Increased mitochondrial mass in ulk1^−/− murine embryonic fibroblasts (MEFs). (A) MEFs derived from ulk1^+/+ (WT) and ulk1^−/− (KO) were stained with Mitotracker Green FM and analyzed by FACS. The histogram shows the distribution of Mitotracker staining in both populations. Mean (n = 5) and standard deviation of the percentage of cells within the marker are shown. The marker was set to include all points above the intersection of the 2 plots to highlight the difference in staining between the 2 populations. (B) MEFs derived from ulk1^+/+ (WT) and ulk1^−/− (KO) were stained with TMRM. The histogram shows the distribution of TMRM staining in both populations before and after incubation with in 50 μM CCCP. Mean (n = 5) and standard deviation of the percentage of cells within the marker are shown. After treatment with CCCP mean FL2 fluorescence was reduced to 5.8 (± 0.3) in WT MEFs and 5.7 (± 0.1) in KO MEFs. (C) Graph showing citrate synthase activity in whole-cell extracts prepared from wild-type (WT) and Ulk1-deficient (KO) MEFs normalized to total protein. The mean (n = 5) and standard deviations are shown. Statistical significance (P < .01) was calculated by Student t test analysis and is marked with an asterisk (*). (D) Representative histogram showing forward scatter distribution of MEFs derived from ulk1^+/+ (WT) and ulk1^−/− (KO).