Ts2r fl

Cell proliferation was assessed after cell transfection. EdU solution (1:1000) was added to the cell culture and incubated for 4 h. The SCs were then fixed with 4% PFA for 30 min. After SC labeling, a Cell-Light EdU DNA Cell Proliferation Kit (C10310, Ribobio) was used to analyze cell proliferation according to the manufacturer’s protocol. Cell proliferation was shown as the ratio of EdU-positive cells, which was quantified with images of randomly selected on a DMR fluorescence microscope (Ts2R-FL, Nikon, Japan).

Cell invasion of 5-8F and 6-10B cells was assessed by Transwell chambers (8-μm pores, Corning Inc., Corning, U.S.A.) coated with Matrigel gel (BD Biosciences, San Jose, CA). The 5-8F and 6-10B cells (1 × 10⁵/cells) were cultivated into the upper chambers of Transwell (Corning Inc., Corning, U.S.A.), which contained 200 μl DMEM, while 500 μl DMEM medium containing 10% FBS was added into the lower wells as the primers. After 48 h at 37°C, 5-8F and 6-10B cells remaining in the upper surface were scraped off by a cotton swab, while those migrated into the lower surface were fixed by 4% pre-cold methanol for 15 min, and stained by 0.1% Crystal Violet solution (Sigma-Aldrich) for 20 min at room temperature. The cells were counted from a minimum of 10 fields per filter under a 200× microscope (Ts2r-FL, Nikon, Japan).

The rice leaves were stained with 1 mg/mL 3,3′-diaminobenzidine (DAB) (Biotopped) solution overnight at 28°C. After incubation with DAB, leaves were fixed and cleared in absolute alcohol with frequent changes of the fresh solution, and then the red-brown precipitate formed by polymerized DAB in the presence of H₂O₂ was examined under a stereomicroscope (MZ62+MSX1; Mshot, Guangzhou, China). Triplicate samples were performed for each treatment. For the callose deposition assay, the rice leaves were fixed and cleared in absolute alcohol with frequent changes of fresh solution, and the chlorophyll was removed. The transparent leaves were washed with 70 mM sodium phosphate buffer three times and then incubated in stain solution (70 mM sodium phosphate buffer; 0.01% aniline blue) (Macklin) for 2 h in the dark. Then, the leaves were observed using an inverted microscope under UV light (340 to 380 nm) (TS2R-FL; Nikon, Tokyo, Japan). The abundance of H₂O₂ accumulation and callose intensities are represented as the percentages of relative DAB and callose intensities, respectively, which were calculated by using Photoshop CS6 and ImageJ based on counting the numbers of DAB staining pixels and callose staining pixels. At least 6 typical photographs were selected from 10 randomly selected seedlings in each treatment. Three replicates were performed for each treatment.