Fig. 10: AMPK-α1/α2 deletion precipitates mitochondrial dysfunction and reactive oxygen species accumulation in pulmonary arterial myocytes. | Nature Communications

Fig. 10: AMPK-α1/α2 deletion precipitates mitochondrial dysfunction and reactive oxygen species accumulation in pulmonary arterial myocytes.

From: AMPK deficiency in smooth muscles causes persistent pulmonary hypertension of the new-born and premature death

Fig. 10

a Images show from left to right a bright-field image of a pulmonary arterial myocyte, then deconvolved 3D reconstructions of a z-stack of images of MitoTracker Green fluorescence, TMRE fluorescence and a merged image from (i) AMPK-α1/α2 floxed (AMPK-α1/α2 FLX) and (ii) AMPK-α1/α2 knockouts (AMPK-α1/α2 KO). a iii Scatter plot shows the mean ± SEM for total (non-deconvolved) TMRE fluorescence (n = 26 cells for AMPK-α1/α2 FLX from 8 independent experiments, n = 3 mice; n = 54 cells for AMPK-α1/α2 KO from 13 independent experiments, n = 3 mice). b i-ii As in A but showing images of MitoTracker Red and Rhodamine 123 fluorescence. b iii, Scatter plot shows the mean ± SEM for total (non-deconvolved) Rhodamine 123 fluorescence (n = 41 cells for AMPK-α1/α2 FLX from 10 independent experiments, n = 5 mice; n = 20 cells for AMPK-α1/α2 KO from 6 independent experiments, n = 3 mice). c Records of Rhodamine 123 fluorescence ratio (F/F0) against time recorded in pulmonary arterial myocytes from (i) AMPK-α1/α2 floxed, (ii) AMPK-α1/α2 KO mice, and (iii) a scatter plot showing the mean ± SEM for the maximum change in Rhodamine 123 fluorescence during application of a mitochondrial uncoupler, FCCP (10 μmol/L; n = 10 cells for AMPK-α1/α2 FLX from 5 independent experiments, n = 3 mice; n = 6 cells for AMPK-α1/α2 KO from 4 independent experiments, n = 3 mice). d i-ii As in (a) but showing images of MitoTracker Green and MitoSOX fluorescence. d iii, Scatter plot shows the mean ± SEM for total (non-deconvolved) MitoSOX fluorescence (n = 24 cells for AMPK-α1/α2 FLX from 5 independent experiments, n = 2 mice; n = 15 cells for AMPK-α1/α2 KO from 4 independent experiments, n = 2 mice). e Images of z-sections and 3D reconstruction for the mitochondrial clusters detected in the cells shown in (a), from (i) AMPK-α1/α2 floxed and (ii) AMPK-α1/α2 KO, together with a (iii) frequency histogram (bin centre = 2, mean value indicated by dotted line) for the values shown in (e iv) and (iv) scatter plot for cluster detection (n = 25 cells for AMPK-α1/α2 FLX from 7 independent experiments, n = 3 mice; n = 39 for AMPK-α1/α2 KO from 9 independent experiments, n = 4 mice). Data are expressed as mean ± SEM. Statistical significance was assessed by two-sided unpaired Mann–Whitney’s test (a iii, c iii), two-sided unpaired Student’s t test (b iii, d iii, e iv) and extra sum-of squares F-test for Gaussian distributions (e iii).

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