Figure 7.

6-OHDA-induced AMPK-ULK1 phosphorylation was associated with PARK7 secretion in WT MEF cells. (A) WT MEF cells were treated with 75 μM 6-OHDA or 2 μM rapamycin for 3 h. Whole cell lysates were immunoblotted using antibodies specific for phospho-AMPK (Thr172), phospho-ULK1 (Ser555), phospho-RPS6KB1 (Thr389), LC3B, or ACTB. Band intensities were quantified by densitometric scanning and p-AMPK:ACTB, p-ULK1:ACTB, p-RPS6KB1:ACTB and LC3B-II:ACTB ratios are shown. n = 3; mean ± S.D.; **, p < 0.01. (B) WT MEF cells were pre-treated with or without 2 mM NAC and were then treated with 75 μM 6-OHDA for 3 h. Whole cell lysates were immunoblotted using antibodies specific for phospho-AMPK, LC3B, or ACTB. Band intensities were quantified by densitometric scanning and p-AMPK:ACTB and LC3B-II:ACTB ratios are shown. n = 3; mean ± S.D.; **, p < 0.01. (c) WT MEF cells were treated with 75 μM 6-OHDA for 3 h and were then subjected to AMP:ATP assay. n = 3; mean ± S.D.; *, p < 0.05. (D and E) SH-SY5Y cells were pre-treated with or without 1 μM MRT68921 for 30 min and were then treated with 75 μM 6-OHDA for 3 h (D), followed by culture in serum-free medium for 2 h (E). Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for LC3, PARK7, or ACTB. LC3B, PARK7 and ACTB band intensities were quantified by densitometric scanning. LC3B-II:ACTB ratio (d) and relative secretion level to vehicle-treated cells (e) are shown. n = 3; mean ± S.D.; **, p < 0.01. (F) WT MEF cells were pre-treated with or without 2 μM MRT68921 for 30 min and were then treated with 75 μM 6-OHDA for 3 h, followed by culture in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or ACTB. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; **, p < 0.01.