sbASOs increase Mecp2 protein expression and induce MDS-like phenotypes in WT mice. (A) Schematic illustration of the experimental workflow, detailing the implantation of osmotic pumps for continuous sbASO administration and subsequent treatment protocols. (B) Western blot analysis of Mecp2 protein expression in the nuclear fraction of the frontal cortex of WT mice treated with sbASO.miR-22 and sbASO.miR-132. Protein quantification was normalized to Histone H3 (H3), with data represented as the mean percentage relative to the vehicle-treated controls. Statistical analysis via one-way ANOVA showed a significant effect of treatment ([*] P = 0.0265), with Fisher's LSD post hoc analysis indicating no significant difference for sbASO.miR-22 (P = 0.6567), but a significant increase for sbASO.miR-132 ([*] P = 0.0137). (C) Western blot analysis of Mecp2 expression in the nuclear fraction of the hippocampus of WT mice treated with sbASO.miR-22 and sbASO.miR-132. Quantification was also normalized to H3, with values shown as mean percentages relative to vehicle controls. One-way ANOVA indicated a significant effect ([**] P = 0.0052), with Fisher's LSD revealing no significant difference for sbASO.miR-22 (P = 0.4535), but a significant increase for sbASO.miR-132 ([**] P = 0.0089). The sample size for each treatment was n = 8–10. Representative western blots showing Mecp2 levels for each treatment (vehicle, 22 = sbASO.miR-22 and 132 = sbASO.miR-132) in both the frontal cortex and hippocampus, with the upper band representing Mecp2 (71 kDa) and the lower band representing H3 (17 kDa), are located directly below each bar graph. (D) Open arm index assessment in the EZM revealed that WT animals treated with sbASO.miR-22 [Bonferroni post hoc (df,20) (**) P = 0.0078] and sbASO.miR-132 [Bonferroni post hoc (df,20) (*)P = 0.0283] exhibited a significant increase in the time spent in the open arm compared to the closed arm of the maze. (E) No significant difference in total distance traveled was detected between treatment groups for sbASO.miR-22 [Bonferroni post hoc (df,20) (P = 0.1734)] and sbASO.miR-132 [Bonferroni post hoc (df,20) (P = 0.6414)]. n = 8–10 per treatment. Outliers were removed using Grubb's tests at α = 0.1. Data are presented as mean ± SEM.
