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. Author manuscript; available in PMC: 2019 Jul 17.
Published in final edited form as: Circulation. 2018 Feb 5;138(3):287–304. doi: 10.1161/CIRCULATIONAHA.117.031258

Fig. 2. MiD49 or MiD51 regulates mitochondrial network, cell proliferation and apoptosis.

Fig. 2

Fig. 2

(A) Mitochondrial fragmentation in PAH PASMC is reversed by silencing of MiD49 or MiD51. Representative images of mitochondrial networks of normal PASMC and PAH PASMC stained with the potentiometric dye TMRM (red). PAH PASMC were transfected with the specified siRNA, infected with Adv-mNeon Green and imaged after 48h following infection. Mitochondria were color coded by their morphology: red: punctate; green: intermediate; purple: filamentous. Scale bar: 10μm.

(B) Silencing of MiD49 or MiD51 reduces mitochondrial fission. Mitochondrial fragmentation was quantified by mitochondrial fragmentation count (MFC) and percentage area of punctate, intermediate and filamentous mitochondria of each image (***P < 0.001; n=15/group).

(C) Mitochondrial network is restored in PAH PASMC by silencing of MiD49 or MiD51. Representative images of the photoactivation experiments confirmed the increase in mitochondrial network in PAH PASMCs co-transfected with specified siRNA and mitochondrial matrix targeted green fluorescent protein (mito-PAGFP) plasmid for 48 h. The cells were also loaded with TMRM (red). Scale bar: 10μm.

(D) Silencing of MiD49 or MiD51 increases mitochondrial networking factor (MNF). Mitochondrial network is quantified by determining mitochondrial networking factor (MNF) which is increased in PAH PASMC following silencing of MiD49 or MiD51 (*P < 0.05, **P < 0.01; n=5/group; AU: arbitrary unit).

(E) Augmenting MiD49 or MiD51 in normal human PASMC induces mitochondrial fission. Representative images of mitochondrial networks of normal human PASMC transfected with the specified plasmid. Cells were loaded with TMRM (red). Scale bar: 10μm.

(F) Augmentation of MiD49 or MiD51 significantly increases mitochondrial fragmentation (*P < 0.05, ***P < 0.001; n=15/group).

(G) Proliferation of PAH PASMC is inhibited by silencing MiD49 or MiD51. Cell proliferation was analyzed 72h post-transfection (*P < 0.05, **P < 0.01; n=3/group).

(H) Proliferation of normal PASMC is increased by overexpressing MiD49 or MiD51. Cell proliferation was analyzed 72h post-transfection (*P < 0.05, **P < 0.01; n=3/group).

(I) Silencing of MiD49 or MiD51 induces cell cycle arrest in the G1/G0 phase. PAH PASMC was transfected with siMiD49 or siMiD51 for 24h, serum starved for 48h, and then serum stimulated for 24h. Cell cycle analyses were performed by flow cytometry following propidium iodide (PI) staining (**P < 0.01; n=3/group).

(J) Silencing of MiD49 or MiD51 increases baseline apoptosis. PAH PASMCs were labeled with Annexin VFITC and PI and assessed by flow cytometry analyses 72h post-transfection (*P < 0.05, **P < 0.01; n=3/group).