Figure 6.

Overexpression of MANF Activates ERK1/2 and STAT3 Signaling in the SVZ Explant Culture

SVZ explants were transduced with LV on DIV1 and analyzed using western blotting at DIV4 or DIV7. (A and B) The expression of myrAKT at DIV4 (A) and DIV7 (B) were detected in LV-myrAKT-transduced SVZ explants. (C) Quantitated level of myrAKT relative to GAPDH (n = 3, two-way ANOVA, time × LV treatment, p < 0.01, ***p < 0.001 compared to GFP, TK). (D and E) Increased expressions of phospho-S6K (P-S6K) was detected at DIV4 (D) and DIV7 (E) in LV-myrAKT-treated SVZ explants. (F) Quantification of P-S6K level relative to GAPDH and increased levels were observed in LV-myrAKT-treated explants (two-way ANOVA, treatment effect p < 0.001, **p < 0.01, ***p < 0.001 compared to GFP TK). (G and H) Increased expression of p-ERK1/2 in MANF-overexpressed SVZ explants at DIV4 (G), but not at DIV7 (H). (I) Quantified expression levels of p-ERK compared to total ERK showed statistically significant elevated levels of p-ERK in LV-MANF at DIV4 compared to LV-GFP and LV-myrAKT groups (two-way ANOVA, p < 0.05, ***p < 0.01, TK) but not at DIV7. (J and K) Overexpression of MANF induced p-STAT3-Ser in SVZ explants at DIV4 (J), but not at DIV7 (K). (L) Statistically significant elevated levels of p-STAT3-Ser in LV-MANF at DIV4 compared to LV-GFP and LV-myrAKT groups (two-way ANOVA, p < 0.05, ***p < 0.001, TK), but not at DIV7. (M–O) There was a modest expression of the UPR-regulated protein GRP78 at DIV4 (M) and DIV7 (N), and it did not differ between groups at either time point (O). TK, Tukey’s post hoc test. Data are presented as mean ± SEM.