R. Campagna et al.
BBA - Molecular Cell Research 1868 (2021) 119082
2.6. Determination of NNMT silencing by real time-PCR
USA). Secondary antibodies used were Alexa Fluor 647-conjugated goat
anti-rabbit and Cy3-conjugated goat-anti-mouse (Jackson Immuno,
Cambridgeshire, UK). For nuclei counterstaining, Hoechst 33258 solu-
tion was used. Images of immunostained cells were taken using a CQ1
(Yokogawa, Musashino, Tokyo, Japan) and CQ 1.04 software, then
analyzed automatically with Columbus 2.4.2 software (Perkin Elmer,
Waltham, MA, USA). Nuclear expression (SIRT1 and pSIRT) was quan-
tified based on counterstaining and specially-design algorthitms of the
Columbus 2.4.2 software. For data normalization, each immunostaining
(n = 6) was performed using cells with similar confluence (≥90%), the
same primary and secondary antibody concentration and constant in-
cubation time for each staining step. As a negative control, cells treated
only with secondary antibodies were used to estimate the background
signal.
Total RNA was isolated through the SV Total RNA Isolation System
(Promega, Madison, WI, USA), according to the manufacturer's protocol.
Total RNA (2 μg) was reverse-transcribed in a total volume of 25 μl for
60 min at 37 ◦C with M-MLV Reverse Transcriptase (Promega, Madison,
WI, USA), using random primers. cDNA samples were used to perform
subsequent real-time PCR analysis. To evaluate NNMT mRNA expression
quantitatively, a real-time PCR assay was performed using a CFX96
Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA,
USA). cDNA, generated as described above, was used as a template. The
primers used were 5′-GAA TCA GGC TTC ACC TCC AA-3′ (forward) and
5′-TCA CAC CGT CTA GGC AGA AT-3′ (reverse) for NNMT, and 5′-TCC
TTC CTG GGC ATG GAG T-3′ and 5′-AGC ACT GTG TTG GCG TAC AG-3′
for β-actin.
Both genes were run in duplicate for 40 cycles at 94 ◦C for 30 s and
58 ◦C for 30 s, using a SsoFast EvaGreen Supermix (Bio-Rad Labora-
tories, Hercules, CA, USA). All samples were tested in triplicate using the
β-actin gene for data normalization. Direct detection of PCR products
was monitored by measuring the fluorescence produced by EvaGreen
dye binding to double-stranded DNA after every cycle. These measure-
ments were then plotted against cycle numbers. The parameter
threshold cycle (Ct) was defined as the cycle number at which the first
detectable increase above the threshold in fluorescence was observed.
Following NNMT gene silencing in EA.hy926 cells, fold changes in
relative gene expression were calculated by 2ꢀ Δ(ΔCt), where ΔCt = Ct
(NNMT) - Ct (β-actin) and Δ(ΔCt) = ΔCt (cells transfected with plasmid
vectors) - ΔCt (mock).
2.9. Cell proliferation assay using electric cell-substrate impedance
sensing
Cells were grown on the surface of planar golden electrodes of an
electric cell-substrate impedance sensing set-up (ECIS), and the resis-
tance of the cell-covered electrode was measured continuously at a
frequency of 4000 Hz. Because of the insulating properties of cell
membranes, the resistance increases with increasing coverage of the
electrode. HAECs and HMEC-1 cells were seeded in 96-well, gold-film
electrode-coated 96W1E + PET arrays (Applied Biophysics). The resis-
tance parameter was constantly monitored up to 125 h after seeding.
2.10. Mitochondrial stress test
2.7. Cell viability assay in menadione-induced endothelial cell injury
For the mitochondrial stress test (MST), HAEC cells were seeded into
Seahorse XFe 96-well plates at a density of 1.5 × 103 per well to produce
confluency on the next day. The cells were treated with JBSF or 5MQ or
not treated for 72 h, and the medium was exchanged after 24 and 48 h
before MST. An assay medium for MST was prepared on the day of the
experiment by supplementation of Seahorse DMEM XF Base medium
(Agilent) with glucose (1 g/l; Merck), glutamine (2 mM; Sigma-Aldrich)
and sodium pyruvate (1 mM; Sigma-Aldrich) and adjusting the pH to 7.4
using 0.1 M NaOH (Sigma-Aldrich). Immediately prior to MST, the cells
were washed twice with the assay medium, then fresh assay medium
was added and cells were incubated for 1 h at 37 ◦C without CO2.
