Supplemental Materials and Methods
Cell growth curve- MDCK cells were seeded at a density of 2×104 per well in
12-well plates and cultured in regular DMEM. Cells were then serum-starved for 16 h,
treated or not treated with ketamine (20 g/ml) and incubated at 37°C for 0, 12, 24, 36
and 48 h. After treatment, the cells were washed twice with ice-cold PBS and
trypsinized with trypsin-EDTA (Gibco-BRL, Life Technologies, Grand Island, NY,
USA). Cell suspensions were then mixed with trypan blue dye (Sigma-Aldrich, St.
Louis, MO, USA) for cell counting using a hemocytometer under a microscope. The
cell growth curves were calculated and established based on the numbers of viable
cells using trypan blue exclusion. The trypan blue-stained cells were classified as
damaged or dead cells.
Supplemental Figure Legends
Supplemental Fig. 1. Ketamine modulates EPC-related protein expression in MDCK
cells.
MDCK cells were treated with different concentrations of ketamine (0-20 μg/ml) for
24 h, and total cell lysates were collected for immunoblot analysis using antibodies
against epithelial junction proteins, including ZO-1 (A), Occludin (B), and E-cadherin
(C), as well as to detect the mesenchymal marker proteins N-cadherin (D),
Fibronectin (E) and Vimentin (F). A set of representative immunoblots is shown in
Figure 4B. The density of each band was measured using a densitometer with
normalization to the loading control (α-tubulin). Quantification values were further
calculated and expressed as the ratio to vehicle (mean±SD) for three independent
1
experiments. ** P<0.01 compared with the vehicle group.
Supplemental Fig. 2. Ketamine induces morphological changes and promotes cell
proliferation in MDCK cells.
(A) Cell morphology of ketamine-treated MDCK cells was analyzed by
phase-contrast microscopy. Control cells were treated with vehicle. MDCK cells were
seeded at a density of 1×104 per well on 6-well plates and cultured in regular DMEM.
Cells were then serum-starved for 16 h and treated or not treated with ketamine (20
μg/ml) in DMEM containing 0.5% FBS for 24, 48 and 72 h. Cell morphology was
visualized on a phase-contrast microscope. In addition, serum was utilized as a
positive control to show the epithelial phenotypic changes of MDCK cells. MDCK
cells were serum-starved for 16 h and then exposed to 10, 20, 30, 40 and 50% serum
for 24, 48, and 72 h. The results showed that 24 h after treatment, serum
concentrations of 20% or higher induced morphological changes in MDCK cells, with
reduced tightness of cell-cell contacts and roundish or spindle-like shapes. Forty-eight
hours after serum exposure, serum concentrations of 20% or higher dramatically
altered the MDCK phenotypes, leading to the loss of cell polarity and loose cell-cell
contacts. These data indicate that the effects of serum on the phenotypic changes of
MDCK cells are dose- and time-dependent. Interestingly, our results further showed
that 72 h of treatment with 20 μg/ml ketamine also induced morphological changes in
MDCK cells that were similar to the effects of serum on cell morphology. These
results suggest that, like serum, ketamine can induce epithelial phenotypic changes in
MDCK cells. Images are representative of at least three independent experiments. Bar,
100 μm. (B) Analysis of the effects of ketamine on the growth of MDCK cells.
MDCK cells were seeded at a density of 2×104 per well in 12-well plates and cultured
in regular DMEM. Cells were then serum-starved for 16 h and treated or not treated
with ketamine (20 g/ml) for 0, 12, 24, 36 and 48 h. For cell number measurement,
2
cells were trypsinized and counted at each time-point using a trypan blue exclusion
assay and a hemocytometer under a microscope. Each experiment was performed in
triplicate. The data are presented as the mean±SD of three independent experiments.
** P<0.01 compared with the vehicle group at the corresponding time-points. The
results showed that ketamine had no significant effect on MDCK cell proliferation
before or at 24 h after the treatment and significantly induced cell proliferation after
36 h or longer treatment. The data indicate that ketamine exhibits an inductive effect
on the proliferation of renal distal tubular cells.
Supplemental Fig. 3. Ketamine induces the phosphorylation of FAK, Akt and
GSK-3 and increases the protein levels of GSK-3downstream targets (-catenin
and Cyclin D1) in MDCK cells.
(A) Quantification of FAK, Akt and GSK-3β phosphorylation status in
ketamine-treated MDCK cells from Figure 6A. Cells were treated with the indicated
concentrations of ketamine (0-20 μg/ml) for 3 h or with ketamine (20 μg/ml) for the
indicated times. Total cell lysates were collected for western blot analysis with
anti-p-FAK, anti-FAK, anti-p-Akt, anti-Akt, anti-p-GSK-3, and anti-GSK-3β
antibodies. A set of representative immunoblot images was shown in Figure 6A.
