Figure 4.

Neomycin treatment reduces PINK1 expression in HEI-OC1 cells. (A) Western blot showing changes of PINK1 protein level and phosphorylated PINK1 in HEI-OC1 cells after treatment with 0.5 mM neomycin for 6 h. The cells were also treated with 20 µM CCCP for 6 h. Immunoblot with anti-PINK1 and anti-p-PINK1 Ser228. (B) Quantification of PINK1 protein level in A, n = 3. (C) Quantification of phosphorylated PINK1 (Ser228) relative to total PINK1 levels in A, n = 3. (D) HEI-OC1 cells expressing exogenous PINK1-FLAG were treated with neomycin (0.5 mM) and/or CCCP for 6 h, then Tris-glycine SDS-PAGE with or without 25 μM phos-tag was performed to detect phosphorylated PINK1. Immunoblot with anti-FLAG antibody. Red asterisks indicate the phosphorylated band of PINK1, FL: full length. (E) Western blot showing the level of phospho-ubiquitin (Ser65) in HEI-OC1 cells treated with 20 μM CCCP and 0.5 mM neomycin for 6 h. Cells were transfected with mCherry-C1 (as the negative control) and mCherry-PRKN. (F) RT-qPCR analysis of Pink1 transcription in HEI-OC1 cells treated with neomycin (0.2 mM, 0.5 mM, 1 mM, 2 mM, and 5 mM) for 6 h, n = 4. (G) Western blot showing the degradation of PINK1 in HEK 293 cells, which were treated with 20 μM CCCP and 0.5 mM neomycin for 3 h followed by CCCP washout (W/O) at the indicated times. PINK1 protein was detected at the indicated times after CCCP washout. (H) RT-qPCR analysis of Pink1 transcription in HEI-OC1 cells treated with 0.5 mM neomycin (6 h, 12 h, and 24 h), n = 4. For all experiments, *p < 0.05, ** p < 0.01, and ***p < 0.001. Data are shown as means ± S.D.