Fig 1: Flow cytometry analysis of apoptosis PPIPK1-expressing cells. A. Untransfected HEK 293F control and stable cell lines expressing PPIP5K1 and IP6K2 were subjected to immunoblot analysis using anti-PPIP5K1 and anti-IP6K2 antibodies. B. Representative data from flow cytometry from three independent experiments. HEK293F untransfected (control) and stable cell lines were incubated either with DMSO or 25 µM etoposide for 16 hours. All attached and detached cells were collected and double stained with Annexin V and propidium iodide (PI) using APOAF kit from Sigma following manufacture’s instructions. Percentage of healthy (unstained), early apoptotic cell (Annexin V only), late apoptotic (Annexin V and PI) and necrotic (PI only) cells were determined. C. Percentages of cells in early apoptosis (black bars) and late apoptosis, as determined from three independent flow cytometry experiments.
Fig 2: Inhibition of TLR4 receptor blocked apoptosis and mitochondrial oxidative damage induced by Prx. (A) Following pretreatment with TAK242 (1 µM) for 1 h, RAW264.7 cells were incubated with Eh-rPrx (5 µg/ml) for 24 h, then suspended in 500 µL of 1× binding buffer (Sigma-Aldrich, APOAF) at a concentration of 106 cells/ml. Following incubation at room temperature for 10 min, 5 µL of Annexin V FITC conjugate (Sigma-Aldrich, APOAF) and 10 µL of propidium iodide solution (Sigma-Aldrich, APOAF) were added to each suspension. Fluorescence of the cells was immediately determined by using a flow cytometer. In all groups, n = 3. Experiments were repeated three times. (B) Quantitative analysis of mitochondrial ROS MFI in RAW264.7 cells treated with rPrx. RAW264.7 cells were pretreated with TAK242 (1 µM) for 1 h or oATP (500 µM) for 2 h. Next, the cells were treated with 5 µg/mL of Eh-rPrx or Tris-HCl (pH 8.0) as the negative control. After 24 h or 36 h of treatment, the live cells were stained with MitoSOX (5 µM) for 10 min, and then with CFSE (2 µM) for counterstaining. The cells were immediately observed and quantified through high-content screening analysis. In all groups, n = 3. Experiments were repeated three times. (C) The fluorescence images of RAW264.7 cells after 36 h of rPrx treatment. Red fluorescence, MitoSOX™ Red; green fluorescence, CFSE; scale bar, 100 µm. In all groups, n = 3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ns, not significant.
Supplier Page from MilliporeSigma for Annexin V-FITC Apoptosis Detection Kit