Fig. 5: CPT1A promotes MFF succinylation and inhibits its ubiquitin-proteasome degradation. | Communications Biology

Fig. 5: CPT1A promotes MFF succinylation and inhibits its ubiquitin-proteasome degradation.

From: Carnitine palmitoyltransferase 1A promotes mitochondrial fission by enhancing MFF succinylation in ovarian cancer

Fig. 5

a Succinylated lysine (succK) was examined in SKOV-3 and OVCAR-3 cells with or without CPT1A knockdown. Bands are derived from the different gels. b ES2 cells exogenously expressing Flag-MFF were infected with the HA-CPT1A lentivirus as indicated. Cells were harvested for immunoprecipitation with an anti-Flag antibody. Succinylated lysine (succK) was examined by Western blotting. The relative succK expression was normalized to Flag (IP) and then compared to control groups. c, d The CPT1A-G710E (G710E, c) or CPT1A-H473A (H473A, d) was transfected into CPT1A-knockdown SKOV-3 cells. CPT1A, MFF, and succinylation and ubiquitination of cellular proteins were analyzed by Western blotting. Beta-tubulin was used as an endogenous control. e Mass spectrum analysis of MFF succinylated peptides. Red arrows point to the parent ion of interest analyzed by tandem mass spectrometry where succinylation occurs. f Conservation analysis of amino acids in the region near the succinylation site among species. g SKOV-3 cells transfected with Flag tagged wild type MFF (Flag-WT), MFF K302R (Flag-K302R) or MFF K302E (Flag-K302E) were immunoprecipitated with an anti-Flag antibody, followed by immunoblotting with anti-Flag, anti-succK, anti-Parkin or anti-ubiquitin antibodes. The relative succK, ubiquitin and parkin expressions were normalized to Flag (IP) and then compared to control groups. h 293TN cells transfected with Flag-MFF-WT, Flag-K302R or Flag-K302E were treated with CHX (10 μM) for up to 12 h. Whole cell lysates were prepared and assessed using anti-Flag antibodies (left). Relative expression of Flag-MFF was normalized to β-tubulin and then compared to 0 h (n = 3 for each group, right). Bands are derived from the different gels. i Representative confocal microscopy images of the mitochondria in SKOV-3 cells with control, CPT1A-knockdown, or exogenous overexpression of CPT1A H473A in conjunction with CPT1A-knockdown (left). The proportion of cells with different mitochondrial morphologies was quantified (n = 3 samples, each sample contains 100 cells, right). Data represent the mean ± SD; ***p < 0.001, n.s. indicates no significant difference compared with the control groups or as indicated. Scale bars, 10 μm.

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