Figure 4.

Klotho suppression of the TLR4-NF-κB signaling in NP cells. (a) NP cells were transfected with HA-con and HA-Klotho plasmid for 24 h, then treated with H₂O₂ (200 μM) for 12 h. The overexpression of HA-tagged Klotho was verified with an anti-HA antibody. Cell lysates were tested for Klotho, TLR4, p-IκBα, and IκBα expressions by Western blotting. (b) Immunofluorescent staining of NF-κB nuclear translocation. NP cells transfected and treated as in Figure 4(a) for 30 min were stained with anti-NF-κB antibody (B), counterstained with DAPI (A), and the figures were merged (C). Scale bar, 20 μm (original magnification ×40). (c) NP cells transfected and treated as in Figure 4(a) for 6 h were tested for IL-1β, NOS2, and IL-18 mRNA levels by qRT-PCR. GAPDH served as an internal control. The results were presented as mean ± SD of three independently performed experiments. ^∗P < 0.05, ^∗∗P < 0.01 vs. control. ^#P < 0.05 vs. H₂O₂.