Fig 1: HKC suppresses TRPC6/CnA/NFAT expression in UUO mice. Representative western blots and quantification of the levels of TRPC6 (A), CnA (B) as well as NFAT (C) in sham, and UUO mice with different treatments. GAPDH was used as an internal control. ## P < 0.01, UUO group vs. sham group; * P < 0.05, ** P < 0.01, UUO + HKC group vs. UUO group. Bar graphs represent the mean ± SEM (n = 3).
Fig 2: Tutin activates CN activity in vitro and in vivo. a Tutin activates CN in vitro, and results were presented as mean ± SD with n = 3. b, c Changes in CN activity after tutin-induced epilepsy. Basal and max CN activity of hippocampus or cortex were assayed (0.5, 1, 2, 6, 12, 24, 72 h). (*P < 0.05, **P < 0.01 vs. control group (basal). #P < 0.05, ##P < 0.01 vs. control group (max.)). Results were represented as mean ± SD with n = 9. d, e Changes in CN activity of hippocampus or cortex after tutin-induced epilepsy. Mice were injected with different doses (0, 1.6, 1.8, 2.0, 2.2 mg/kg) of tutin, CN activity of hippocampus or cortex was assayed (2, 12, 24 h) (*P < 0.05, **P < 0.01 vs. saline group). Results were expressed as mean ± SD with n = 8. f, g CNA expression in hippocampus or cortex by Western blot. Results were represented as mean ± SD with n = 8
Fig 3: CNA knockdown reduces the intensity of seizures and neuronal loss. a Experimental flowchart was shown. b CNA expression in hippocampus by Western blot. Quantitative analysis of CNA in hippocampus of mice, and results were expressed as mean ± SD with n = 6, **P < 0.01, ***P < 0.001, ****P < 0.0001. c Behavioral episodes in mice with CNA knockdown induced by tutin (n = 20, *P < 0.05, **P < 0.01). d Distribution of maximum Racine score within 2 h after seizures, and results were represented as mean ± SD with n = 20 (*P < 0.05, **P < 0.01). e Nissl staining of hippocampal CA1, CA3 and cortical neurons after seizures (scale bar: 100 µm). f Statistical analysis of Nissl-stained cells of hippocampal CA1, CA3 and cortical neurons after seizures; Data are shown as mean ± SD with n = 8, *P < 0.05, **P < 0.01 vs.Tutin (-) -shRNA (-) group, #P < 0.05, ##P < 0.01 vs. Tutin (+) -shRNA (-) group
Fig 4: Tutin binds to CN. a Tutin increases the CNA thermal stability in living cells by temperature-dependent CETSA (n = 3). b Tutin increases the CNA thermal stability in living cells by concentration-dependent CETSA. Results were expressed as mean ± SD with n = 3. c The MST dose-response curve shows the interaction between tutin and CN. Results were expressed as mean ± SD with n = 3. d Deuterium uptake of peptides targeted by tutin. HDX graphs of CN alone (black curves) and bound both to tutin (red curves). e Structural overview of a predicted CN-tutin complex model (CNA: gray, CNB: orange, tutin: yellow, residues: green). Zoom-in view of the predicted CN-tutin interface. f Interface residues (Arg 254 and Ala 283) in CN are shown and labeled by the names and positions of residues
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