transfections c2c12 mouse myoblast cells  (ATCC)

Journal:

Article Title: The Phosphatidylinositol 3-Kinase/Akt Cassette Regulates Purine Nucleotide Synthesis * S⃞

doi: 10.1074/jbc.M806707200

Figure Lengend Snippet: Effect of PI3K inhibitors or Akt1 siRNA on de novo purine synthesis. a–c, C2C12 cells were incubated for 2 h with either 0.2% DMSO (white bars, No add), 20 μm LY294002 in DMSO (LY2; black bars), or 20 μm LY303511 in DMSO (LY3; gray bars); some cells received 100μm folic acid (FA; b, vertical striped bar) or 100 μm 5-formyltetrahydrofolate (5-fTHF; c, horizontal striped bar). In a, serum-starved cells were incubated for 14 h in DMEM containing 0.1% FBS and then received either phosphate-buffered saline (Serum-starved), 10% serum (Serum-stimulated), or 10 nm insulin at the same time as receiving LY294002 or LY303511. After the 2-h incubation with drugs, rates of de novo purine synthesis were measured for 2 h by incubating cells with either 10 μCi of [14C]formate (a and b) or 10 μCi of [14C]glycine (c). Below the bar graph of a is a Western blot for total Akt and Akt phosphorylated on Ser-473 (pSer473) and Thr-308 (pThr308). Experimental conditions for the blots are the same as for the corresponding bars: lane 1, control; lane 2, control plus LY294002; lane 3, control plus LY303511; lane 4, serum-starved; lane 5, serum-starved plus LY294002; lane 6, serum-starved plus serum; lane 7, serum-starved plus serum and LY294002; lane 8, serum-starved plus insulin; and lane 9, serum-starved plus insulin and LY294002. d, C2C12 cells were transfected with either a control siRNA directed against green fluorescent protein (GFP) or one of two different Akt1 siRNAs (Akt1 siRNA1 in the main figure and Akt1 siRNA2 in the inset). In the main figure, cells additionally were transfected with either vector DNA or a myristoylated (Myr) human Akt1 cDNA resistant to the effects of the Akt1 siRNA. After 48 h, rates of de novo purine synthesis were measured using [14C]formate as described in a and b. Below the bar graphs are Western blots for Akt and tubulin; experimental conditions for the blots are the same as for the corresponding bars. e, Tsc2-deficient (white bar) and matched (black bar) mouse embryo fibroblasts were incubated with 0.2% DMSO (No add), 20μm LY294002, or 20μm LY303511 for 2 h, and then rates of purine synthesis were measured as described in a. In a–c and e, asterisks indicate p < 0.05 when comparing LY294002-treated cells with non-LY294002-treated cells. Asterisks in d indicate p < 0.05 when comparing cells transfected with Akt1 siRNAs with cells transfected with green fluorescent protein siRNA. Data in the bar graphs are the mean ± S.E. (error bars) of three independent experiments performed in duplicate.

Article Snippet: Cell Culture and DNA Transfection —C2C12 mouse mesenchymal cells were from the American Tissue Culture Collection (ATCC), and tuberin-deficient mouse embryo fibroblasts were provided by D. J. Kwiatkowski ( 13 ); both cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Incubation, Saline, Western Blot, Control, Transfection, Plasmid Preparation

Journal:

Article Title: The Phosphatidylinositol 3-Kinase/Akt Cassette Regulates Purine Nucleotide Synthesis * S⃞

doi: 10.1074/jbc.M806707200

Figure Lengend Snippet: Effect of PI3K inhibitors on early and late steps of de novo purine synthesis. C2C12 cells were treated for 2 h with 0.2% DMSO (Vehicle; white bars), 20 μm LY294002 (LY2; black bars), 20 μm LY303511 (LY3; gray bars), or 10 nm rapamycin (diagonal striped bars). During the last 45 min of the 2-h incubation, the cells received 10 μm azaserine (a and b) and 100 μm AICA-riboside (b). [14C]Formate incorporation into FGAR (a) and purine nucleotides (b) was then measured over 2 h. Asterisks indicate p < 0.05 when comparing LY294002-treated cells with vehicle-treated cells. Data are the mean ± S.E. (error bars) of three independent experiments performed in duplicate.

