(A) Identification and MACAW alignment of the PARIS DNA-binding sequence as determined by CASTing. Darker colors represent a greater degree of overlap of the segment pairs (bottom right - % overlap). *Duplicate sequence tags.
(B) Relative luciferase activity of the 1-kilobase human
PGC-1α (−992 to +90) compared to
Renilla luciferase ± PARIS or ± parkin or ± familial mutant Q311X parkin,
n = 3. Location of IRS, CRE motifs and oligos for human ChIP (arrows) in the
PGC-1α promoter construct (top of panel). Immunoblot analysis confirms the expression of FLAG-PARIS, MYC-Parkin and MYC-Q311X parkin (right panel).
(C) EMSA of GST-PARIS of
32P-labeled WT (WT-
32P) IRS oligonucleotides (IRS1-WT, IRS2-WT, IRS3-WT) of the human
PGC-1α promoter and
32P-labeled mutant (T→

) (MT-
32P) IRS oligonucleotides (IRS1-MT, IRS2-MT, IRS3-MT). Unlabeled WT (WT cold) IRS oligonucleotides disrupt the GST-PARIS-DNA protein complexes with the WT-
32P IRS oligonucleotides,
n = 3. Unlabeled mutant probes (MT cold) has no effect on mutant (MT-
32P). Arrow indicates specific PARIS-shifted probe; NS, nonspecific; FS, fragmented PARIS-shifted probe; FP, free probe.
(D) PARIS occupies the endogenous
PGC-1 α promoter as determined by ChIP assay with anti-PARIS polyclonal antibodies in SH-SY5Y cells,
n = 3.
(E) PARIS occupies the endogenous mouse
PGC-1 α promoter as determined by ChIP in mouse whole brain,
n = 3. Mouse specific IRS primers are indicated in .
(F) ChIP assay of endogenous PARIS binding to the IRS region of the human
PGC-1a promoter in human PD and aged-matched control (CTL) striatum, control
n = 3; PD
n = 4.
(G) Quantitation of ChIP in panel F. Human specific IRS primers for D and F are indicated in panel B.
(H) Real-time qRT-PCR of PGC-1α, GFP and β-actin following transient transfection of GFP, GFP-PARIS or GFP-C571A PARIS mutant,
n = 4.
(I) Immunoblot analysis of PGC-1α, GFP and β-actin following transient transfection of GFP, GFP-PARIS or GFP-C571A PARIS mutant,
n = 4.
(J) Quantitation of the immunoblots in panel I normalized to β-actin,
n = 4.
(K) Immunoblot analysis of parkin, PARIS, PGC-1α and β-actin in double knockdown experiments via lentiviral transduction of shRNA-parkin and/or shRNA-PARIS in SH-SY5Y cells,
n = 3.
(L) Quantitation of the immunoblots in panel K normalized to β-actin,
n = 3, sh = shRNA.
(M) Relative mRNA levels of PGC-1α normalized to GAPDH,
n = 3. Quantitative data = mean ± S.E.M., *
p < 0.05, **
p < 0.01, ***
p < 0.001, unpaired two-tailed Student’s
t-test (G), ANOVA with Student-Newman-Keuls post-hoc analysis (B, H, J, L, M).
See also , .