Figure 4

DROSHA is degraded by autophagy in motor neurons. (A) Stability of DROSHA protein in 10DIV WT and SMA motor neurons. 20 μg/ml of cycloheximide was treated for 6 hours: n = 8 (WT) and n = 10 (SMA) (B) Western blot analysis of DROSHA levels in 10DIV WT motor neurons treated with 25 μM MG132, 10 μM nutlin-3, 25 μM calpeptin, 55 mM KCl or 100 μM NMDA for 24 hours: n = 5. (C) Western blot analysis and quantification of LC3-I, LC3-II and p62 levels in 10DIV motor neurons. (D) Western blot analysis and quantification of DROSHA levels in 10DIV WT motor neurons treated with autophagy inducers, 100 nM rapamycin, 100 nM Torin-1, and 10 μM 10-NCP for 24 hours: n = 6. (E) Western blot analysis and quantification of DROSHA levels in 10DIV SMA motor neurons treated with blockers of autophagic flux, 5 mM 3-methyladenine (3-MA) or 1 nM bafilomycin A for 24 hours: n = 8 (3-MA treated) and n = 9 (non-treated and bafilomycin A treated). (F) Western blot analysis shows Atg5 knockdown (KD) efficiency in SMA motor neurons. 50 pmol of siRNAs (Atg5 or negative control) were transfected and KD efficiency was confirmed in 48 hours: n = 3. (G) Atg5 KD elevated DROSHA levels in SMA motor neurons after 72 hours of transfection: 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.