Rhodamine phalloidin

Stably transfected FPR2-RBL cells were seeded in 12-well plate with round coverslips for 24 h. Then the cells were treated with or without C18 (10 μM) for 30 min, and stimulated with WKYMVm (final concentration of 1 μM) for another 15 min at 37°C. Subsequently, remove cell medium and wash the cells once with PBS, then fixed cells with 4% Paraformaldehyde for 15 min at room temperature, remove fixative solution and washed twice with PBS, blocked with 5% BSA+PBS for 1 h, and then stained with rhodamine phalloidin (Cytoskeleton, USA) and Hoechst according to the manufacture's protocol. In brief, stain the cells with 100 nM rhodamine phalloidin, incubate at room temperature in the dark for 30 min wash the cell coverslips three times in PBS, and stain DNA for 5 min with of 2 Mg/ml Hoechst. Rinse the coverslips and invert on a drop of anti-fade mounting media on a glass slide and seal each side with nail polish. Coverslips with the cells were mounted on glass slides and cell images were taken immediately with a 40× oil objective. The images were analyzed by ImageJ 1.49U (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2018).

Different groups of adherent cells were washed with phosphate-buffer edsaline (PBS), fixed with 4% paraformaldehyde in PBS for 20 min, and permeabilized with 0.1% TritonX-100 for 5 min at room temperature. After further washing, the cells were incubated with rhodamine-phalloidin (Cytoskeleton, Denver, CO, USA) for 30 min and a Hoechst 33342 solution (Sigma–Aldrich, St. Louis, MO, USA) was used to counter stain the nucleus for 10 min at room temperature in a dark room. Other cells were incubated with a mouse polyclonal antibody directed against Nrf2 or FN over night at 4 °C after blocking with 10% goat serum. Then the cells were incubated in the dark at room temperature for 1h with a secondary antibody (Alexa Fluor 488/Alexa Fluor555, Invitrogen, Carlsbad, CA, USA). The nucleus was stained with Hoechst33342 as aforementioned. Cells were placed under alaser scanning confocal microscope LSM710, CarlZeiss, Germany) for observation and image acquisition.

DLD-1 and DLD-1/TRAIL cells were seeded into the wells of a Lab-Tek II Chamber Slide System (Thermo Fisher Scientific Inc., Waltham, MA), each well containing 1.0 ml of culture medium plus 10% (w/v) fetal bovine serum. After 48 h of treatment, the cells were immunostained with anti-DR5 antibody according to the immunofluorescence protocol of Cell Signaling Technology. The nuclei were stained with Hoechet33342, and for actin labeling the cells were incubated with the fluorescent F-actin probe Rhodamine Phalloidin (Cytoskeleton, Denver, CO). The cells were observed with a BIOREVO fluorescence microscope (Keyence, Osaka, Japan).

After 1 and 3 d of incubation, cells on each sample were fixed with 2.5% glutaraldehyde (v/v) for 30 min and dehydrated in gradient ethanol of 50%, 75%, 90%, and 99% for 15 min each. Then, the samples were dried, sprayed with Pt, and observed by SEM (Phenom ProX, Thermo Fisher Scientific).
After 1 and 3 d of incubation, cells on each sample were fixed with 4% paraformaldehyde (v/v) for 30 min, permeabilized with 0.2% Triton X-100 (v/v, Amresco, USA) for 5 min, stained with rhodamine phalloidin (Cytoskeleton Inc., USA) for 30 min and DAPI-Fluoromount-G (Southern Biotech Co., USA) for 5 min, and then observed by laser scanning confocal microscope (LSM 710, Zeiss Co., Germany).

To further determine the effect of RGD, the spreading morphology of osteoblasts was observed [17 (link), 23 (link)]. Briefly, after culturing for 24 h, the osteoblast-loaded scaffolds in both FTA group and CTA group were fixed with 4% paraformaldehyde for 15 min. Subsequently, the samples were incubated with rhodamine phalloidin (Cytoskeleton Inc., Denver, CO, USA) for 1 h at 37°C to label the cytoskeleton. Finally, the spreading morphology of osteoblasts on different scaffolds was observed under a confocal microscope with the wavelengths of 488 and 517 nm for excitation and emission, respectively.

Pharmacological agents used: angiotensin II (Ang II), chloroquine (CQ), Xanthine oxidase (XO), and rapamycin (Rapa) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Rhodamine phalloidin was acquired from Cytoskeleton (Denver, CO, United States). Mito-TEMPO and 4-PBA were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, United States). Antibodies against LC3A/B, ATG5, p62, mTOR, p-mTOR, ATG5, ATG7, Beclin 1, PERK, XBP1, CHOP, β-actin, and GAPDH were purchased from Cell Signaling Technology (Boston, MA, United States). GRP78, GSK3β, p-GSK3β (Ser9), β-TrcP, and NRF2 were purchased from Abcam (Cambridge, MA, United States). NOX2, NOX4, and HO-1 were provided by Proteintech (Wuhan, China).