PINK1 Deficiency Figure 1. PINK1 Expression & Up-regulation. A) PINK1 expression in human dopaminergic neurons. Red; PINK1, green; TH, blue; hoescht. B) Western blot showing endogenous PINK1 protein expression in control (c) and PINK1 kd (with 90% mRNA knockdown) human neuronal lysates after 5 days differentiation. Anti-PINK1 (Novus) was used to detect PINK expression; b-actin levels are shown as loading control. C) Histogram showing relative fold change in PINK1 mRNA expression levels between NSCs, 5 day differentiation (dd5), 30 day differentiation (dd30). PINK1 expression levels from pre-immortalised neural stem cells from 8 week old ventral mesencephalon (Midbrain P5) and adult human brain (Human Cb) are shown for comparison. Values represent mean of three replicate samples6s.e.m. D) Histogram showing expression of PINK1 mRNA in human neurons in four control (c1-4) and four PINK1 kd (kd1-4) selected clones relative to an endogenous control gene. RQs were normalised to that of c1; values represent mean of 3 replicate samples6standard error (s.e.m). E) Immunofluorescence images showing dopaminergic cells in control (i) and PINK1 kd (ii) human neuronal cultures after 5 days differentiation. Red; TH, green; bIII tubulin, blue; Hoecsht. doi:10.1371/journal.pone.0002455.g001 #1029 resulting in .90% kd consistently in both the SHSY5Y and the human NSCs. (Figure S1 B). shRNA directed against a different gene (Lamin AC) did not reduce PINK1 mRNA levels (see S1B), confirming that this method did not result in non- specific PINK1 kd. Results shown are normalized for housekeep- ing gene, LRPO. However RT_PCR for other genes including two other control genes (b-actin and GAPDH) as well as other PD genes (parkin and DJ-1) did not show significant alterations in expression between control and PINK1 kd (data not shown).. Knockdown of endogenous PINK1 mRNA was maintained after differentiation as human neurons (Figure 1D) even taking into account the ,100 fold increase in PINK1 expression described above. Analysis of protein levels by Western blotting confirmed reduced levels of protein expression in #1029 clones compared PLoS ONE | www.plosone.org 5 June 2008 | Volume 3 | Issue 6 | e2455