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    2020-08-30

    r> plate and fluorescein was quantified (ex/em 490/520 nm) against a standard curve in PBS using a Tecan Plate Reader. The average of three biological repeats was used to measure the amount of fluorescein up-take.
    2.11. Fluorescein-modified peptide uptake and retention studies
    20,000 BODIPY493 / 503
    were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. Non-toxic concentrations of peptides were used (< 0.8 μM). The EMT6/AR-1 cells were treated with the fluorescein-modified peptides (0.8 μM) for 2, 5, or 24 h in full media. The fluorescein retention study was completed by incubating with either peptides alone or co-incubation of Str-H8R8 (0.8 μM) with free VES (20 μM) for 24 h, followed by 24 h in fresh media. A similar DMSO concentration was used as a control. After treatment, cells were washed 3 times with PBS, and harvested with trypsin. Cell fluorescence was analyzed using a BD Accuri C6 flow cytometer with excitation wavelength of 488 nm and emission filters of 533/30 nm (fluorescein, FL-2 channel). Cell debris and doublets were gated out using FSC-A vs FSC-H, and at least 10,000 events were col-lected. The mean fluorescence intensity in the FL-1 channel (ex/em 488/533(30) nm) for three biological repeats was used to measure the amount of fluorescein uptake.
    2.12. Mitochondrial membrane polarization assay
    Mitochondrial membrane potential was assayed using the JC-1 probe (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine io-dide) (ex/em 488 nm/533–585 nm) (Biotium Inc., CA, USA) [21]. 20,000 cells were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. The cells were treated the next day for 2 or 5 h in full media. Control treatments and peptides were incubated at 8 μM, in the range of the IC50 of both Str-H8R8 and VES-H8R8. A similar DMSO concentration was used as a control. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 50 μM was used as a positive control for mitochondrial depolarization. Following treatment, the cells were washed with PBS 3 times and then incubated at 37 °C with 10 μM of JC-1 in full media for 30 min. The EMT6/P cells were incubated with 2 μM of JC-1 in full media to avoid fluorescent saturation in the flow cytometer. The cells were then washed 3 times in PBS, trypsinized, and placed on ice before measuring fluorescence in a flow cytometer within 1 h. Cell debris and doublets were gated out using FSC-A vs FSC-H, and at least 10,000 events were collected. A gate was set according to DMSO and CCCP treated cells in the FL-2 channel (ex/em 488 nm/585(40) nm) to measure the proportion of JC-1 aggregate fluorescence versus JC-1 monomer fluorescence in the FL-1 channel (ex/em 488 nm/533(30) nm). To assess the mitochondrial permeability transition pore formation, cy-closporine A was used as previously described [21]. Averages were ob-tained from three biological repeats. Changes in the mitochondria mem-brane potential (ΔΨm) were expressed using the following equation:
    Relative Mitochondrial Membrane Potential (m)
    JC1aggregate = JC1monomer Treatment x 100%
    JC1aggregate
    JC1monomer Control
    2.13. Real-time investigation of Oxygen Consumption Rate (OCR) and Extracellular Adcification rate (ECAR)
    Analyses of bioenergetics processes were performed in intact EMT6/ P and EMT6/Ar-1 cells using the Seahorse XF Analyzer (Agilent, CA, USA). An optimized cell density of 10,000 cells/well were seeded in a XF96 V4 cell culture microplate and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured  Journal of Controlled Release 305 (2019) 210–219
    as cells were incubated with 30 μM of VES-H8R8 or Str-H8R8 for 2 h before inhibitors were added. An optimized concentration of 1 μM of carbonyl cyanide p-triflouromethoxyphenylhydrazone (FCCP) was used. Basal respiration, proton leak, ATP production and maximum-respiratory rate were calculated as reported before and averaged from three biological repeats [22].
    Reactive oxygen species (ROS) in cells were detected using 5-(and-6)-carboxy-2,7-dichlorodihydrofluorescein diacetate (CDCFDA) (AAT Bioquest, CA, USA) as previously described [23]. 20,000 cells were seeded into 48-well plates and allowed to adhere for 24 h at 37 °C in a 5% CO2 and 95% air humidified incubator. The cells were treated the next day for either 2 or 5 h in full media. Control treatments and pep-tides were incubated at 8 μM, in the range of the IC50 of both Str-H8R8 and VES- H8R8. A similar DMSO concentration was used as a control. Hydrogen peroxide (H2O2) at 1 mM was used as a positive control. Following treatment, cells were washed with PBS 3 times, and then incubated at 37 °C in full media containing 2 μM CDCFDA for 30 min. The cells were washed 3 times with PBS, harvested, and placed on ice before measuring fluorescence using a flow cytometer within 1 h. Cell debris and doublets were gated out using FSC-A vs FSC-H, and 10,000 events were collected. The mean fluorescence intensity was collected in the FL-1 channel (ex/em 488/533(30) nm) and averaged from three biological replicates. The results were expressed as fold increase in mean fluorescence intensity of treated group relative to DMSO control. To assess the radical scavenger capability of free vitamin E, 100 μM of vitamin E was incubated simultaneously with peptide treatments (8 μM) for 5 h and ROS was measured as stated above.