Changes in oxygen consumption rate (OCR) and extracellular acidifi-
cation rate (ECAR) were recorded over time. After four basal measure-
To assess cell viability, cells were cultivated in full medium and
treated with menadione (Sigma Aldrich, Saint Louis, MO, USA) for 24,
48 or 72 h and 5MQ or JBSF-88 when indicated. To confirm that effects
of NNMT inhibitors are not related to their toxicity 5MQ and JBSF-88
(3–15 uM) incubated with HAEC and HMEC-1 for 2, 24 and 72 h were
tested by MTT assay and there was no statistically significant effects on
the cell viability for all the concentrations and time-points used in this
study. Additionally, cells were treated with specific SIRT1 inhibitor EX-
527 (Sigma Aldrich, Saint Louis, MO, USA) to examine the role of
sirtuin-dependent pathways in menadione-induced endothelial injury.
Cells were washed with PBS before fixing (with a solution of 4% para-
formaldehyde at room temperature) or before live cell imaging and
incubated with a solution of YO-PRO™-1 Iodide (Thermo Fisher Scien-
tific, USA) diluted 1:1000 in the dark, at 37 ◦C for 30 min. For nuclei
counterstaining, a Hoechst 33258 solution (Sigma Aldrich, Saint Louis,
MO, USA) was used. Imaging was performed with a confocal quantita-
tive image cytometer (CQ1) (Yokogawa, Musashino, Tokyo, Japan).
Images were analyzed via Columbus 2.4.2 software (Perkin Elmer,
Waltham, MA, USA) and the number of cells and/or number of cells
positive to the YO-PRO™-1 iodide stain was calculated.
ments, oligomycin (1
followed by three measurements, then FCCP (1.5
was added from port B, followed by three measurements, and finally,
μ
g/ml; Calbiochem) was added from port A,
μM; Sigma-Aldrich)
rotenone (0.5 μM; Sigma-Aldrich) with antimycine A (0.5 μM; Sigma-
Aldrich) were added from port C, followed by three measurements.
Concentrations of oligomycin, FCCP, rotenone and antimycine A had
been optimized in preliminary experiments. After the assay, cells were
washed once with DPBS (Gibco), 10 μl of 0.1% Triton X-100 (Sigma-
Aldrich) in DPBS was added per well, and cells were frozen at ꢀ 80 ◦C.
On the next day, the protein content was measured using a Bicinchoninic
Acid Protein Determination Kit (Sigma-Aldrich). The data were
normalized for protein content and analyzed using the Seahorse XF Cell
Mito Stress Test Report Generator (Agilent). The following parameters of
mitochondrial function were analyzed: basal respiration, proton
leakage, ATP production, maximal respiration, spare respiratory ca-
pacity and non-mitochondrial oxygen consumption.
2.8. Expression of SIRT1, pSIRT1, p53, acetyl-p53 and NNMT by
immunocytochemistry
Cells were plated in 96-well format on black Corning multiplates
with a clear bottom. After the treatment, cells were fixed with a 4%
formalin solution for 10 min, washed with PBS and then incubated with
a blocking solution containing 5% normal goat serum (Sanquin,
Amsterdam, Netherlands) to minimalize non-specific binding of anti-
bodies. For indirect immunohistochemical detection, cells were incu-
bated overnight with the following primary antibodies: NNMT
polyclonal antibody, p53 monoclonal antibody, Acetyl(Lys382)p53
monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA),
SIRT1 monoclonal antibody (Merck Millipore, Burlington, MA, USA),
Phospho-SIRT1 (Ser47) polyclonal antibody (Cell Signaling Technology,
2.11. Statistical analysis
Data were analyzed using GraphPad Prism software version 6.00 for
Windows (GraphPad Prism Software, San Diego, CA, USA). Differences
between groups were determined using the Mann-Whitney nonpara-
metric test, or Kruskal-Wallis one-way ANOVA, followed by a post-hoc
Dunn test. A p-value <0.05 was considered statistically significant.
4
Campagna, Roberto
