Y.-S. Cheng, et al.
FreeRadicalBiologyandMedicine160(2020)719–733
(75,000 cells/dish) were seeded into 35-mm cell culture dishes in
DMEM/F12 for three days. After cells became confluent, they were
starved for 3 h. The cells were then pre-incubated in HH for 1 h fol-
lowed by 24 h incubation in 20 μM of HOHA lactone. Aliquots (20 μL)
of ARPE-19 cell lysates were assayed to determine total intracellular
GSH levels using a 96-well microplate format described earlier [22]. In
these experiments, all of the reagents were prepared in 0.1 M potassium
phosphate buffer with 5 mM EDTA disodium salt, pH 7.5 (KPE buffer).
Briefly, 20 μL of KPE buffer, GSH standards, or samples were added to
the respective microplate wells, followed by the addition of 120 μL of a
freshly prepared 1:1 mixture of DTNB (2 mg/3 mL) and glutathione
reductase (10 U/3 mL). Then, 60 μL of NADPH (2 mg/3 mL) was
quickly introduced into the wells and the plate was mixed well. The
absorbance was read immediately at λ = 412 nm in a microplate
reader (SpectraMax M2, Molecular Devices). Measurements were taken
every 20 s for 5 min (15 readings in total from 0 to 300 s). The total
GSH concentration in the samples was determined by linear regression
to calculate the values obtained from a standard curve.
hypotonic Tris buffer (pH 7.6), divided into aliquots and stored at
−80 °C. The cell mitochondrial solution was thawed and subjected to
three cycles of freeze-thawing in liquid nitrogen/37 °C water bath to
disrupt the mitochondrial membranes just before use. The mitochon-
drial protein concentration was determined using the Pierce 660 nm
Protein Assay kit ThermoFisher Scientific (Waltham, MA).
Assays for mitochondrial enzyme activities. Mitochondrial re-
spiratory chain enzymatic activities (complexes I-IV) were assessed as
previously described [23] and accommodated to 96-well microplate
format.
Complex I activity: To isolated mitochondrial fraction (2 μg of
protein) was added the assay medium containing potassium phosphate
buffer (50 mM, pH 7.5), fatty acid-free BSA (3 mg/mL), KCN (300 μM),
NADH (100 μM) in distilled and deionized water (200 μL). After reading
the baseline at 340 nm for 2 min, ubiquinone 1 (60 μM final con-
centration) was quickly introduced. The decrease in reaction mixture
absorbance was recorded at λ = 340 nm for 60 min. In parallel, the
same quantity of reagents and samples but with the addition of rote-
none solution (10 μM final concentration) was used.
Determination of RPE cellular ATP. To measure cellular ATP
level, CellTiter-Glo (Promega, Madison, WI, USA) was used according
to manufacturer's directions. In brief, after equilibrating to room tem-
perature, equal volume of the CellTiter-Glo reagent was added to the
cell medium of HOHA lactone treated cells in a 96 well white poly-
styrene cell culture microplate (Greiner BioOne CELLSTAR white plate
with solid bottom, Cat. # 655,083) and mixed for 2 min on an orbital
shaker. The microplate was incubated for additional 10 min at room
temperature to stabilize the luminescence and was immediately mea-
Complex II activity: To isolated mitochondrial fraction (1 μg of
protein) was added the assay medium containing potassium phosphate
buffer (25 mM, pH 7.5), fatty acid-free BSA (1 mg/mL), KCN (300 μM),
succinate (20 mM), 2,6-dichlorophenolindophenol (DCPIP) sodium salt
(80 μM) and distilled water. The mixture was incubated at 37 °C for
10 min, and then the baseline was recorded at 600 nm for 2 min. Then
decylubiquinone (DUB, 50 μM final concentration) was applied and the
decrease in absorbance at 600 nm was recorded for 60 min.
sured with
a
luminescence microplate reader (SpectraMax M2,
Complex III activity: To isolated mitochondrial fraction (1 μg of
protein) was added the assay medium containing potassium phosphate
buffer (25 mM, pH 7.5), KCN (500 μM), EDTA (100 μM), oxidized cy-
tochrome c (75 μM), Tween-20 (0.025% (vol/vol)) and distilled water.
After reading the baseline at λ = 550 nm for 2 min, decylubiquinol
(100 μM final concentration) was introduced. The increase in absor-
bance at 550 nm was recorded for 15 min.
Molecular Device).
