Figure 5.

ApoE deficiency inhibits α-syn release in SH-SY5Y cells. (A) Differentiated NT and ApoE KD #1, #2/A53T α-syn-EGFP OE SH-SY5Y cells were cultured in serum-free media for 1 day, and culture media were collected. The cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (B) The α-syn in culture media was analyzed by ELISA (n = 3). (C) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (D) A53T α-syn-EGFP OE SH-SY5Y cells were transfected with plasmids for ApoE isoforms (ApoE2, ApoE3, and ApoE4) and differentiated by 50 μM RA for 5 days. The cells were then cultured in serum-free media for 1 day and culture media were collected. Cells were lysed with RIPA buffer and culture media were 50-fold concentrated. The samples were then analyzed by Western blot. Values were derived from three independent experiments (n = 3). (E) Culture media were analyzed by α-syn RT-QUIC. Values were derived from triplicate and a representative of three independent experiments (n = 3). (F) SH-SY5Y cells in the lower chamber were cocultured with differentiated NT, ApoE KD #1, or #2/A53T α-syn-EGFP OE SH-SY5Y cells for 24 h. The cells in the lower chamber were immunostained with an anti-EGFP antibody. Values were derived from three independent experiments (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 against the control; one-way ANOVA. Blue indicates DAPI staining. Scale bar indicates 20 μm.