Fig 2: AQP4 regulates sodium‐calcium exchange through Nav1.6 to inhibit LPS‐induced astrocyte PPAR‐γ moving into the nucleus. a) Representative families of VGSC current traces in AQP4+/+ and AQP4−/− primary astrocytes which were stimulated by LPS or not (upper). The membrane potential was held at ‐80 mV, and the currents were elicited by 5 ms test pulses ranging from ‐80 to +40 mV in 5 mV steps. Current‐voltage relationship of VGSC currents in primary astrocytes (lower left). Mean current density of VGSC at ‐25 mV in primary astrocytes under different treatments (lower right). n = 8–13 cells from four independent experiments. b) Representative fluorescence images of primary astrocyte incubated with Fluo‐4 AM dye in different groups. Scale bar, 100 µm. c) Measurement of Fluo‐4 AM fluorescence intensity by microplate reader after AQP4+/+ and AQP4−/− primary astrocytes were treated with TTX, KB‐R7943 or EGTA, ATX II followed by LPS challenge or not. n = 6. d) Representative protein bands of PPAR‐γ in the nucleus after AQP4+/+ and AQP4−/− primary astrocytes treated with TTX, KB‐R7943, EGTA, or ATX II followed by LPS challenge or not (left). Quantitative analysis for PPAR‐γ/laminB1 ratio of relative protein expression in nucleus of all groups, n = 5. (right). Data are presented as mean ± SD. a) ** p < 0.01, **** p < 0.0001, two‐way ANOVA with Tukey's post hoc test; c,d) * p < 0.05, *** p < 0.001, **** p < 0.0001, one‐way ANOVA with Tukey's post hoc test.

Fig 3: PC Scn8a mutant mice exhibit histological changes in cerebellum. (A,B) Abnormal PC morphology and gradual reduced PC number and molecular layer thickness in Scn8a mutant cerebellum (Scale bar upper row 200 μm. bottom row 100 μm). (C,D) The quantification of PC density and the quantification of PC soma area was performed in the IV–V. PC loss was not apparent until 5 months of age, and showed ongoing loss till 9 months (C). At 9 months, mutant Purkinje cell size decreased significantly (D). (E) The thickness of the molecular layer (ML) was normal in mutant cerebellum at 4 months of age, whereas it decreased afterward at 5 months (n ≥ 3 per group. Data shown are means ± SEM. **P < 0.01, ***P < 0.001, two-way ANOVA, Bonferroni's post-hoc analysis).

Fig 4: Downregulation of overexpressed genes with hypermethylated gene body CGI in Alb-R26Met HCC cells interferes with their tumorigenic properties both in vitro and in vivo. a Western blots showing SCN8A, ACTN1, SRD5A2, NFkB2 and NEURL1B protein levels in stable clones established after transfection of Alb-R26Met HCC14 cells with plasmids carrying a shRNA sequence targeting the corresponding gene. Protein levels were compared to control cells (ctr). ACTIN was used as a loading control in all western blots. The asterisk indicates nonspecific bands detected using anti-SRD5A2 antibodies. b–e Biological assays to assess functional properties of Alb-R26Met HCC14 cells carrying a shRNA sequence targeting candidate genes. Effects were compared to HCC14 cells either untransfected or transfected with a control shRNA (shCtrl). b Graph reporting the number of colonies formed in anchorage-independent growth assays using 2 different shRNA targeting sequences for each candidate gene. Note a decrease in colony number formation of cells with downregulated candidate genes compared with control cells. c Graphs reporting the number (left) and the size (right) of colonies formed in anchorage-dependent growth assays. Whereas no significant changes in colony numbers were detected, note a significant decrease in colony size when the candidate gene is downregulated. d Graph reporting number of spheres formed in tumour sphere assays. Note that downregulation of candidate genes significantly reduces sphere number formation. e Graph reporting the tumour volume of mice injected either with Alb-R26Met HCC14 control cells or with Alb-R26Met HCC14 cells carrying a shRNA sequence targeting candidate genes. Note that downregulation of candidate genes significantly interferes with the in vivo tumorigenic properties of Alb-R26Met HCC14 cells. Significant differences between groups are indicated on the top. *P < 0.05, **P < 0.01, ***P < 0.001 (nd: no determined)

Fig 5: Abnormal social and repetitive behaviors and impaired reversal learning in PC Scn8a mutant mice. (A) Mutant mice spent comparable time sniffing non-social stimuli whereas less time sniffing social odor cues (n = 11 per group). (B) Reduced sociability of KO mice is indicated by reduced time interacting with a female conspecific (Control: n = 8; Mutant: n = 10). (C) PC Scn8a mutant mice spent more time self-grooming (Control: n = 10; Mutant: n = 11). (D,E) On Day 1 to Day 3, mutants display normal acquisition learning of the escape platform location in the water T maze as indicated by total correct trials (D) and trials needed for 5 consecutive correct responses (E). However, on reversal day 1 and day 2, PC Scn8a mutant mice have significantly fewer correct trials (D), and take more trials to achieve five consecutive correct responses (Control: n = 9, Mutant: n = 12). RD, Reversal Day. Data shown are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Two-way ANOVA, Bonferroni post-hoc analysis in (A), Student's unpaired t-test in (B–E).