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. 2009 May 13;4(5):e5515. doi: 10.1371/journal.pone.0005515

Figure 4. ASYN-induction is toxic to differentiated SH-SH5Y cells due to CMA blockade and macroautophagy induction.

Figure 4

(A, B) ASYN (WT, ΔDQ/WT, A53T or ΔDQ/A53T)-expressing SH-SH5Y cells, were differentiated in RA (+/−dox). Samples were collected at 5, 7, 9, 12, 14 days after RA addition and survival was assessed by counting the number of intact nuclei. Rate of survival, presented in each case as the percentage of the (−) over the (+) dox condition, is shown for WT/ΔDQ and WT ASYN (A), and for A53T/ΔDQ and A53T ASYN (B) cells. All presented data are the mean of 3 independent experiments. Within each experiment triplicate samples per condition were assessed. (*p<0.05, **p<0.01, ***p<0.001, Student's t-test comparing WT or A53T ASYN-expressing cells and their corresponding ΔDQ mutants). (C, D) Suppression of macroautophagy with 3MA (C) or with ATG 5 siRNA (D), rescues differentiated SH-SY5Y cells from ASYN-induced death. (C) ASYN (WT, ΔDQ/WT, A53T)-expressing SH-SY5Y cells, were differentiated (+/−dox) for 5 days before 3MA addition. 36 hrs later, survival was assessed as in A, B. (*p<0.05, student's t-test, comparing WT or A53T-expressing cells+/−3MA). (D) WT or A53T ASYN cells and bgal cells were differentiated for 5 days (+/−dox) and transfected with scrambled (scr) or ATG 5 siRNA together with EGFP. Cell death was assessed 72 hrs later by counting the percentage of EGFP-positive transfected cells that were also Ethidium Homodimer-positive. At least 100 EGFP-positive cells were counted per well per condition. The data are presented as mean±SE of 3 independent experiments. Within each experiment triplicate samples per condition were assessed (### p<0.001, one way ANOVA followed by the Student-Newman-Keuls' test, comparing WT or A53T ASYN cells+/−dox; *p<0.05, **p<0.01, comparing WT or A53T ASYN-induced cells (−dox) transfected with ATG 5/EGFP to the control scr/EGFP transfected cells).