Detection of CEP in ARPE-19 cells. ARPE-19 cells (10,000 cells/
chamber) were plated on an 8-chamber slide (Lab-Tek II Chamber Slide
System, Nunc, Rochester, NY) in DMEM/F12 supplemented with 10%
FBS and then incubated at 37 °C under 5% CO2 for three days. After 5 h
of starvation in basal DMEM/F12 cell culture medium, the cells were
incubated with HH for 1 h, and were subsequently added HOHA lactone
to a final concentration of 20 μM. After 24 h incubation in a humidified
CO2 incubator at 37 °C under 5% CO2, the cell culture medium was
aspirated from each chamber and the chambers were washed twice
with DPBS. The cells were fixed with cold acetone (−20 °C) for 12 min
at −25 °C. After washing with ice-cold PBST three times, the slides
were blocked with 3% BSA in PBST for 1 h at 23 °C. The cells were
probed with rabbit anti-CEP polyclonal antibody (in 3% BSA in PBST,
18 μg/mL) overnight at 4 °C, and washed exhaustively with PBST the
next day. The slides were treated with Texas Red-X goat anti-rabbit
antibody (1:100 dilution in 3% BSA in PBST; T-6391, ThermoFisher
Scientific, Waltham, MA) overnight at 4 °C, washed exhaustively with
PBST. The slides were further incubated with 1:20 diluted in PBS stock
solution of Flash Phalloidin™ Green 488 (0.2 U/μL, Biolegend, San
Diego, CA) in the dark for 30 min at 23 °C. After washing with PBST,
slides were mounted with DAPI containing Fluoromount-G (Southern
Biotech, Birmingham, AL). All images were acquired with a Leica DMI
6000 B inverted fluorescent microscope using a Retiga EXI camera.
Image analysis was performed using Metamorph imaging software
(Molecular Devices, Downington, PA). The images were acquired at
20× magnification.
Complex IV activity: To isolated mitochondrial fraction (1 μg of
protein) was added the assay medium containing potassium phosphate
buffer (25 mM, pH 7.0), KCN (300 μM), reduced cytochrome c (50 μM)
in distilled and deionized water (200 μL). The decrease in absorbance at
λ = 550 nm was recorded for 15 min.
HPLC analysis of HOHA lactone scavenging activity. Both
10 mM or 100 mM solutions of the compounds and 0.5 mM solution of
HOHA lactone in DPBS were prepared. In a screw cap-equipped vial
were mixed 10 μL of the compound solution and 100 μL of the HOHA
lactone solution (final molar ratio between compound and alde-
hyde = 1:2 or 1:20). The resulting mixture was sealed and incubated at
37 °C. At time (T) = 0, 15, 30, 45 and 60 min a 20 μL sample was
pooled from the vial and injected into the HPLC system. Reverse-phase
HPLC was conducted using a Shimadzu UFLC system equipped with a
5 μm 4.6 × 250 mm Phenomenex Luna C18 column (isocratic mode;
mobile phase, H2O/methanol/formic acid 70:30:0.1; flow rate 1 mL/
min). Peak areas corresponding to the unreacted HOHA lactone were
integrated and the residual concentration was calculated according to
the formula: HOHA lactone residual concentration (%) = (A0/
At) × 100 where A0 is the peak area at time 0 and At is the peak area at
each sampling time. The data were plotted into a graph reporting the
residual % or consuming % concentration of compound vs. time in-
terval (for each compound the analysis was run in duplicate; values are
Isolation of mitochondria from ARPE-19 cells. ARPE-19 cells
were sub-cultured in T-150 flasks and trypsinized once they reached
confluency (10–15 × 106 cells). After being washed twice with DPBS
and centrifuging at 1,000g for 5 min at 4 °C, the cell pellet was flash-
frozen in liquid nitrogen, then thawed and suspended in 1 mL of 10 mM
ice-cold hypotonic Tris buffer (pH 7.6). After the suspension was
homogenized carefully with a Teflon tissue homogenizer on ice, 200 μL
of 1.5 M sucrose solution was added, vortexed thoroughly and cen-
trifuged at 600g for 10 min at 4 °C. The supernatant was collected and
centrifuged additionally at 14,000g for 10 min at 4 °C. The resulting
mitochondrial pellet was resuspended in 0.5 mL of 10 mM ice-cold
the mean
SD).
Quantitation of HH uptake by LC-MS/MS. ARPE-19 cells
(200,000 cells/dish) were seeded in a 60-mm dish and were grown to
confluency. After being starved in basal DMEM/F12 cell culture
medium overnight, the cells were treated with 0–800 μM HH for 24 h.
Before harvest, the cells were washed extensively with HBSS to remove
any residual HH. The cell pellets were frozen in dry ice and resuspended
in methanol. The suspensions were then vortexed and sonicated to
extract the cellular HH.
722