Fig 1: Suppression of Gatm promotes cyst expansion by increasing ROS. (A) 2',7'-dichlorofluorescein diacetate (DCFDA) was used to measure cellular ROS activity, including hydroxyl, peroxyl and other ROS. The intensity of DCFDA was measured using a microplate reader. (B) MitoSOX staining to detect mitochondrial superoxide (O2-) levels by confocal microscopy. IMCD cells were transfected with Gatm siRNA (20 nM). CoCl2 was used as a positive control. NT, non-treated. (C) Co-staining with MitoSOX Red and MitoTracker Green, which accumulates in active mitochondria. Images were obtained by confocal microscopy at 400× magnification and 1.5× zoom. (D) Cyst formation in control or Gatm siRNA treated IMCD cells in a Matrigel 3D culture system. Cyst area was measured after 4 days in 3D culture. Data are presented as the mean ± SD of triplicate samples from three independent experiments. **p<0.01.
Fig 2: Schematic of the proposed novel mechanism of ADPKD progression. miR-132-3p is up-regulated in ADPKD. Increased miR-132-3p levels inhibit Foxo3 and subsequently down-regulate Gatm, leading to elevated ROS levels and oxidative stress, which promotes cyst growth in ADPKD.
Fig 3: Gatm is down-regulated in ADPKD and regulated by FOXO3. (A) Schematic of the comparative analysis of mitochondrial genes and differentially expressed genes (DEGs) in Pkd1 cKO mice for the identification of differentially expressed mitochondrial genes. (B, C) Validation of the mRNA expression levels of Gatm using qRT-PCR in the (B) kidney tissues of Pkd1f/f and Pkd1f/f:HoxB7-Cre mice and (C) non-ADPKD and ADPKD patients. (D) The protein expression levels of GATM in mIMCD and HRCE cells transfected with Foxo3 or control siRNA (20 nM) by western blotting. (E) GATM protein expression in the mitochondrial and the cytosolic fractions of mIMCD cells transfected with Foxo3 or control siRNA (20 nM) by western blotting. (F) Relative luciferase activity of four Gatm promoter region constructs in mIMCD cells. Several deletion constructs of the Gatm promoter region inserted into the pGL3-base luciferase reporter vector are shown on the right. (G) Relative luciferase activity of the p0.2 kb construct in mIMCD cells transfected with Foxo3 or control siRNA. Data are the mean ± SD of triplicate samples from three independent experiments. *p<0.05, **p<0.01, ***p<0.001.
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