Fig. 1

StarD13 is a potential tumor suppressor in lung adenocarcinoma. a Representative fluorescent micrographs of formalin-fixed normal lung tissue from biopsy (upper) or lung adenocarcinoma tissue (lower), paraffin embedded, sectioned and then stained with DAPI (left panels) and immunostained with anti-StarD13 antibody (middle panels). The right hand panels show the merged channels. The graph is a quantitation of the immunohistochemistry. The mean fluorescent intensity/pixel was measured using ImageJ software and expressed to the corresponding tissues. Data are the mean −/+ SEM from 3 different experiments (with 4 tissues each) and *p < 0.05. Scale bar is 100 μm. b Data analyzed from Oncomine website. mRNA of the indicated number of samples (indicated in the legend) were quantified for expression levels of StarD13 in Landi Lung. c A549 and WI38 cells were lysed and immunoblotted by western blot analysis for StarD13 (upper gel) or for β-actin (lower gel) for loading control. The graph is a quantitation (using the ImageJ software) of the bands from the StarD13 gel normalized to the corresponding bands in the actin gel. Data are the mean −/+ SEM from 3 different experiments and *p < 0.05. d A549 cells were transfected with luciferase control siRNA or with 2 oligos of StarD13 siRNA for 72 h. The cells were then lysed and immunoblotted by western blot analysis for StarD13 (upper gel) or for β-actin (lower gel) for loading control. The graph is a quantitation (using the ImageJ software) of the bands from the StarD13 gel normalized to the corresponding bands in the actin gel. Data are the mean −/+ SEM from 3 different experiments and *p < 0.05. e Cell proliferation was determined using WST-1 reagent. Cell viability of siRNA-transfected A549 cells was expressed as fold increase from control (luciferase-transfected). Data are the mean −/+ SEM from 3 different experiments and *p < 0.05. d The micrographs are phase contrast representative images of luciferase siRNA- or StarD13 siRNA-transfected WI38 cells grown in culture for 72 h. Cell proliferation for siRNA-transfected WI38 cells was determined using WST-1 reagent and cell viability expressed as fold increase from control (luciferase-transfected). Data are the mean −/+ SEM from 3 different experiments and *p < 0.05. g A549 cells were transfected with luciferase or StarD13 siRNA permeabilized and stained with 30 μL propidium iodide for 10 min. Cells were analyzed using a C6 flow cytometer, which indicated the distribution of the cells into their respective cell cycle phases based on their DNA content. G0/G1 cells were 2n; S-phase cells were > 2n but <4n while G2/M were 4n. Cell DNA content was determined by CellQuest software. The graph is a quantitation expressed as percentage of cells in G1 and combined percentage of cells in S + G2. Data is the mean +/− SEM from 3 different experiments. h A549 cells were transfected with either control luciferase siRNA or with StarD13 siRNA. The cells were then trypsinized and stained with 5 μL of Annexin V FITC and 10 μL of Propidium Iodide. The fluorescence of the cells was determined immediately with a flow cytometer. Cells, which are early in the apoptotic process, will stain with the Annexin V FITC Conjugate alone. Live cells will show no staining by either the Propidium Iodide solution or Annexin V FITC conjugate. Necrotic cells will be stained by both the Propidium Iodide solution and Annexin V FITC conjugate. The graph is a quantitation expressed as percentage of cells. Data is the mean +/− SEM from 3 different experiments and *p < 0.05. i A549 transfected with Luciferase or StarD13 siRNA were grown in suspension and mixed with Matrigel to form spheres, as described in the Materials and Methods. The graphs are a quantitation of sphere diameter (μm) and area (μm²) of the spheres. Data is the mean +/− SEM from 50 spheres/condition and *p < 0.05