Quantification of p-FAK, p-Akt, p-GSK-3β and their corresponding total protein
levels was performed using a densitometer with normalization to their total protein
levels. The levels of FAK, Akt and GSK-3β phosphorylation were further calculated
and expressed as ratios with respect to vehicle. The results were calculated from three
independent experiments and showed that ketamine significantly induced the
phosphorylation of FAK, Akt and GSK-3β in a dose-response manner. Moreover,
ketamine increased the phosphorylation of FAK, Akt and GSK-3β as soon as 3 h after
treatment with 20 μg/ml ketamine and reached a plateau. (B) Immunoblot analysis of
GSK-3 downstream targets in ketamine-treated MDCK cells. Cells were treated with
3
different concentrations of ketamine (0-20 μg/ml) for 24 h, and total cell lysates were
collected for immunoblot analysis using antibodies against GSK-3 downstream
targets including -catenin and Cyclin D1. The density of each band was measured
using a densitometer and normalized to the loading control (α-tubulin). Quantification
values were calculated and expressed as ratios relative to vehicle. All data are
presented as the mean±SD of three independent experiments. * P<0.05; ** P<0.01
compared with the vehicle group. The results showed that ketamine induced β-catenin
and Cyclin D1 protein levels in MDCK cells, indicating that ketamine can affect
GSK-3β activity, leading to increased levels of β-catenin and Cyclin D1 protein.
Supplemental Fig. 4. Ketamine induces GSK-3β phosphorylation and modulates
EPC-related protein expression in MDCK cells.
(A) Immunoblot analysis of Akt and GSK-3β phosphorylation levels, as well as
c-Myc protein levels in ketamine-treated MDCK cells in the presence or absence of
PI3K/Akt or GSK-3β inhibitor. MDCK cells were pretreated with 2.5 M of a
PI3K/Akt inhibitor, LY294002, or 5 M of a GSK-3β inhibitor, 3F8, for 1 h and then
treated or not treated with ketamine (20 μg/ml) for 3 h. Total cell lysates were
collected for western blot analysis with anti-p-Akt, anti-Akt, anti-p-GSK-3,
anti-GSK-3β, anti-c-Myc and anti-α-tubulin antibodies. The experiments were
independently performed three times. Quantifications of p-Akt, p-GSK-3β, their
corresponding proteins and c-Myc protein levels were performed using a densitometer.
Akt and GSK-3β phosphorylation levels were further normalized to their total protein
levels, while the levels of c-Myc were normalized to the loading control -tubulin.
The results were further calculated and expressed as ratios relative to vehicle. The
results showed that ketamine significantly induced the phosphorylation of Akt and
GSK-3β, as well as the protein level of c-Myc in MDCK cells. Inhibition of PI3K/Akt
signaling by LY294002 blocked the effect of ketamine on Akt and GSK-3β
4
phosphorylation and c-Myc expression. Moreover, GSK-3β inhibition by 3F8
increased c-Myc protein levels, similar to the results of ketamine treatment. A
combination of ketamine and 3F8 had no additive effect on Akt and GSK-3β
phosphorylation levels or on c-Myc expression. Taken together, these results indicate
that ketamine can induce the activation of PI3K/Akt/GSK-3β signaling in MDCK
cells. (B) Quantification of the immunoblot results from the examination of the effect
of PI3K/Akt or GSK-3β inhibition on the levels of ketamine-modulated EPC-related
proteins. MDCK cells were pretreated with 2.5 M of the PI3K/Akt inhibitor
LY294002 or 5 M of the GSK-3β inhibitor 3F8 for 1 h and then treated or not
treated with ketamine (20 μg/ml) for 24 h. Total cell lysates were collected for
immunoblot
analysis
with
anti-ZO-1,
anti-Occludin,
anti-E-cadherin
and
anti-Vimentin antibodies. A set of immunoblot images was shown in Figure 6B. The
density of each band was measured using a densitometer with normalization to the
loading control (α-tubulin). The quantification results were further statistically
calculated and expressed as ratios relative to vehicle. All data are presented as the
mean±SD of three independent experiments. ** P<0.01 compared with the vehicle
group;
##
P<0.01 compared with the ketamine group. The results showed that
ketamine significantly reduced the levels of junctional proteins (ZO-1, Occludin and
E-cadherin) and increased the level of the mesenchymal protein Vimentin. Inhibiting
PI3K/Akt signaling with LY294002 suppressed the effects of ketamine on the
EPC-related proteins. Interestingly, treatment with a selective GSK-3β inhibitor, 3F8,
reduced ZO-1, Occludin and E-cadherin protein levels and enhanced Vimentin protein
levels, similar to ketamine treatment. The 3F8 treatment had no additive effect on
ketamine-suppressed ZO-1, Occludin and E-cadherin, or ketamine-induced Vimentin,
suggesting a common mechanism of action through GSK-3β inactivation in ketamine
signaling to induce EPCs in renal distal tubular cells.