Article Snippet: Cell Culture and DNA Transfection —C2C12 mouse mesenchymal cells were from the American Tissue Culture Collection (ATCC), and tuberin-deficient mouse embryo fibroblasts were provided by D. J. Kwiatkowski ( 13 ); both cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Incubation

Journal:

Article Title: The Phosphatidylinositol 3-Kinase/Akt Cassette Regulates Purine Nucleotide Synthesis * S⃞

doi: 10.1074/jbc.M806707200

Figure Lengend Snippet: Effect of PI3K inhibitors on PRPP availability and the pentose phosphate pathway. a and b, C2C12 cells were treated for 3 h with either 0.2% DMSO (Vehicle; white bars in a and open triangles in b), 20 μm LY294002 (black bars and filled triangles), or 20 μm LY303511 (gray bar). Cells were serum-starved, serum-stimulated, and treated with insulin as described in the legend to Fig. 2. Cellular PRPP availability was assessed by measuring [8-14C]adenine incorporation into adenine nucleotides over 30 min. The culture medium contained 25 mm glucose in a (DMEM with high glucose) and the indicated glucose concentrations in b. c, C2C12 cells were incubated for 2 h with 0.2% DMSO (Vehicle; white bar), 20 μm LY294002 (LY2; black bar), or 20 μm LY303511 (LY3; white bar), and then [6-14C]glucose was added to the cells for 2 h. The cells were extracted, the extracts were fractionated by high performance liquid chromatography, and radioactivity in ATP-containing fractions was measured. d, HEL cells were incubated for 2 h with 0.2% DMSO (Vehicle; white bar), 20 μm LY294002 (black bar), or 20μm LY303511 (white bar) in a tightly capped tube fitted with a center well containing NaOH. [1-14C]Glucose was added, and CO2 was collected in the NaOH over 2 h. Asterisks in a and d indicate p < 0.05 when comparing LY294002-treated cells with vehicle-treated cells. Data in the bar graphs are the mean ± S.E. (error bars) of three independent experiments performed in duplicate.

Article Snippet: Cell Culture and DNA Transfection —C2C12 mouse mesenchymal cells were from the American Tissue Culture Collection (ATCC), and tuberin-deficient mouse embryo fibroblasts were provided by D. J. Kwiatkowski ( 13 ); both cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: Incubation, High Performance Liquid Chromatography, Radioactivity

Journal:

Article Title: The Phosphatidylinositol 3-Kinase/Akt Cassette Regulates Purine Nucleotide Synthesis * S⃞

doi: 10.1074/jbc.M806707200

Figure Lengend Snippet: Effect of PI3K inhibitor on adenine and guanine nucleotide synthesis and on purine nucleotide synthesis by the salvage pathway. C2C12 cells were treated for 2 h with either 0.2% DMSO (Vehicle; white bars), 20 μm LY294002 (LY2; black bars), or 20 μm LY303511 (LY3; gray bars). [14C]Formate incorporation into adenine (a) and guanine nucleotides (b) or [8-14C]hypoxanthine incorporation into purine nucleotides (c) was then measured over a 2-h period. Asterisks indicate p < 0.05 when comparing LY294002-treated cells with vehicle-treated cells. Data are the mean ± S.E. (error bars) of three independent experiments performed in duplicate.

Article Snippet: Cell Culture and DNA Transfection —C2C12 mouse mesenchymal cells were from the American Tissue Culture Collection (ATCC), and tuberin-deficient mouse embryo fibroblasts were provided by D. J. Kwiatkowski ( 13 ); both cell types were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Techniques: