Lyso tracker red

Dulbecco’s Modified Eagle Medium (DMEM) sugar-free was purchased from Beijing Solarbio Technology Co., Ltd. Metformin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The ROS detection kit (DCFH-DA), the nuclear staining kit (dihydrochloride, 4,6-diamino-2-phenyl indole), the lysosomal red fluorescent probe (Lyso-Tracker Red), the POD detection kit, the CAT detection kit, the SOD detection kit, the OXD detection kit, the cell viability detection kit, and the quantitative protein kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. The reactive oxygen inhibitor was purchased from Aladdin Reagent Shanghai Co., Ltd.

Liensinine was prepared for different concentrations (0, 2.5, 5, 10, or 20 μM) and added into the wells for 48 h. After liensinine treatment and incubation, cells were collected and loaded with Lyso-Tracker Red (LTR) (C1046, Beyotime, China) at 37°C for 30 min. After washing 3 times with PBS, the fluorescence intensities were measured by a flow cytometry.

Cadmium chloride was purchased from Sigma-Aldrich (202,908, St. Louis, MO, Unites States). Puerarin (purity >99%) was purchased from Selleck Chemicals (S2346, Houston, TX, Unites States). Dulbecco's modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) and insulin–transferrin–selenium-A supplement (ITS-A) were purchased from Gibco (Grand Island, NY, United States). The Dansylcadaverine (MDC) Kit was purchased from Solarbio (G0170, Beijing, China). Lyso-Tracker Red (LTR) was purchased from Beyotime (C1046, Shanghai, China). Bafilomycin A1 (Baf) and DQ-BSA-Red (Self-Quenched BODIPY FL Conjugate of bovine serum albumin) were purchased from Invitrogen (Carlsbad, CA, United States). All of the other materials were of analytical grade.

Cadmium chloride, taurine, anti-LAMP2, anti-LC3B, and anti-p62/SQSTM1 were purchased from Sigma-Aldrich (202,908, St. Louis, MO, USA). DMEM and FBS were obtained from Gibco (Grand Island, NY, USA). Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-rat IgG, Lyso-Tracker Red (LTR), and Hanks’ Balanced Salt Solution were obtained from Beyotime (Shanghai, China); Anti-β-actin, anti-Atg7, anti-Beclin-1, anti-CTSB, and anti-rabbit IgG were obtained from Cell Signaling Technology Inc (Danvers, MA, USA). anti-LAMP2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DQ-BSA-Green was purchased from Invitrogen (Carlsbad, CA, USA). Anti-STX17, anti-SNAP29, and anti-VAMP8 were obtained from Abcam (UK). All of the other materials were of analytical grade.

To observe the distribution of CTSB in the cytoplasm, we incubated slides with primary rabbit monoclonal antibodies against CTSB (CST, #31718, 1:200) after fixation and blocking, and lysosomes were labeled with Lysotracker Red (Beyotime). Alexa Fluor 488 secondary antibodies (Proteintech, SA00006-2) diluted at 1:400 in blocking buffer where then added in the dark. We analyzed the 4,6-diamidino-2-phenylindole (DAPI) counterstained slides under a fluorescence microscope (40 × 10).

DC2.4 cells were seeded in a 6-well plate at a density of 1 × 10⁵ cells/mL and then incubated in complete RPMI-1640 medium overnight. After pre-treatment with GSLS-NPs, G-Solution or empty NPs at a final concentration of 100 μg/mL for 4 h, the cells were incubated with the inactivated PEDV antigen for 24 h. Untreated cells served as the NC. After removing the supernatant, the cells were washed three times with PBS and fixed with Immunol Staining Fix Solution (P0098, Beyotime, Shanghai, China). A monoclonal antibody (1:100) against the PEDV nucleocapsid protein was then added and incubated for 1.5 h at 37 °C. DyLight 488-conjugated goat anti-mouse IgG (E032210-01, EARTHOX, San Francisco, CA, USA) (1:200) was used as the secondary antibody. LysoTracker Red (C1046, Beyotime, Shanghai, China) and DAPI (C1005, Beyotime, Shanghai, China) were used to stain the lysosomes and nuclei, respectively. The cells were observed under an inverse confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan). The images were acquired using Olympus confocal software (FV10-ASW 3.1). In addition, a monoclonal antibody against the PEDV nucleocapsid protein was labeled with the fluorescent dye APC by using an APC conjugation kit (ab201807, Abcam, Shanghai, China). The relative fluorescence intensity of intracellular PEDV was quantified on a FACSCanto^TM (BD Biosciences, San Diego, CA, USA).