Hoechst 33258

Apoptotic EPCs were identified by using Hoechst 33258 staining (Beyotime, China). We seeded EPCs at densities of 6 × 10⁵ cells/well in wells of a 60 mm plate. After applying the variously composed treatments, we fixed EPCs with 4 % paraformaldehyde for 10 min, then washed samples with PBS three times, followed by staining with 2 μg/mL Hoechst 33258 (Beyotime, China) in Hank’s balance salt solution for 5 min. Changes in the morphology of apoptotic nuclei were evaluated under fluorescence using microscopy (Olympus BX51) at an absorbance wavelength of 460 nm.

Kidney cryosections (8 μm) were immersed into cold acetone for 15 min and then blocked with 5% BSA for 1 h at 25 °C. After incubated with primary antibodies against Ly-6G or F4/80 (1:100, Abcam, Cambridge, UK) overnight at 4 °C, sections were incubated with Dylight488-conjugated secondary antibody (1:200, Biolegend, San Diego, CA, USA), and then the nuclei were co-stained with Hoechst 33258 (5 μg/mL, Beyotime, Shanghai, China). The cryosections were tested using fluorescence microscopy (Olympus, Tokyo, Japan). For ICC experiments, the indicated coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% TritonX-100, and blocked with 5% BSA. The slips were incubated with primary antibodies against 3-NIT, 4-Hydro, Drp1, Mfn1, or MFF (1:100, Abcam, Cambridge, UK) overnight at 4 °C. In some experiments, mitochondria were labeled with Mito-Tracker Red CMXRos before fixation (Beyotime, Shanghai, China) according to the provided protocols. The next day, slips were stained with Cy3/ Dylight488-conjugated secondary antibody (1:200, Biolegend, San Diego, CA, USA), and then nuclei were co-stained with Hoechst 33258 (5 μg/mL, Beyotime, Shanghai, China). Quantification of positively stained cells was performed as described previously.

Apoptotic NP cells were identified by Hoechst 33258 staining (Beyotime, Haimen, China). NP cells were seeded at a density of 6 × 10⁵ cells/well in a 60-mm plate. After treatment with TBHP and other drugs, NP cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS three times and stained with 2 µg/ml Hoechst 33258 (Beyotime, Haimen, China) in Hank’s balanced salt solution for 5 min. The morphologic changes in apoptotic nuclei were assessed under a fluorescence microscope (Olympus Fluoview, Tokyo, Japan) with excitation at 350 nm and emission at 460 nm.

Apoptosis was analyzed by three methods: 1) Flow cytometry: Apoptotic cells were analyzed using the Annexin-V-FITC/PI kit (BD, San Diego, USA) by a FACSCalibur flow cytometer (BD) according to the manufacturer's instructions. Briefly, cells (2 × 10⁵/well) were cultured in 6-well plates in the appropriate medium for 6 h prior to treatment with GEM (100 nM) and/or ADI (1 mU). Following incubation for the indicated times, cells were trypsinized and centrifuged, washed with PBS, and stained with Annexin V and PI in the dark. Samples were analyzed, and the percentage of apoptotic cells was evaluated. 2) In situ Annexin V/PI staining: Following the pretreatment as indicated in the flow cytometry, cells were washed with PBS and stained with 5 μL of anti-Annexin V-FITC and 5 μL of PI in 500 μL of binding buffer in the dark for 15 min and then examined using a fluorescence microscope. 3) Hoechst 33258/PI double staining: After treatment as indicated previously, cells were washed with PBS and stained with 0.1 mL of Hoechst 33258 (Beyotime Biotechnology, Nantong, China) and PI for 15 min. Stained cells were photographed under a fluorescence microscope.

A tube formation assay was performed to evaluate the BM-EPC function as described before (Yu et al., 2016 (link)). Briefly, a 96-well plate (Corning, Tewksbury, MA, United States) was coated with 50 μL/well of growth factor–reduced matrix gel (BD Biosciences, San Diego, CA, United States; cat. no. 356231) for 1 h. The cells were then placed on the Matrigel-coated plate with 5 × 10⁵/ml concentration and maintained at 37°C with 5% CO₂. After 8 h of incubation, images of the forming tubes were acquired under ×50 magnification using a light microscope. The number of tubes was calculated.
The adhesion ability assay was used to assess the EPC function as previously described (Han et al., 2017a (link)). A total of 5 × 10⁴ BM-EPCs were placed on the mouse vitronectin (1 μg/ml) coated 96-well plate per well. After 2 h incubation at 37°C with 5% CO₂, nonadherent cells were softly removed by phosphate-buffered saline (PBS). Then, adherent cells were fixed with 2% paraformaldehyde for 15 min at RT and stained by Hoechst 33258 (10 μg/ml; Beyotime, Shanghai, China; cat. no. C1011). The stained cells (blue color) were observed using a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of ×400. Each well was counted in 3 random fields.

B. emeiensis was picked from Mountain Emei, Sichuan, China, at the altitude of 1300–1500 m. The rhizomes of B. emeiensis were washed with distilled water to remove dirt and dried naturally. Then the rhizomes were grounded into fine powder sieving with a mesh of 60 and stored at 4 °C.
Dimethyl surtoxide (DMSO), ascorbic acid (Vc), sodium chloride, chloroform, magnesium sulfate, calcium chloride, and others were purchased from Kelong Chemical Factory (Chengdu, China). Reactive oxygen species (ROS) reagent, Hoechst 33258 was bought from Beyotime Biotechnology (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) and agar powder were obtained from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Rhodamine 123, Fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), phosphate buffered solution (0.0067 M, PBS) and trypsin were from Hyclone (Logan, UT, USA).