Mito red

HeLa cells at a density of 2 × 10⁴ cells/well seeded on a confocal culture dish were treated with 0, 1, and 10 μM of β-cryptoxanthin for 24 h. The cells were collected from the lower wells, rinsed with PBS, fixed with 4% (v/v) paraformaldehyde for 15 min, then permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min, washed two times with ice-cold PBS, blocked in 3% (w/v) bovine serum albumin (BSA) for 40 min, and incubated with primary antibody (Asp175; Cell signaling technology, Danvers, USA) at 4 °C. After 3 h, HeLa cells were rinsed five times with PBS, before secondary antibody staining with fluorescein (FITC) donkey anti-rabbit IgG (H + L). The cells were finally washed two times with PBS [10 (link)]. Additionally, Mito-Red (Santa Cruz Biotechnology, South Korea) and Hoechst 33258 (Dojindo, South Korea) staining were performed to detect the integrity of the mitochondrial membrane and apoptotic nuclei, respectively. These confocal culture dishes were mounted with a fluorescent mounting media and analyzed with a Zeiss LSM-800 microscope (Carl Zeiss, Germany). Images were taken with the Zen-Black Edition software (Zen 2.3 SP1, Version: 14.0, Carl Zeiss, Germany).

C2C12 cells were obtained from Sigma (#91031101) along with Dulbecco's Modified Eagle Medium (DMEM; #D6429), fetal bovine serum (FBS; #F0926), horse serum (#H1270), and insulin (#I9278). Recombinant IL-15 was from GenScript (#Z03309-50) and GW-6471 (#11697) and GSK-3787 (#15219) were obtained from Cayman Chemicals. Trizol (#15596), SYBR green (#A25742) and a SuperScript VILO reverse transcription kit (#11754050) were purchased from ThermoFisher. The mitochondrial dye, Mito Red, was from Santa Cruz Biotechnology (SC-#301164). Male C57BL6 mice were kept on a 12 : 12 light dark cycle and fed a standard diet and water ad libitum. Gastrocnemius muscle was obtained following euthanasia. All animal procedures were approved by the Institutional Animal Care and Use Committee at Chapman University.

CII was synthesized similarly as reported in a previous study [24 ,28 (link),29 ]. Sodium fluorescein was purchased from Mallinckrodt, Inc (St. Louis, MO). Ethanol, hydrochloric acid (HCl), sodium hydroxide (NaOH), and dichloromethane (DCM) were purchased from VWR (Radanar, PA). MCF-7 model cell line was purchased from ATCC^® (HTB-22) (Manassas, VA). Dulbecco’s Modified Eagle Media (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, acetic acid, dibasic sodium phosphate, coverslips (12 mm borosilicate glass), Permount^™ mounting media, and tissue culture plates were purchased from Fisher Scientific (Walthram, MA). Paraformaldehyde and MitoRed were purchased from Santa Cruz. Phosphate buffered saline (PBS, pH 7.4) was purchased from ThermoFisher (Walthram, MA). Glycine was purchased from Eastman Organic (Kingsport, TN). Sodium acetate was purchased from Fluka (Walthram, MA). Citric acid was purchased from Alfa Aesar (Haverhill, MA). Distilled deionized water of 18.2 MΩ cm was obtained from ultrapure distilled water purifier (ELGA).