Fig 2: Foxo3a regulates the Egr1/Creb1 transcription factors activity and expression. a, b Neuro-2a cells were treated for 48–72 h with control or Mapk10 shRNA, lysates were prepared, and immunoblotting was performed with pFoxo3a (Ser253), pFoxo3a (Thr32), and Gapdh antibodies (a) and immunostaining was performed with p14-3-3 beta/zeta (Ser184/186) antibody (b). c p14-3-3 beta/zeta (Ser184/186) quantification, in N2a cell line after treatment with control or Mapk10 shRNA for 48–72 h (Control shRNA, n = 39; Mapk10 shRNA, n = 41). d Immunostaining was performed with Foxo3a antibody in Neuro-2a cells after either treatment with control or Mapk10 shRNA for 72 h. e RNA was isolated from control and Foxo3a siRNA-treated Neuro-2a cells and qRT-PCR was performed for genes as indicated (n = 5 in each group). f, g A Luciferase reporter assay for the Egr1 (f) and Creb1 (g) activity was performed on N2a cells treated with either control siRNA or Foxo3a siRNA (48 h) and 90 min of hypoxia (n = 4 in each group). h Schematic diagram of ischemia-induced JNK3 pathway during blood flow recovery. Statistically significant differences between groups are indicated (*P < 0.05 by Student’s t test). The data are mean ± SEM. Scale bar, 5 µm (b) and 20 µm (d). Source data are provided as a Source Data file

Fig 3: MAPK10 expression was decreased in A549 and H1299 cells and NSCLC tumor tissues. Compared with BEAS-2B cells, MAPK10 (A) mRNA and (B and C) protein expression was significantly decreased in A549 and H1299. (D-H) Compared with normal samples, MAPK10 expression was significantly decreased in NSCLC tissues, according to data from Starbase V3.0 project, reverse transcription quantitative PCR and western blotting. **P<0.01, A549, H1299 vs. BEAS-2B and tumor tissues vs. normal tissues; ***P<0.001, tumor tissues vs. normal tissues. MAPK10, mitogen-activated protein kinase 10; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.

Fig 4: JNK2 or JNK3 shRNA reduce while JNK activation increases a-SNC secretion from neurons. a PC12 cells expressing a-SNC/p25a and JNK1, JNK2, or JNK1+2 shRNA were lysed after 48 h transgene induction and whole cell lysates and conditioned media (TCA) were subjected to western blotting against JNK1, JNK2, and a-SNC. Showing representative images from two to three independent experiments (we performed only two experiments with JNK1+2 shRNA expressing cells). b Differentiated SH-SY5Y cells expressing a-SNC/p25a and JNK1-3 shRNA were lysed after 48-h transgene induction, and whole cell lysates and conditioned media (TCA) were western blotted as indicated. The blots shown are representative of three independent experiments. c Differentiated SH-SY5Y cells expressing a-SNC/p25a were transfected with plasmids encoding constitutively active (FLAG-MKK7B2-JNK1-3) JNK isoforms or a kinase dead version of JNK1 (AFP) as control. The day after transfection, whole cell lysates and conditioned media (TCA) were western blotted as indicated. The blots shown are representative of three independent experiments. d Quantification of a a-SNC present in conditioned medium. Data are shown as mean + SEM (N = 3) percent relative to control transfected with scrambled (Scr) shRNA (*p < 0.05; ***p < 0.001 compared to “Scrambled”). e Quantification of b a-SNC present in conditioned medium. Data are shown as mean + SEM (N = 3) percent relative to control transfected cells (**: p < 0.01 compared to “Scramble”). f-h Quantification of (C) a-SNC present in conditioned medium (TCA), endogenous (Endo) p-JNK, and FLAG-MKK7B2-JNK1-3 (FLAG) p-JNK. Data are presented as mean + SEM (N = 3) normalized to control cells transfected with an empty vector (*p < 0.05; **p < 0.01; ***p < 0.001 compared to “Control”). i Calculated ratio of p-JNK of endogenous to FLAG-MKK7B2-JNK1-3 densitometric western blot signals

Fig 5: Expression of total JNK isoforms in chronic epilepsy. A. Western blot showing total expression of JNK1 isoform (phosphorylated plus non-phosphorylated) in chronic epilepsy and naïve control conditions. JNK1 expression was not significantly changed in the epileptic condition. B. Expression of JNK2 was reduced in epilepsy for both the 54 and 46 kDa variants. C. Expression of only the 54 kDa variant of JNK3 was significantly reduced under epileptic conditions. D. Quantification of JNK isoform expression in the epileptic condition as a percentage of control expression shows significant downregulation of both JNK2 variants as well as the 54 kDa JNK3 variant.