Figure 3
Figure 3. Retention of mitochondria in mature red blood cells of ulk1−/− mice. Peripheral blood from 21 ulk1+/+ (WT) and 19 ulk1−/− (KO) mice ranging in age from 8 weeks to 5 months was analyzed by fluorescence-activated cell sorting (FACS) analysis. (A) Representative contour plot of WT (left panel) and KO (center panel) cells stained with Mitotracker Green FM (y-axis) and CD71-PE (x-axis), with histogram (right panel) showing direct comparison of Mitotracker Green fluorescence (FL1) of the 2 populations (WT vs KO). (B) Representative contour plot of WT (left panel) and KO (center panel) cells stained with thiazole orange (y-axis) and CD71-PE (x-axis), with histogram (right) showing direct comparison of thiazole orange fluorescence (FL1) of the 2 populations. (C) Graph showing percentage of erythroid cells in individual WT and KO animals staining positively with Mitotracker Green FM (MG+), thiazole orange (TO+), and CD71 (CD71+). The population means and standard deviations (error bars) are graphically depicted and show the following statistically significant differences in percentage of Mitotracker Green–positive (3.7% ± 1.2% in WT vs 16.5% ± 6.4% in KO), thiazole orange–positive (7.0% ± 2.1% in WT vs 15.8% ± 4.4% in KO), and CD71+ (3.1% ± 1.2% in WT vs 8.0% ± 3.4% in KO) erythroid cells. Statistically significant differences (P < .001) between WT and KO populations were identified by Student t test analysis and are marked by an asterisk (*). (D) Representative histograms of WT (left panel) and KO (right panel) red blood cells stained with TMRM. The markers denote positive TMRM staining. Each TMRM-stained sample was split into 2 tubes, and the mitochondrial decoupler, CCCP, was added to one of the tubes immediately prior to analysis. The percentage of TMRM-positive cells before (1.4% in WT vs 13.2% in KO) and after (0.1% in WT vs 0.4% in KO) incubation in 50 μM CCCP is shown. (E) Representative electron micrographs of WT (top panel) and KO (bottom panel) red blood cells. ↗ point to intact mitochondria. Ribosomes are seen in a subset of the red cells (bottom right panel).

Retention of mitochondria in mature red blood cells of ulk1−/− mice. Peripheral blood from 21 ulk1+/+ (WT) and 19 ulk1−/− (KO) mice ranging in age from 8 weeks to 5 months was analyzed by fluorescence-activated cell sorting (FACS) analysis. (A) Representative contour plot of WT (left panel) and KO (center panel) cells stained with Mitotracker Green FM (y-axis) and CD71-PE (x-axis), with histogram (right panel) showing direct comparison of Mitotracker Green fluorescence (FL1) of the 2 populations (WT vs KO). (B) Representative contour plot of WT (left panel) and KO (center panel) cells stained with thiazole orange (y-axis) and CD71-PE (x-axis), with histogram (right) showing direct comparison of thiazole orange fluorescence (FL1) of the 2 populations. (C) Graph showing percentage of erythroid cells in individual WT and KO animals staining positively with Mitotracker Green FM (MG+), thiazole orange (TO+), and CD71 (CD71+). The population means and standard deviations (error bars) are graphically depicted and show the following statistically significant differences in percentage of Mitotracker Green–positive (3.7% ± 1.2% in WT vs 16.5% ± 6.4% in KO), thiazole orange–positive (7.0% ± 2.1% in WT vs 15.8% ± 4.4% in KO), and CD71+ (3.1% ± 1.2% in WT vs 8.0% ± 3.4% in KO) erythroid cells. Statistically significant differences (P < .001) between WT and KO populations were identified by Student t test analysis and are marked by an asterisk (*). (D) Representative histograms of WT (left panel) and KO (right panel) red blood cells stained with TMRM. The markers denote positive TMRM staining. Each TMRM-stained sample was split into 2 tubes, and the mitochondrial decoupler, CCCP, was added to one of the tubes immediately prior to analysis. The percentage of TMRM-positive cells before (1.4% in WT vs 13.2% in KO) and after (0.1% in WT vs 0.4% in KO) incubation in 50 μM CCCP is shown. (E) Representative electron micrographs of WT (top panel) and KO (bottom panel) red blood cells. ↗ point to intact mitochondria. Ribosomes are seen in a subset of the red cells (bottom right panel).

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