5
Supplemental Fig. 5. Examination of the effects of the well-known GSK-3β inhibitor
LiCl on Akt and GSK-3β phosphorylation levels, EPC-related protein levels, and
TEER in MDCK cells.
(A) Immunoblot analysis of the effects of the GSK-3β inhibitor LiCl on Akt and
GSK-3β phosphorylation and protein levels in ketamine-treated MDCK cells. MDCK
cells were pretreated with 10 mM LiCl for 1 h and then treated or not treated with
ketamine (20 μg/ml) for 3 h. Ten millimolar NaCl was applied to MDCK cells as a
negative control to exclude ionic strength or osmotic effects. Total cell lysates were
collected for western blot analysis with anti-p-Akt, anti-Akt, anti-p-GSK-3 and
anti-GSK-3β antibodies. The experiment was independently performed three times,
and a representative blot is shown in the upper panels. p-Akt and p-GSK-3β levels
were quantified using a densitometer with normalization to their corresponding total
protein levels. The results were further calculated and expressed as ratios relative to
vehicle and showed that LiCl increased GSK-3β phosphorylation levels with no effect
on Akt. NaCl exhibited no significant effect on either Akt or GSK-3β, suggesting that
the ionic strength or osmotic effect of LiCl does not play a significant role in the
increased phosphorylation levels of GSK-3β. (B) Examination of the effects of the
GSK-3β inhibitor LiCl on ZO-1, Occludin, E-cadherin and Vimentin protein levels in
ketamine-treated renal distal tubular cells. MDCK cells were pretreated with 10 mM
LiCl for 1 h and then treated or not treated with ketamine (20 μg/ml) for 24 h. Ten
6
millimolar NaCl was applied to treat MDCK cells as negative control to exclude ionic
strength or osmotic effects. Total cell lysates were collected for immunoblot analysis
with anti-ZO-1, anti-Occludin, anti-E-cadherin and anti-Vimentin antibodies. The
density of each band was analyzed using a densitometer with normalization to the
loading control (α-tubulin). The experiment was performed independently three times.
Quantification values were further statistically calculated and expressed as ratios
relative to vehicle. The results showed that GSK-3β inhibition with LiCl significantly
reduced the levels of junctional proteins (ZO-1, Occludin and E-cadherin) and
increased the level of the mesenchymal protein Vimentin, similar to the effects of
ketamine on these proteins. Again, NaCl had no significant effect on these proteins,
suggesting that the LiCl-altered levels of these EPC-related proteins were not due to
ionic strength or osmotic effects. (C) Examination of the effects of LiCl on the TEER
of polarized MDCK cells in the presence or absence of ketamine. A polarized MDCK
monolayer on a filter was pretreated with 10 mM LiCl for 1 h and then treated or not
treated with ketamine (20 µg/ml) on both the apical and basolateral surfaces for 24 h.
TEER was then measured using a Millicell-ERS Volt-ohm Meter resistance apparatus.
Ten millimolar NaCl was used to treat MDCK cells as negative control to exclude
ionic strength or osmotic effects. TEER values were calculated statistically with
normalization to vehicle cells. All data are presented as the mean±SD of three
7
independent experiments. ** P<0.01 compared with the vehicle group. The results
showed that GSK-3β inhibition with LiCl exhibited a similar effect as ketamine in
reducing the TEER values of polarized MDCK cells, and NaCl had no effect on the
TEER values of the polarized cells. Taken together, these results indicate that the
inhibition of GSK-3β by LiCl can promote the EPC of renal distal tubular cells by
altering junctional proteins and increasing the permeability of polarized cells.
LiCl-induced EPC is apparently not due to the ionic strength or osmotic effect of this
compound.
Supplemental Fig. 6. Ketamine induces proinflammatory cytokines production via
PI3K/Akt/GSK-3β signaling in MDCK cells.
MDCK cells were pretreated with 2.5 μM of the PI3K/Akt inhibitor LY294002 for 1
h and then treated or not treated with ketamine (20 μg/ml) for 24 h. Otherwise, cells
were exposed to 5 μM of the GSK-3β inhibitor 3F8 for 24 h. The mRNA levels for
TNF-, IL-6 (B) and TGF-1 (C) in response to individual treatment was
measured using quantitative RT-PCR. The data were normalized to vehicle cells and
are presented as the mean±SD from at least three independent experiments. ** P<0.01
compared with the vehicle group; ## P<0.01 compared with the ketamine group.
8