Hoechst 33342 solution

After HHP treatment, 1 × 10⁵ cells per sample were washed with PBS, resuspended in fresh DMEM/ 10% FBS, and seeded in a 35-mm glass-bottom dish (Matsunami Glass Ind., Ltd., Osaka, Japan), after which they were stored for 30–60 min at 37 °C in a 5% CO₂ incubator to encourage attachment to the dish bottom. For apoptosis/necrosis observation, the medium was gently discarded, and cells were washed 2 times with 200 μl of Assay Buffer. The cells attached to each glass-bottom dish were then incubated with a mixture of 200 μl Assay Buffer, 2 μl Apopxin Green Indicator, 1 μl 7-AAD and 1 μl CytoCalcein 450 (ab176749; Abcam) at RT for 30–60 min. For imaging ROS production, 1 ml of 5 μM MitoSOX reagent working solution (Molecular Probes) was administered to each glass-bottom dish, which was then incubated at 37 °C for 10 min. After gently washing 3 times with PBS, those cells were counterstained with Hoechst 33342 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and incubated at RT for 15 min. Finally, images were acquired using a confocal laser scanning microscope (Fluoview FV3000; Olympus Co., Tokyo, Japan).

Effects of the polyphenols on Raldh2 promoter activity in differentiated THP-1 cells were evaluated by monitoring changes in enhanced green fluorescent protein (EGFP) fluorescence derived from THP-1 (Raldh2p–EGFP) cells using the IN Cell Analyzer 2200 (Cytiva, Tokyo, Japan). THP-1 (Raldh2p–EGFP) cells were seeded in 96-well blackplates (Greiner Bio-one, Tokyo, Japan) at a density of 6 × 10⁵ cells/mL, treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA), and cultured for 48 h. After culturing, polyphenols were directly added to the cells at the final concentration of 10 μM and cells were further cultured for 24 h. Cells were then fixed with 4% formaldehyde for 15 min at room temperature. After washing the cells with phosphate-buffered saline (PBS), the cells were stained with 1 μg/mL Hoechst 33,342 solution (Dojindo, Kumamoto, Japan) for 20 min. The relative EGFP fluorescence intensity per cell was measured using IN Cell Analyzer 2200.

Thin blood smears from cultured parasites were prepared and fixed as previously described (29 (link)). Blocking was done with 10% normal goat serum (Invitrogen)–PBS at 37°C for 30 min. To confirm the expression of Cas9, blood smears were immunostained with mouse anti-FLAG monoclonal antibody (M20008; Abmart, Shanghai, China) at 1:100 dilutions in PBS with 0.05% Tween 20 (T-PBS) and incubated at 37°C for 60 min. To evaluate myc epitope tagging of SBP3, the slides were immunostained with mouse anti-myc monoclonal antibody (9B11; Cell Signaling Technology, Danvers, MA, USA) at a 1:500 dilution. After 3 washes with T-PBS, the blood smears were incubated with Alexa Fluor 488-conjugated secondary goat anti-mouse IgG antibody (Invitrogen) (1:500 dilutions) at 37°C for 30 min. The smears were washed again 3 times with T-PBS and incubated with 1 μg/ml of Hoechst 33342 solution (Dojindo, Kumamoto, Japan) at 37°C for 20 min. After 3 washes with T-PBS, the smears were examined under a Nikon A1R confocal laser scanning microscope (Nikon, Tokyo, Japan).