Total RNA and protein lysates from quadriceps were isolated from C57/Bl6 wild-type male mice 5 hrs post one bout treadmill running (n=6) or sedentary control (n=6) as described in experimental procedures. A. Total RNA was analyzed by realtime RT-PCR; Data represented means±SEM. *** indicates p <0.005 vs. control, *, p <0.05. Similar results were observed in more than three independent experiments. B. Protein lysates were probed by immunoblot as indicated, with total eIF2α as a loading control. The first two lanes are positive controls using lysates from primary myotubes treated with 100nM thapsigargin or vehicle for 8hrs. C. Exercise training leads to adaptation in skeletal muscle. Untrained mice (naïve) or trained mice (trained: five sessions of 1hr treadmill run/wk for 4weeks), together with age-matched sedentary control mice were sacrificed 5 hrs after one bout equal distance treadmill running. N=6 animals per group. Total RNA from quadriceps was isolated and analyzed by realtime RT-PCR with CytC as a positive control; Data represented means±SEM. *** indicates p <0.005 trained vs. naïve, **, p <0.01, *, p <0.05. ^### indicates p <0.005, rest vs. trained : run, ^##, p <0.01, ^#, p <0.05. Statistical comparison between rest vs. naïve : run is similar to Figure 1A, and not shown in this panel for visual clarity.

(A) PGC-1α is induced upon ER stress in primary myotubes. Wild-type primary myotubes were pretreated with 10μM BAPTA or vehicle for 1hr and followed by vehicle or 100nM Thapsigargin (TG) treatment for 6hrs. Wildtype primary myotubes were treated with 5mM DTT or 10mM 2DG for 6hrs. Total RNA was isolated and the expression of PGC-1α was quantitated by realtime RT-PCR, normalizing against tbp expression. Data represent means±SD from biological triplicates. * indicates p <0.05 TG+BAPTA vs. TG. (B, C) UPR markers are induced by PGC-1α in skeletal muscles and in primary myotubes. B. Total RNA from gastrocnemius muscle isolated from MCK-PGC-1α and littermate control mice was analyzed by real-time RT-PCR for UPR markers expression with CytC as a positive control. N=4–5 animals per group. Data represent means±SEM. *** indicates p <0.005 vs. control, **, p <0.01, *, p <0.05. C. Total RNA from primary myotubes infected with adenovirus expressing PGC-1α or GFP as a control was assayed by realtime RT-PCR for UPR markers expression with CytC as a positive control. Data represent means±SD from biological triplicates. *** indicates p <0.005 vs. control.

(A, B). PGC-1α-deficient myotubes respond to ER stress. A. Total RNA was isolated from wildtype and PGC-1α−/− primary myotubes treated with 10 or 100nM TG for 8hrs. RT-PCR was used to simultaneously detect both spliced (s) and unspliced (us) Xbp1 mRNA. The image is presented in black and white inverted form for greater visual clarity. B. Wild-type and PGC-1α−/− primary myotubes were treated with 100nM TG for 1hr, followed by cell lysis and immunoblot using antibody that detects phosphorylated form of eIF2α and total eIF2α as a loading control. C. PGC-1α−/− primary myotubes are defective in UPR markers upregulation upon ER stress. Total RNA from wildtype and PGC-1α−/− primary myotubes treated with 100nM TG for 16hrs was assayed by realtime RT-PCR for UPR markers expression, normalizing against tbp expression. Data represent means±SD from biological triplicates. *** indicates p <0.005 +/+:TG vs. −/−:TG, **, p <0.01, *, p <0.05. D. MKO-PGC-1α mice are defective in upregulating ER chaperones and experience exacerbated ER stress after repetitive exercise challenges. Total RNA from quadriceps of MKO-PGC-1α and wildtype control mice either sedentary or after four bouts equal distance treadmill running (once a day for 4 days, recover for one day after the last run) was isolated and analyzed by realtime RT-PCR; N=7–8 animals per group. *** indicates p <0.005 MKO-PGC-1α : Run vs. Control : Run; *, p <0.05. ^### indicates p <0.005, control : rest vs. control : run, ^##, p <0.01, ^#, p <0.05.

A. C2C12 myoblasts were transfected with expression plasmids for XBP-1 coded by spliced form of Xbp-1 mRNA (XBP-1s), cleaved form of ATF6α (ATF6α-N), ATF4 or expression vector control, respectively, together with expression plasmid for PGC-1α, or vector and rat BiP promoter-luciferase construct. The cells were subsequently differentiated for 2 days and harvested and luciferase activity was measured, normalizing against renilla. Data represent means±SD from biological triplicates. B. Coimmunoprecipitation of ATF6α and PGC-1α. Cultured COS cells were transfected with plasmids as indicated. Total lysates from transfected cells were subjected to immunoprecipitation using beads specific for Flag tag. Both lysates and precipitates were analyzed by immunoblotting with antibodies specific for the Flag and HA epitope tag. (C and D) Total RNA from ATF6α+/+ and −/− primary myotubes (C) and IRE1α+/+ and −/− primary myotubes (D) infected with adenovirus expressing PGC-1α or GFP as a control was assayed by realtime RT-PCR for UPR markers expression. Data represent means±SD from biological triplicates.

A. Total RNA from quadriceps of ATF6α +/+ and −/− mice, 1hr or 1 day after equal distance treadmill running and littermate sedentary control mice was isolated and analyzed by realtime RT-PCR; Data represented means±SEM, *** indicates p <0.005 +/+ vs. −/−, *, p <0.05. B. Whole blood was collected by cardiac puncture from mice in (A) and serum creatine kinase activity was determined. Data represented means±SEM, *** indicates p <0.005 +/+ vs. −/−. C. ATF6α−/− mice shown exercise intolerance compared to wild-type control mice after repetitive treadmill runnings. Adult male ATF6α−/− mice and wild-type control (3–4 months old, n=4–12 per group) were subjected to repetitive exhaustive treadmill running once a day for 4 days as described in Experimental Procedures. Distances run on day 1 and day 4 of both genotypes were calculated from the individual performances. Data represented means±SEM. * indicates p <0.05 +/+ vs. −/−.

A. MKO-PGC-1α and control mice either with CHOP-null alleles or not were subject to treadmill running till exhaustion as described in Experimental Procedures. Distance was calculated from individual performances. N=15–22 animals per group. ** indicates p <0.01, PGC-1α MKO:CHOP−/− vs. PGC-1α MKO:CHOP+/+. B. Whole blood was collected by cardiac puncture from mice in (A) and serum creatine kinase activity was determined. Data represented means±SEM, * indicates p <0.05 PGC-1α MKO:CHOP−/− vs. PGC-1α MKO:CHOP+/+.