Extended Data Fig. 6: Quantitative analysis of PHB complexes.
(a) Representative TEM micrograph of a U2OS mitochondrion. The IBM to CM ratio was determined by measuring the total IBM length (red) and summing the lengths of all cristae, multiplied by 2 due to the dual-membrane composition of a crista. The U2OS wt cells exhibited an IBM to CM ratio of 0.64 to 1 (n = 32 mitochondria). (b) Quantitative analysis of prohibitin abundance in cells. Increasing amounts of purified PHB1 and PHB2 were used as a reference to estimate the total number of prohibitin molecules in U2OS cells. Purified prohibitins and U2OS cell extracts were loaded on the same gel and blotted for antibody-based detection of PHB1 (top) or PHB2 (bottom). Note that purified PHB1 and PHB2 run at a higher molecular weight than endogenous prohibitins due to the His-tag and linker sequence. (c) Determination of total mitochondrial network length by image analysis. U2OS cells stained with Mitotracker Deep Red FM revealed a mitochondria network length per cell of 1082 ± 360 µm (mean ± SD, n = 34 cells). A representative image is shown with a network length of 1029 µm. (d) Representative TEM micrograph of a U2OS mitochondrion. Average distance between crista membranes determined from individual mitochondria was determined as 74.1 ± 15.1 nm, SD, n = 36 mitochondria. (e) Representative TEM micrograph of a U2OS mitochondrion used to determine the total mitochondrial length and size of voids between groups of cristae. Measurements revealed an average gap size of 21 ± 8% of the mitochondrial tubules (n = 98 mitochondria). (f) Line profile along the white dashed line in (e). This mitochondrion shows an average inter-crista distance of approximately 79 nm. Cristae are indicated with black arrowheads. (g) Representative EM micrograph of U2OS cells. Data were used to measure the diameter of mitochondria. Scale bar in A, 500 nm. Scale bar in C, 10 µm. Scale bar in D, E 300 nm. Scale bar in G, 2 µm. Source data are available.
