Fig 1.
Analysis of CCCP-induced MFN ubiquitination in cultured fibroblast cells.
Western blot analysis of fibroblasts from healthy controls (C1 and C2) and two early-onset PD patients with mutations in PINK1 or PRKN. Cells were left untreated or were treated for 2 hours with 20 μM CCCP. Blots were developed with antibodies against MFN1 and MFN2. Antibodies against SDHA were used as loading control. Asterisks indicate bands representing ubiquitinated MFN proteins.
Fig 2.
Analysis of CCCP-induced MFN ubiquitination in Jurkat cells.
(A) Jurkat cells left untreated or treated for 2 hours with 20 μM CCCP were analysed by western blotting with antibodies against MFN1 and MFN2. (B) Western blot analysis of Jurkat cells treated with CCCP for increasing periods of time (0.5–24 hours), developed with antibodies against MFN1 and MFN2. Asterisks indicate bands representing ubiquitinated MFN proteins.
Fig 3.
Analysis of CCCP-induced MFN ubiquitination in different cell types.
SH-SY5Y, fibroblast, Jurkat and PBMC samples were left untreated or were treated with 20 μM CCCP for 2 hours and analysed by western blotting with antibodies against MFN1 and MFN2. Asterisks indicate bands representing ubiquitinated MFN proteins.
Fig 4.
CCCP induces mitochondrial depolarisation in Jurkat and PBMC samples.
Fluorescent micrographs of Jurkat and PBMC samples loaded with the red fluorescent, mitochondrial membrane potential-dependent dye TMRM before and after addition of 20 μM CCCP (+ CCCP). Nuclei were counterstained fluorescent blue with Hoechst 33343.
Fig 5.
Analysis of Parkin expression in different cell types.
(A) Reverse transcriptase qPCR analysis of PRKN mRNA levels in Jurkat, control fibroblast (Fib), SH-SY5Y and PBMC samples. qPCR was performed on equal amounts of cDNA from each cell type and Ct values were expressed relative to the Jurkat sample. (B, C) Western blot analysis of fibroblast, SH-SY5Y, Jurkat and PBMC cultures with an antibody against Parkin. (D) Quantification of Parkin signal in the different cell types. Signal is expressed relative to SDHA and normalised to that detected in Jurkat cells. Graphs display mean ± SEM.
Fig 6.
Analysis of PINK1 expression in different cell types.
(A) Reverse transcriptase qPCR analysis of PINK1 mRNA levels in Jurkat, control fibroblast (Fib), SH-SY5Y and PBMC samples. qPCR was performed on equal amounts of cDNA from each cell type and Ct values were expressed relative to the Jurkat sample. (B) Western blot analysis of fibroblasts, SH-SY5Y, Jurkat and PBMC cultures left untreated and treated with 20 μM CCCP for 24 hours with an antibody against PINK1 (Novus Biologicals, BC100-494). The band corresponding to PINK1 is indicated by the arrow. (C) Quantification of PINK1 signal from CCCP-treated cells. Signal is expressed relative to β-actin and normalised to levels detected in Jurkat cells. (D) Quantification of SDHA signal from untreated and 24-hour CCCP-treated Jurkat, fibroblast, SH-SY5Y and PBMC samples. Signal is expressed relative to β-actin and normalised to the untreated condition. Graphs display mean ± SEM; * = P<0.05, Student’s t-test; n.d. = not detected; n.s. = not significant.
Fig 7.
Parkin does not accumulate on CCCP-induced depolarised PBMC mitochondria.
Fluorescent micrographs of Jurkat and PBMC samples, treated with DMSO (vehicle) or CCCP, followed by immunostaining for Parkin (fluorescent green) and the mitochondrial marker TOMM20 (fluorescent red). Nuclei were counterstained fluorescent blue with DAPI. Arrows indicate Parkin co-localising with mitochondria in Jurkat cells.
Fig 8.
Analysis of PINK1 –Parkin signalling pathway in lymphoblastoid cell lines and CD3/CD28 activated PBMCs.
(A) Phase contrast microscopy images of Jurkat cells, a lymphoblastoid cell line (LCL) and CD3/CD28 Dynabead-activated PBMCs. (B) Western blot analysis of MFN1 and -2 ubiquitination following CCCP treatment of activated PBMCs and LCLs. (C) Western blot analysis of PINK1 expression in activated PBMCs and LCLs following prolonged CCCP treatment. (D) Western blot analysis of Parkin expression in resting and CD3/CD28 Dynabead-activated PBMCs, and LCLs.