Fig 2: Lentiviral-Mediated Pls3 Expression Rescues Axonal Deficit in Smn-Deficient Motor Neurons(A) Western blot showing NSC34 cells transduced with LV-PLS3 at MOI 5, 10, 20, 40, or 80 and probed with anti-human PLS3 antibody; a-tubulin was the loading control (n = 3). Untransduced (UT) cells were considered as negative control. (B) A dose-response curve of LV-Pls3 at 5 days posttransduction, along with fold change in Pls3 protein levels for each MOI used. (C) Motor neurons isolated from E13 wild-type (WT), carrier (HEMI), and SMN?7 (KO) embryos were transduced with LV-Pls3 or LV-GFP at MOI 80. Cells were labeled for Pls3 (blue), tubulin (red), or GFP (green). Scale bars, 20 µm. (D) Impact of LV-Pls3 treatment on axonal length in Smn-deficient motor neurons. The bar chart shows the effect of LV-Pls3 treatment on axonal length in wild-type, carrier (HEMI), and SMN knockout (KO) embryos. One-way ANOVA (*p < 0.05, ***p < 0.001) was considered to be statistically significant. Data generated from four independent experiments (n = 4). Average neurite lengths ± SEM (n = 100 neurites).

Fig 3: PLS3 Immunoreactivity in the Brain and Spinal Cord Sections of WT and KO P10 Pups(A) Immunohistochemical staining showing PLS3 expression in the brain from P10 wild-type (WT) and SMN?7 mice (KO) (n = 3 per group). Brain sections from WT showing immune reactivity of PLS3 associated with a large number of neurons in the brain cortex (brown) (arrows). SMN?7 mice (KO) brain sections showed reduced reactivity of PLS3 associated with neurons (arrows). Scale bar, 50 µm. PLS3 immunoreactivity in the cervical (B) and lumbar (C) spinal cord from P10 wild-type (WT) and SMN?7 (KO) mice (n = 3). WT spinal cord sections showing PLS3 immuno-reactivity in large number of neurons in the ventral horn of the spinal cord (brown) (arrows), when compared to sections from SMN?7 mice KO section, which showed minimal reactivity of PLS3 associated with neurons (arrows). Scale bar, 50 µm.

Fig 4: Plastin 3 Protein Levels in Postnatal Day 1, 5, and 10 SMN?7 MiceRepresentative images of western blot and fold changes in PLS3 protein levels in brain, spinal cord, and muscle tissues from wild-type (WT), carrier littermates (HEMI), and SMN?7 (KO) postnatal day 1 (P1) (A and B), P5 (C and D), and P10 (E and F) neonate mouse pups (n = 3). The membrane was probed with mouse anti-PLS3 antibody; GAPDH was used as the house keeping control (n = 3). Error bars represent SEM. One-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.