1. Volume 25, Issue 5, Pages 1176-1185.e5 (May 2017)
PPARβ Is Essential for Maintaining Normal Levels of PGC-1α and Mitochondria and for the Increase in Muscle Mitochondria Induced by Exercise 
Jin-Ho Koh, Chad R. Hancock, Shin Terada, Kazuhiko Higashida, John O. Holloszy, Dong-Ho Han 
Cell Metabolism 
Volume 25, Issue 5, Pages e5 (May 2017)
DOI: /j.cmet
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2. Cell Metabolism 2017 25, 1176-1185.e5DOI: (10.1016/j.cmet.2017.04.029)
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3. Figure 1
Rat Tibialis Anterior Muscle Electroporation with myc-PPARb-GFP
Muscles were electroporated with PPARβ labeled with GFP and photographed 10 days later.
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4. Figure 2
Overexpression of PPARβ in Skeletal Muscle Induces Increases in Expression of PGC-1α Protein and Mitochondrial Enzyme Proteins
(A) Electroporation of mouse tibialis anterior muscles with myc-PPARβ results in an increase in PGC-1α expression that is prevented by co-electroporation with a PPARβ shRNA.
(B) Ten days after electroporation, PPARβ overexpression in muscle results in increased expression of NRF-1, phospho-AMPK, MEF2A, GLUT4, and some mitochondrial respiratory chain enzymes, with no increase in PGC-1α (relative to electroporation with an empty vector [EV]). Values are means ± SE (n = 4). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
(C) Three weeks after electroporation of muscle with a PPARβ construct, PGC-1α and citrate synthase were also increased. Values are means ± SE (n = 4). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
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5. Figure 3
Immunofluorescence Evaluation of the Effects of PPARβ Overexpression in Mouse Tibialis Anterior Muscle
(A) Mitochondrial respiratory chain enzymes are increased 10 days after muscle electroporation with PPARβ compared to empty vector (EV). Scale bar, 50 μm.
(B) PPARβ overexpression results in large increases in expression of myosin heavy chains (MHCs) I and IIA and decreases in MHCs 2B and 2X. There was also an increase in hybrid fibers. Scale bar, 50 μm.
(C) Quantification of western blot results of MHC isoforms. Values are means ± SE (n = 4). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
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6. Figure 4 PPARβ Is a Transcription Factor for NRF-1
(A) ChIP assay showing that PPARβ binds to the NRF-1 promoter. Values are means ± SE (n = 7 independent treatments of C2C12 cells, IgG group; n = 14, PPARβ group). ∗p < verse IgG. Significance was determined using Student’s t test.
(B) PPARβ overexpression induces an increase in NRF-1 promoter activity. Values are means ± SE (n = 6 independent treatments of C2C12 cells per group). ∗p < versus EV. Significance was determined using Student’s t test.
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7. Figure 5
PPARβ Mediates an Increase in CaMKKβ Expression via an Increase in NRF-1
(A) PPARβ overexpression in muscles 21 days after electroporation increases expression of both CaMKKα and β. Values are means ± SE (n = 4). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
(B) ChIP assay showing that NRF-1 binds to the CaMKKβ promoter. Values are means ± SE (n = 4 independent treatments of HEK cells per group). ∗p < versus IgG. Significance was determined using Student’s t test.
(C) NRF-1 overexpression induces an increase in CaMKKβ promoter activity. Values are means ± SE (n = 5 independent treatments of C2C12 cells per group). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
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8. Figure 6 PPARβ Protects PGC-1α against Ubiquitination and Degradation
(A) Overexpression of PPARβ in muscle results in a large increase in endogenous PPARβ. Values are means ± SE (n = 4). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
(B) Muscles were electroporated with myc-PPARβ. Three weeks later, muscles were harvested and homogenized. Flag-PGC-1α and ubiquitin were measured in the homogenates by western blot. PPARβ overexpression resulted in a marked reduction in PGC-1α ubiquitination and an increase in PGC-1α protein. Values are means ± SE (n = 4). ∗p < versus EV. Significance was determined using Student’s t test.
(C) C2C12 myoblasts were co-transfected with Flag-PGC-1α and myc-PPARβ. After 48 hr of differentiation, myotube extracts were prepared, Flag-PGC-1α was immunoprecipitated, and PPARβ and ubiquitin bound to PGC-1α were measured by western blot. Values are means ± SE (n = 4 independent treatments of C2C12 cells per group). ∗p < 0.01 versus EV. Significance was determined using Student’s t test.
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9. Figure 7
Partial Knockdown of PPARβ Reduces the Adaptive Response to Swimming Exercise Training for 2 Weeks
Rat epitrochlearis were electroporated with a PPARβ shRNA empty vector (EV). Three weeks later, rats were exercised by means of swimming and muscles were harvested 24 hr after the exercise bout. Values are means ± SE (n = 6). ∗p < 0.05, Sed versus Ex; #p < 0.05, EV versus shPPARβ. Significances was determined using two-way ANOVA; post hoc analysis was conducted with Fisher least significant difference (LSD). Sed, sedentary; Ex, exercise.
Cell Metabolism  , e5DOI: ( /j.cmet )
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