Journal: BMC Cancer
Article Title: Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine
Figure Lengend Snippet: a 3-BrPA potentiates Prima-1 toxicity against A549 cells in 5 mM glucose. A549 cells (4X10 3 ) were seeded in tissue culture 96 well plates in complete medium containing 20 mM glucose and 10% fetal bovine serum, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum for 48 h. Relative proliferation /toxicity was assayed fluorometrically in octuplicate by the Alamar Blue method by quantitating the conversion of resazurin to fluorescent resorufin [ 8 ]. This revealed that 50 μM Prima-1 cooperated with 3-BrPA rather than with CHC to suppress A549 cell growth. b Prima-1 decreases SLC2A1-GLUT1 in A549 cells. Sparse cells were seeded in 5 cm tissue culture plates (5 × 10 5 cells per plate) in complete Dulbecco’s medium containing 20 mM glucose supplemented with 10% serum for 18 h, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum whenever indicated (+) for 24 h. After RNA extraction with TRIZOL and quantification in a Qubit® 2.0 Fluorometer, cDNAs were prepared for end-point PCR analysis as indicated under Methods.essentially similar results were obtained in cells treated with Prima-1 in 5 mM glucose (not shown). Cells treated in parallel with those used for RNA analysis were used for GLUT1 protein immune blot [ 40 ]. c Prima-1 activates p21CDKN1A gene expression in A549 cells in 5 mM glucose. qPCR was used to determine relative expression of the p21CDK1N1 gene in control and treated cells, after RNA extraction, cDNA preparation and qPCR, as indicated under Methods. *denotes significance between treated cells relative to controls
Article Snippet: Real-time and end-point RT-PCR Cells were seeded in 5 cm-well plates (3 × 105 cells per plate) in complete Dulbecco’s medium containing 20 mM glucose supplemented with 10% serum for 24 h. Cells were washed 3X with PBS and treated as indicated in medium supplemented with physiological 5 mM glucose and 5% dialyzed serum for 24 h. RNA extraction was performed using TRIZOL® (Life Technologies, Cat # 15596–026) and quantification was determined using a Qubit® 2.0 Fluorometer (Life Technologies, Cat #Q32866) with a Qubit™ RNA Assay Kit (Life Technologies, Cat # Q32852).
Techniques: RNA Extraction, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction