GRAPHICAL ABSTRACT RESULTS cont Our findings OXPHOS Kinetics 150 caution against 100 Colon 50 CT26.WT the use of of max JO2 % Heart 0 indiscriminate OligomycinFCCP FCCP[1µM] FCCP[2µM] FCCP[3µM] [4µM] GATPGATP (-54.16)GATP (-58.93)GATP (-60.64) (-61.49)FCCP [0.5µM] Δ Δ Δ Δ mitochondrial Figure 3 Respiration in permeabilized colon and heart strips and in permeabilized cells, depicted as a RESULTS % of max respiration. inhibitors for Mitochondrial Bioenergetics ATP/O ratio OXPHOS proteome

Figure 1 Figure 2 Pooled OXPHOS Expression cancer treatment. OXPHOS Kinetics (per mg cellular ) Respiratory Capacity (per mg cellular protein) OXPHOS 8000 8000 * Efficiency 100 * Colon * * * ns * * * 6000 CT26.WT 6000 * * ns 50 Heart * * 2.0 * * * ns 4000 * * 4000 * * * 1.5

(pmol/s/mg) 0

* Max of Percent (pmol/s/mg) 2 Ilya Boykov 2 2000 * * 2000 CI CII CIII CIV CV JO * JO * * * * * 1.0 * * Mentor: Kelsey Fisher-Wellman * (pmol/s/mg) Mitochondrial Complex * 2 0 0 0.5 Figure 4: Mitochondrial complex enrichment Department of Physiology JO depicted as % of max content for each P/M P/M CytC CytC PCRPCRPCR Basal BasalClamp Oligo complex Oct/Suc Rot/Anti Oct/Suc Rot/Anti 0.0 1uM FCCP2uM FCCP3uM FCCP4uM FCCP 1uM 2uMFCCP 3uMFCCP 4uMFCCP FCCP 0.5uM FCCP 0.5uM FCCP Complex IV proteome P/O Colon P/O Heart Figure 5 In situ quantification of mitochondrial OXPHOS Kinetics Respiratory Capacity P/O CT26.WT (per mg mitochondrial protein) (per mg mitochondrial protein) Cox7c 20000 Figure 2: Mitochondrial bioenergetics reveals disparate 20000 COX2 * * * * * P/O ratio in mitochondria Cox6b1 * * with 100uM ADP Cox6c OXPHOS kinetics between mouse 15000 * * 15000 * energized with Succinate Cox7a1 * * COX1 1 colorectal cancer cells and healthy * and Octanoyl-L-carnitine. * * * * Cox7a2 10000 * N=7/group PM supported tissues 10000 Cox4i1 * * P/O is in supplemental Cox5b 1 2 1 2 1 2 (pmol/s/mg*) * 2 Ilya N Boykov ,Margaret Am Nelson , Kelsey L McLaughlin , (pmol/s/mg*) figures Cox5a 5000 2 * 5000 Coa3 1 2 1 2 JO * James T Hagen , Hannah S Coalson , Kelsey H Fisher- * * * JO * * Cmc1 0 Wellman1 2 * * Ndufa4 0 1 0 Coa4 Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, Cox6a1 United States. P/M CytC P/M 2 Basal CytC PCRPCRPCROligo Figure 5 Complex IV Cox6a2 East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, United Oct/Suc Rot/Anti BasalClamp 1uM FCCP2uM FCCP3uM FCCP4uM FCCP Oct/Suc Rot/Anti Cox7a2l States. 0.5uM FCCP 1uM 2uMFCCP 3uMFCCP 4uMFCCP FCCP proteome. We investigated 0.5uM FCCP Coa6 intrinsic differences in the Coa7 -1 For references and Figure 1 A-D Respiration data from the PCR titration and FCCP titration protocols in mitochondrial proteome Cox11 permeabilized colon and heart strips and in permeabilized cells. Data was normalized across the groups. In this Cmc2 more data scan figure we depict complex Cox14 to total protein (A-B) or to mitochondrial protein (C-D). N=7/group. Heart’s respiratory Cox17 here: capacity is greatly elevated when compared to colon and CRC. Statistics: two-way IV per group. ANOVA in relationship to colon. *P<0.0332 N=4/group Colon Heart Cells

Results: Despite minimal differences between CRC and normal mouse colon, in cardia myofibers, both total respiratory capacity and OXPHOS conductance were >5-fold higher when adjusted to total protein and >2-fold when adjusted to mitochondrial protein.