of 100 nW. The fluorescence was detected at 650–680 nm (PSII-maximum) and 710–750 nm (PSI-maximum) with internal hybrid detectors. The total image size was 9.2 * 9.2 μm, with 128*128 pixels.

Cells were plated for 24 h at a density of 1.5 × 10⁴ cells/well and then treated with pmOGT-HaloTaq or pHaloTaq as described in the cell culture and treatment section. 48 h after transfection, the medium of the cells was replaced for 30 min with medium containing cell-permeable HaloTaq^® Coumarin Ligand (Promega^TM). The medium was then replaced for 30 min with an equal volume of fresh, warm culture medium containing the cell-permeant dye Mito Tracker^TM Red FM (Thermo Scientific^TM), which accumulates in active mitochondria in living cells. To remove excess dye from the cells, the monolayers were washed twice with fresh warm culture medium for 15 min. DiOC18(3), which intercalates into the plasma membrane, was used to stain living cells. The fluorescence emission signals were recorded at 25 °C and 63× magnification using a Leica TCS SP8 system.