Fig 2: AAV-shRNA targeting HSP27 increased cell apoptosis after SAH in rats. (A) Schematic of AAV vector encoding shRNA and experimental design. (B–D) Coronal sections from the sham (n = 6), SAH + vehicle (n = 6), SAH + negative control of shRNA (SAH + NC, n = 7), and SAH + HSP27 shRNA (SAH + shRNA, n = 6) group reperfusion on day 2 after SAH, subjected to immunostaining for the neuronal marker NeuN (red) or HSP27 (red) or TUNEL (green) in the basal cortex. (B) Representative images showing AAV-eGFP-shRNA expression (indicated by EGFP, green) in the neurons (NeuN staining, red) in the basal cortex, scale bar = 50 μm. (C,D) Quantification was performed by counting the HSP27 or TUNEL positive cells per mm2 region in the basal cortex, scale bar = 50 μm. Data are mean ± SD, *p < 0.05, ANOVA with Bonferroni's multiple comparisons test.

Fig 3: Change of HSP27 level in rat SAH model. (A) Representative images of rat brain at indicated time from the sham and SAH groups. (B) Samples of CSF was collected (6, 12, 72 h, n = 6; 24 h, n = 7), HSP27 concentration was measured with HSP27 ELISA Kit and expressed in ng/ml. (C,D) Coronal sections from the sham and SAH group reperfusion (12 and 72 h) subjected to immunostaining for the neuronal marker NeuN (green) or macrophages/microglia marker Iba-1 (green) and HSP27 (red) in the basal cortex. Quantification was performed by counting the HSP27/NeuN or HSP27/Iba-1 positive cells per mm2 region in the basal cortex, n = 6, scale bar = 50 μm. Data are mean ± SD, *p < 0.05, ANOVA with Bonferroni's multiple comparisons test.

Fig 4: RBFOX3 regulated hTERT promoter activity and expression in HCC cells. (A) Relative hTERT promoter activity in RBFOX3 overexpressing QGY7703 cells and RBFOX3 knockdown Hep3B cells. (B) Relative hTERT mRNA level was determined by quantitative real-time PCR analyses in RBFOX3 overexpressing QGY7703 cells and RBFOX3 knockdown Hep3B cells. (C) hTERT protein expression was up-regulated in RBFOX3 overexpressing QGY7703 cells, and down-regulated in RBFOX3 knockdown Hep3B cells. (D) The expression of total RBFOX3 proteins in N9 microglia (N9 MG) cell line, glioma cell lines (U138, U251 and U373), and HCC cell lines (Hep3B, QGY7703 and HepG2) were analyzed by Western blot. GAPDH was used as a loading control. (E) hTERT protein expression was detected with RBFOX3 knockdown in N9 MG and U251 cell lines. (F) ChIP assays were performed in neuronal cells and HCC cells using RBFOX3 antibody and the hTERT promoter primers. The PCR products were separated in 2% agarose gels. The IgG was used as a negative control. (G) Relative hTERT mRNA and protein levels were determined in RBFOX1 knockdown Hep3B cell lines by quantitative real-time PCR and Western blot, respectively. (H) Relative hTERT mRNA and protein levels were determined in RBFOX2 knockdown Hep3B cell lines by quantitative real-time PCR and Western blot, respectively. Results were shown as means ± SD, *p<0.05 by two-tailed Student's t-test. All experiments were performed in at least 3 independent trials.

Fig 5: Kv1.1 cKO mice display impairments in contextual fear conditioning. (A) Behavioral paradigm for assessing learning and memory in Kv1.1 cKO mice and Kv1.1 WT mice. Tamoxifen was injected for five consecutive days in ∼4-mo-old mice. Contextual fear conditioning started at ∼6.5 wk post tamoxifen injection. Mice were perfused at ∼8 mo of age. (B) Protocol for contextual fear conditioning. On days 1 to 3, the mice were shocked in the fear context. Percent freezing before the shock (0 min to 3 min) and after the shock (3 min to 4 min) were measured. (C) Quantification of percent freezing before shock on days 1 to 3, revealing a modest impairment of contextual fear learning and memory in Kv1.1 cKO mice (n = 16) compared to Kv1.1 WT mice (n = 19). Linear mixed model with REML and Geisser–Greenhouse correction followed by Sidak’s multiple comparisons test on days 1 to 3: genotype effect (F1, 33 = 7.5, P = 0.010), day effect (F1.8, 61 = 197, P < 0.0001), and genotype × day interaction (F2, 66 = 2.9, P = 0.060); Sidak’s multiple comparisons: day 1 (P = 0.75), day 2 (P = 0.098), and day 3 (P = 0.010). (D) Quantification of percent freezing after shock on days 1 to 3, revealing no difference between Kv1.1 cKO mice and Kv1.1 WT mice. Linear mixed model with REML and Geisser–Greenhouse correction followed by Sidak’s multiple comparisons test on days 1 to 3: genotype effect (F1, 33 = 0.54, P = 0.47), day effect (F1.7, 55 = 59, P < 0.0001), and genotype × day interaction (F2, 66 = 0.077, P = 0.93); Sidak’s multiple comparisons: day 1 (P = 0.88), day 2 (P = 0.77), and day 3 (P = 0.98). (E) Quantification (cells per cubic millimeter) of GFP+, NeuN+ cells to assess the amount of adult-born granule cells in the dentate gyrus of Kv1.1 cKO and Kv1.1 WT behavioral cohorts. Neurogenesis was decreased in Kv1.1 cKO mice compared to Kv1.1 WT mice (P = 0.037). Unpaired two-tailed Student’s t test with Welch’s correction. P < 0.10 indicated; *P < 0.05. Data are presented as mean ± SEM.