Fig. 2.

Alcat1 is required for maintenance of mitochondrial function and morphology. (A) Alcat1 was depleted using one of several (numbered 1–5) candidate lentiviral shRNA constructs for 48 h in MLE cells. Cell lysates were analyzed by immunoblotting for Alcat1 or for one of several additional control proteins (Cls1, cardiolipin synthase1; Fbxo28, F-box only protein 28; β-actin). The lower panel shows plots of the densitometry analysis of blots in A. The densitometry analysis values were normalized to those of β-actin. Scr, scrambled. (B) shRNA against Alcat1 (Alcat1 shRNA, shRNA construct 3 shown in Fig. 2A) or scrambled RNA constructs were introduced into cells for 48 h, and the cells were subjected to MitoTracker staining, or DAPI in order to visualize the nucleus. Scale bar: 10 μm. (C) JC-1 staining of Alcat1-shRNA- and LPS-treated MLE cells. Cells were electroporated with scrambled RNA or Alcat1 shRNA plasmid for 48 h with or without LPS. A plasmid encoding Alcat1 was also overexpressed in cells (Alcat1 express). Cells were treated with LPS for 5 min before staining with JC-1 (2 µM) for 20 min. Cells were washed with warm PBS five times before analysis by using confocal microscopy. Scale bar: 10 μm. (D) Densitometry analysis of images detailed in C. *P<0.05, green versus red staining (Student's t-test). (E) Cells were infected with lentiviral Alcat1 shRNA or scrambled RNA with or without LPS treatment, and the oxygen consumption rate was measured using a seahorse XF analyzer. Data in each panel represents n=3 separate experiments. Means±s.e.m. are shown.