Figures and Legends
Figure 1. Effect of α-Klotho (A,B) and LPS (C,D) treatment on cell
viability (A,C) and cytotoxicity (B,D) of mouse glial cells. The primary
culture of glial cells was subjected to 24-hour treatment with LPS in
different concentrations. Cell viability and cytotoxicity were assessed
by MTT and LDH assays, respectively. One-way ANOVA analysis, followed by
Tukey’s post-test, * p <0.05. Results are presented as mean ±
SEM of 7 independent experiments.
Figure 2. Effect of α-Klotho on
LPS-induced TNF-α secretion in mouse glial cells. Glial culture was
pre-treated with serum-free medium (control) or with α-Klotho protein in
different concentrations (0.1, 0.5, 1 and 2 nM) and times, 1 (A), 4 (B)
and 24 (C) hours, and then
challenged with 1 µg/mL / mL LPS
for 8 hours. The supernatant was collected to measure TNF-α levels.
One-way ANOVA analysis, followed by Tukey’s post-test, * p
<0.05, ** p <0.01. Results are presented as mean ±
SEM of 7 independent experiments.
Figure 3. Effect of α-Klotho on IL-1β, IL-6 and IFN-γ levels in mouse
cell glia challenged with LPS. Glial culture was pretreated with
serum-free medium (control) or with 1 nM α-Klotho for 24 hours, and then
challenged with 1 µg/mL / mL LPS for 8 hours. The supernatant was
collected to measure the levels of IL-1β (A), IL-6 (B), IFN-γ (C).
One-way ANOVA analysis, followed by Tukey’s post-test, * p
<0.05, ** p <0.01, **** <0.0001. Results
are presented as mean ± SEM of 7 independent experiments.
Figure 4. α-Klotho rescue the NF-κB activation induced by LPS in nuclear
fraction. (A ) Purified astrocytes from cortical glial cells
were treated 24h with vehicle (control) (PBS) or 1 nM α-Klotho.
Inflammatory stimulation with LPS 1 μg/ml was then performed and the
cells fixed with methanol 4 hours later stained with RelA antibody and
DAPI. (B ) The RelA (p65)-positive nuclei were counted and
divided by the total number of nuclei, and the graph shows the
comparison between the LPS group and for α-Klotho + LPS group expressed
by the ratioin abitray units of RelA (p65) translocated to the nucleus
over the total amount of RelA (p65) (n = 5). One-way ANOVA analysis,
followed by Tukey’s post-test, **p<0,01; ***p<0,001;
****p<0,0001. (C,D ) Effect of α-Klotho on p65 subunit
NFkB translocation in astrocytes cells.Nuclear (15 mg) and cytosol (20
mg) proteins were extracted from primary cultured cells: (C)
Representative Western blotting autoradiographs of RELA (p65) nuclear
and cytosolic and b-actin; (D) Densitometric analysis (arbitrary units,
A.U.) of p65 nuclear/b-actin and p65 cytolosic/b-actin ratios of groups
presented in C panel (n = 5). One-way ANOVA analysis, followed by
Tukey’s post-test, **p<0,01; ***p<0,001;
****p<0,0001. (E) Nuclear fraction was used to perform the
EMSA assay to measure NF-kB activity. (F ) Densitometric
analysis comparing NF-κB activity of contro, α-Klotho, LPS and α-Klotho
– LPS groups (n = 5). One-way ANOVA analysis, followed by Tukey’s
post-test,***p<0,0001.(G ) A super-shift was also
performed to show which NF-κB subunits are involved in this phenomenon.
Figure 5. Effects of GCM -induced cytotoxicity of mouse neurons. The
primary embryonic culture of neurons was subjected to 24-hour treatment
with GCM in different concentrations (25% and 50%). Cytotoxicity was
assessed by the LDH assay. One-way ANOVA analysis, followed by Tukey’s
post-test, * p<0.05. Results are presented as mean ± SEM of 5
independent experiments.
Figure 6. Effects of GCM and GCM-KL -induced cytotoxicity of primary
cortical mouse neurons. The primary embryonic culture of neurons was
submitted to 24-hour treatment with GCM or GCM-KL in different
concentrations, 25% (A) and 50% (B). Cytotoxicity was assessed by the
LDH assay. One-way ANOVA analysis, followed by Tukey’s post-test, * p
<0.05, ** p <0.01. Results are presented as mean ±
SEM of 5 independent experiments.
Figure 7. Representative schedule of the anti-inflammatory and
neuroprotective effect of α-Klotho protein. LPS induces GCM to produce
pro-inflammatory mediators that can lead to neuronal death (A). α-Klotho
protein decreases the production of pro-inflammatory mediators induced
by LPS in GCM (B), and it can have a protective effect on neurons from
the neurotoxic effects of LPS induced by GCM. Figure was “Created with
BioRender.com.”