Figure 4. AAV9-STAS Decreases p53S18 Phosphorylation in Vulnerable SMA Motor Neurons and Stasimon Deficiency Is Sufficient to Induce p53S18 Phosphorylation in NIH 3T3 Cells.
(A) ChAT (red) and p53 (green) immunostaining of L5 spinal cords from uninjected WT mice and SMA mice injected with AAV9-GFP or AAV9-STAS at P11. L5 MMC motor neurons are indicated by dotted circles and magnified in the insets. Scale bar: 100 μm.
(B) Percentage of p53+ L5 MMC motor neurons in the same groups as in (A). Data represent means and SEM from WT (n = 5), SMA+AAV9-GFP (n = 5), and SMA+AAV9-STAS (n = 5). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. ***p < 0.001; ns, not significant.
(C) ChAT (red) and p-p53S18 (green) immunostaining of L5 spinal cords from the same groups as in (A). L5MMC motor neurons are indicated by dotted circles and magnified in the insets. Scale bar: 100 μm.
(D) Percentage of p-p53S18+ L5 MMC motor neurons in the same groups as in (A). Data represent means and SEM from WT (n = 4), SMA+AAV9-GFP (n = 5), and SMA+AAV9-STAS (n = 6). Statistics were performed with one-way ANOVA with Tukey’s post hoc test. **p < 0.01, ***p < 0.001.
(E) Schematic of the experimental design. NIH 3T3 cells (WT or StasRNAi) were treated with either control MOs or with splice-switching antisense MOs targeting the 5′ splice sites of Mdm2 exon 3 and Mdm4 exon 7. Nuclear accumulation of p53 and p-p53S18 was then assessed by immunofluorescence analysis.
(F) RT-PCR analysis of Mdm2 and Mdm4 alternative splicing in NIH 3T3 cells treated with control MOs as well as Mdm2 and Mdm4 MOs either alone or in combination.
(G) Immunofluorescence analysis of total p53 (green) in WT and StasRNAi NIH 3T3 cells treated with the indicated MOs. Nuclei were counterstained with DAPI (blue). Scale bar: 25 μm.
(H) Percentage of p53+ NIH 3T3 cells from the same treatment groups as in (G). Data represent means and SEM (n ≥ 3). Statistics were performed with two-tailed unpaired Student’s t test. ns, not significant.
(I) Immunofluorescence analysis of p-p53S18 (green) in WT and StasRNAi NIH 3T3 cells treated with the indicated MOs. Nuclei were counterstained with DAPI (blue). Scale bar: 25 μm.
(J) Percentage of p-p53S18+ NIH 3T3 cells from the same treatment groups as in (I). Data represent means and SEM (n ≥ 3). Statistics were performed with two-tailed unpaired Student’s t test. ***p < 0.001; ns, not significant.
See also Figure S3.