Tritc phalloidin

To evaluate the angiogenic characteristics of DPSCs, following in vitro experiment was performed to assess the formation of capillary-like structures. 96-well plates were pre-coated with gelatin methacryloyl (GelMA) hydrogel as previously described (Chen et al., 2012 (link)). In brief, DPSCs and human umbilical vein endothelial cells (HUVECs, ATCC, United States) were plated onto hydrogel-treated 96-well plates and cultured with an endothelial cell growth medium-2™ (EGM-2™, Lonza Bioscience, Switzerland) supplemented with 2% FBS, .4% hFGF-B, .1% VEGF, .1% hEGF, .1% R3-IGF-1, .1% Heparin, .1% ascorbic acid, .1% gentamicin/amphotericin-B, and .04% hydrocortisone. The cultured medium was changed every 2 days. After differentiation for 7 days, the cells were fixed with 4% PFA and treated with Phalloidin-TRITC (1:200, Solarbio, China), and the nuclei were stained by 4′6-Diamidino-2-phenylindole-dihydrochloride (DAPI, Beyotime Institute of Biotechnology, Shanghai, China). The images were taken using a fluorescence microscope (Eclipse 80i, Nikon, Japan), and the vascular tube length and number were calculated using NIS-Elements AR 3.1 (Nikon).

To investigate the effect of Nspd on angiogenic differentiation of DPSCs, GelMA hydrogels was used as a scaffold structure (Chen et al., 2012 (link)). Complete endothelial cell growth medium-2^TM (EGM-2^TM, containing 2% FBS, 0.4% hFGF-B, 0.1% VEGF, 0.1% hEGF, 0.1% R3-IGF-1, 0.1% Heparin, 0.1% ascorbic acid, 0.1% gentamicin/amphotericin-B, and 0.04% hydrocortisone) (Lonza Bioscience, Switzerland) with various concentrations of Nspd were added to the confluent DPSCs and GelMA in 96-well plates. After 7 day differentiation, cells were washed with PBS and fixed with 4% PFA for 15 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 for 20 min and washed three times in PBS. Then the cells were incubated with Phalloidin-TRITC (Solarbio Science & Technology Co., Ltd., China) (1:200 with 1% BSA) for 1h in the dark at 37°C, followed by incubation with DAPI for 5 min. Phalloidin-TRITC was used to stain F-actin, one of the cytoskeletons, which is important to cell-to-cell and cell-to-matrix adhesion. DAPI was used to stain nuclei. The samples were analyzed through fluorescence microscopy (Nam et al., 2017 (link)).

Huh7 cells were seeded in 24-well plates containing clean coverslips. After washing with PBS 3 times, cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-100, then blocked with 5% BSA for 1 h at room temperature. Subsequently, the coverslips were incubated with primary antibody (FAM21C: 1:50, Biorbyt, UK) at 4°C overnight. The next day, the primary antibody was discarded and the cells were washed 3 times with PBS and incubated with Alexa Fluor 488-conjugated secondary antibodies (1:200, Proteintech, China) in a wet box for 1 h. Subsequently, cells were stained with TRITC Phalloidin (1:200, Solarbio, China) for 30 min at room temperature to detect the actin cytoskeleton. Finally, 10 μL of antifluorescence quencher containing DAPI was used to stain the nuclei. Cells were examined by fluorescence microscopy and photographed (40x magnification).

Sodium Hyaluronate (MW = 100 kDa) was provided by Shandong Freda Biochem Co., Ltd. (Shandong, China). Dowex^® 50WX8-200, tetrabutylammonium hydroxide (TBA-OH), benzyl bromide, TritonX-100 and hyaluronidase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anhydrous dimethyl sulfoxide (DMSO), ethyl acetate, and Hexafluoroisopropanol (HFIP) were obtained from Aladdin Reagent Company (Shanghai, China). Doxorubicin hydrochloride (Dox•HCl, >99%) and cell counting kit-8 (CCK-8) were brought from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). Fluorescein diacetate (FDA), propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), and TRITC-phalloidin were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). DMEM medium, RPMI-1640 and fetal bovine serum were provided by Gibco (Cleveland, TN, USA). Penicillin-streptomycin and trypsin were purchased from HyClone (Logan, UT, USA). All other chemicals were used without further purification.