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. 2021 Jul 9;12:4220. doi: 10.1038/s41467-021-24469-y

Fig. 3. Prok2 protects mitochondrial function in an Acsl4-dependent manner.

Fig. 3

20 μM Erastin for 24 h is used to induce ferroptosis in these studies. a Mito-Tracker staining exhibits a fusiform structure, a small rod-like form in neurites, and an interconnected network in the cytoplasm in the normal primary neurons. Erastin treatment disrupts the mitochondrial network and mitochondria appear as small fragmented punctiform structures and small circles. Prok2 overexpression reduces mitochondrial circularity (b) and increases mitochondrial length (c). Data presented as mean ± SD (n = 5 experiments). d Immunofluorescence assessment of the expression and intracellular distribution of Tomm20 and Prok2. Prok2 overexpression increases Tomm20 positivity and promotes migration of mitochondria to neurites. Scale bar is 10 μm. e Representative electron microscopy images show shrunken mitochondria and outer membrane rupture upon exposure to Erastin (red arrow), which is inhibited by Prok2 overexpression. Scale bar is 500 nm. f–l Expression levels of Acsl4, Gpx4, Tfam, Tomm20, and MT-ND1 (mitochondrial DNA copy number) in Acsl4 deficient (shAcsl4) and control (shCtrl) primary neurons. GAPDH is used as control. Data presented as mean ± SD (n = 3 experiments). m ATP levels are decreased upon exposure to Erastin. Overexpression of Prok2 prevented the decrease in mtDNA copy number and ATP levels in Erastin-treated cells. Data presented as mean ± SD (n = 5 experiments). n Representative TEM images illustrating that Erastin administration does not cause marked changes of mitochondrial morphology in shAcsl4 primary neurons. Scale bar is 500 nm. For all panels, n indicates biologically independent repeats. P value was determined by two-tailed unpaired Student’s t test for comparations between two groups. Source data are provided as a Source Data file.