Figure 3

Neuronal activity decreases DROSHA levels in motor neurons. (A) Western blot analysis of DROSHA levels in 10DIV WT motor neurons treated with 55 mM KCl or 300 nM tetrodotoxin (TTX) for 24 hours: n = 5. (B) Western blot analysis of DROSHA levels in 10DIV SMA motor neurons treated with 10 nM riluzole or 300 nM TTX for 96 hours: n = 9 (C) Images show that DROSHA is detected in nucleus and cytoplasm of motor neurons. Cytoplasmic DROSHA intensity is reduced in SMA: n = 113 (WT) and n = 98 (SMA) from 9 (WT) and 8 (SMA) biological samples (D) Western blot analysis and quantification of DROSHA levels in nucleus and cytoplasm of WT and SMA motor neurons: n = 7 (WT) and n = 6 (SMA) (E) Western blot analysis and quantification of DROSHA levels in nucleus and cytoplasm of depolarized neurons. Neurons were depolarized with 55 mM KCl. LaminB and ACTB confirm that subcellular fractionation was successful: n = 5 (F) Primers to amplify exon 7 related splicing variants (G) PCR shows that exons 5/6/7/8 containing splicing variant was the major form in motor neurons and SMN deficiency does not alter the exon 7-related splicing. PCR was performed 39 cycles and PCR product was confirmed with Sanger sequencing: n = 4 (H) qRT-PCR confirms that the amount of transcripts containing exon 7 did not alter in SMA: n = 4, Data are represented as mean±SEM. Statistical significance is determined with t-test, *p < 0.05, **p < 0.01 and ***p < 0.001. ns = not significant.