fetal bovine serum fbs  (Equitech-Bio)

Journal: Scientific Reports

Article Title: GADD34 inhibits activation-induced apoptosis of macrophages through enhancement of autophagy

doi: 10.1038/srep08327

Figure Lengend Snippet: Loss of GADD34 increased cellular damage and apoptosis in RAW 264.7 cells treated with LPS and Tyr/Cys-deprivation. (a). Knockdown of GADD34 by shRNA was evaluated. Real-time PCR analysis of GADD34 expression in GADD34-deficient (shGADD34) and control RAW 264.7 cells (shControl) after treatment with LPS and Tyr/Cys-deprivation for 8 h. Graph shows the relative expression as means ± SE of three independent experiments. (b). GADD34-deficient or control RAW 264.7 cells were treated with DMEM + 10%FBS (control) or LPS (1 μg/mL) in Tyr/Cys-deprivation medium for 8 h or 14 h. Cells were stained with 7-AAD and PE-labeled Annexin V and assessed by flow cytometry. (c). GADD34-deficient or control RAW 264.7 cells were treated with LPS with or without Tyr/Cys-deprivation for 14 h. Cells were stained with 7-AAD and PE-labeled Annexin V and assessed by flow cytometry. Relative % of Annexin V + 7AAD − apoptotic cells shown as means ± SE of three independent experiments. (d). BMDMs from GADD34 KO or WT were treated with LPS (1 μg/mL) combined with Tyr/Cys-deprivation for 8 h. Cells were stained with 7-AAD and PE-labeled Annexin V and assessed by flow cytometry. (e). GADD34-deficient or control RAW 264.7 cells were treated with LPS (+L; 1 μg/mL) and/or Tyr/Cys-deprivation for 24 h. Cell lysates were immunoblotted with anti-caspase 3 antibody and detected full length caspase 3 (35 kDa) and cleaved caspase 3 (17, 19 kDa). (f). GADD34-deficient or control RAW 264.7 cells were treated with DMEM + 10% FBS (control) or LPS (1 μg/mL) with or without Tyr/Cys-deprivation medium for 24 h. After treatment, cells were collected and counted by trypan-blue dye exclusion using a Burker-Turk cell count chamber. Data are means ± SE of cell number from three independent experiments. Data are representative of three independent experiments (b, d, e). * p

Article Snippet: RAW 264.7 cells were cultured in DMEM (Sigma, D5766) supplemented with 10% heat-inactivated FBS (Equitech-Bio Inc.).

Techniques: shRNA, Real-time Polymerase Chain Reaction, Expressing, Staining, Labeling, Flow Cytometry, Cytometry, Cell Counting

Journal: Scientific Reports

Article Title: GADD34 inhibits activation-induced apoptosis of macrophages through enhancement of autophagy

doi: 10.1038/srep08327

Figure Lengend Snippet: Expression of GADD34 induced by amino acid-deprivation and LPS. (a). Deprivation of grouped amino acids. RAW 264.7 cells were cultured with the indicated amino acid deprivation media for 4 h. Cell lysates were immunoblotted with anti-GADD34 and anti-β-actin antibodies. Β-actin was used as a control. Arrow indicates GADD34-specific bands. Asterisk indicates non-specific bands. Intensities of the bands were measured by densitometry. Graph shows the immunoblot relative intensities as means ± SE of 3 independent experiments. FBS; DMEM + 10% FBS, +15 A.A. (amino acid); Basal medium containing 15 amino acids ( Table 1 ) without FBS. Gly/Ser/Tyr/Cys deprivation; Arg/Gln/His deprivation; Ile/Leu/Lys/Met deprivation; Phe/Thr/Trp/Val deprivation. (b). Deprivation of single or two amino acids. Assay was performed as in a. (c). Effects of amino acid deprivation. Expression of GADD34 was analyzed for Leu/Lys, Arg or Trp deprivation compared to Tyr/Cys deprivation. (d). RAW 264.7 cells were stimulated with LPS (1 μg/mL) in DMEM + 10% FBS (control) or Tyr/Cys-deprivation medium for 4 h. GADD34 expression was determined by immunobloting. (e). Time course (0–24 h) of GADD34-expression in RAW 264.7 cells incubated in Tyr/Cys-deprivation medium with or without LPS (1 μg/mL). (f). BMDMs were stimulated with LPS (1 μg/mL) in control or Tyr/Cys- deprivation medium for 0–24 h. (g). Human macrophage THP1 cells were stimulated with LPS, deprived of Tyr/Cys or both for 8 h. The expression of GADD34 was determined by real-time PCR. Β-actin was used as an internal control. (h). GADD34 KO or WT mice were starved and injected with LPS (5 μg/g body weight). Expression of GADD34 in bone marrow was analyzed after 24 h starvation and LPS stimulation. The original immunoblots are presented in Supplementary Figure 3 . All immunoblots are representative of three independent experiments (a–f, h). Graph shows the relative expression as means ± SE of three independent experiments (a–d, g). * p

Article Snippet: RAW 264.7 cells were cultured in DMEM (Sigma, D5766) supplemented with 10% heat-inactivated FBS (Equitech-Bio Inc.).

Techniques: Expressing, Cell Culture, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Mouse Assay, Injection

Journal: The Journal of Biological Chemistry

Article Title: BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1 *

doi: 10.1074/jbc.M110.118653

Figure Lengend Snippet: Inhibition of Ras-dependent cell proliferation and survival by GATA-1. A , Ba/F3/N-RasE12/G1ERT cells were seeded at a density of 100/μl and cultured in RPMI supplemented with 1% FBS without IL-3 in the presence or absence of 1 μ m 4-HT.

Article Snippet: A murine IL-3-dependent hematopoietic cell line, Ba/F3, was maintained in RPMI (nacalai tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX) and 0.3 ng/ml rmIL-3.

Techniques: Inhibition, Cell Culture