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.”