wi38 normal lung fibroblast cells human  (ATCC)

Journal: Biology Open

Article Title: Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response

doi: 10.1242/bio.038257

Figure Lengend Snippet: Inhibition of glutamine-dependent anaplerosis with AOA induces inhibition of cell proliferation and cell cycle arrest in WI38 cells. WI38 cells were treated with vehicle or AOA in the absence or presence of 5 mM αKG for the indicated time-period with medium changed at 2-day intervals. (A) Effect of AOA on cell proliferation and cell cycle progression. Left panel: the relative cell numbers were calculated by normalizing against the value of day 0. Right panel: cell cycle analysis was conducted after a 2-day treatment period using flow cytometry with Propidium Iodide staining of DNA. The percentages of cells in various phases of the cell cycle are presented. (B) Effect of AOA on mTORC signaling. Cell lysates were prepared after a 1-day treatment period and analyzed for the activation status of mTORC1 and mTORC2 by immunoblotting and densitometry analysis of P-S6K1-T389/S6K1 and P-4E-BP1-S65/β-actin, and P-AKT-S473/AKT with β-actin served as a loading control. (C) Effect of mTOR inhibitors on AOA-regulated mTORC1/2 activities. Cells were first treated with 3 mM AOA and then given vehicle (DMSO), 1 nM rapamycin or 30 nM Torin1 1 h before the end of the 24-h culture period. Cell lysates were analyzed by immunoblotting as described in B. (D) Effects of AOA on energy status. Intracellular ATP content and ADP/ATP ratio were determined by ApoGlow Assay Kit. All quantitative data are expressed as the mean±s.e.m. ( n =3) of three independent experiments. Different lowercase letters indicate significant difference among treatment groups at the same time-point ( P <0.05). Asterisk (*) designates a significant difference compared with the respective vehicle control ( P <0.05).

Article Snippet: The WI38 normal human embryonic lung fibroblast cell line and the U2OS human osteosarcoma cell line (ATCC, Manassas, USA) were grown in minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotic (100 U ml −1 penicillin and 100 μg ml −1 streptomycin) at 37°C in 5% CO 2 .

Techniques: Inhibition, Cell Cycle Assay, Flow Cytometry, Staining, Activation Assay, Western Blot, Control

Journal: Biology Open

Article Title: Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response

doi: 10.1242/bio.038257

Figure Lengend Snippet: Anaplerotic factors pyruvate or oxaloacetate, similar to αKG, prevent AOA-induced effects on cell proliferation, mTORC1 and mTORC2 activity in WI38 cells . WI38 cells were treated with vehicle or AOA in the absence or presence of 3 mM pyruvate, oxaloacetate or 5 mM αKG for the indicated time-period with medium changed at 2-day intervals. (A) Effect of anaplerotic factors on AOA-induced inhibition of cell proliferation. Cell numbers were assessed and calculated as described in Fig. 1 A. (B) Effect of anaplerotic factors on AOA modulation of mTORC signaling ( n =3). After 24-h treatment, cell lysates were prepared and analyzed for the activation status of mTORC1 and mTORC2 respectively indicated by P-S6K1-T389/S6K1 and P-AKT-S473/AKT as described in Fig. 1 B. All quantitative data are expressed as the mean±s.e.m. ( n =3) of three independent experiments. Different lowercase letters indicate significant difference among treatment groups at the same time-point ( P <0.05). * and # designate a significant difference compared with the respective vehicle control and AOA group, respectively ( P <0.05).

Article Snippet: The WI38 normal human embryonic lung fibroblast cell line and the U2OS human osteosarcoma cell line (ATCC, Manassas, USA) were grown in minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotic (100 U ml −1 penicillin and 100 μg ml −1 streptomycin) at 37°C in 5% CO 2 .

Techniques: Activity Assay, Inhibition, Activation Assay, Control

Journal: Biology Open

Article Title: Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response

doi: 10.1242/bio.038257

Figure Lengend Snippet: Prolonged blockade of glutamine-dependent anaplerosis with AOA triggered cellular senescence in WI38 cells. WI38 cells were treated with vehicle or AOA in the absence or presence of anaplerotic factors αKG, pyruvate or oxaloacetate for the indicated time-period as described in Fig. 1 A. (A) Effect of AOA on cellular senescence. The senescent cells were assessed using the SA-β-gal and SAHF staining assays. Upper panel: SA-β-gal positive cells were counted in at least 10 microscopic fields in each of the triplicate cultures of all treatment groups. The percentage of SA-β-gal positive cells was calculated relative to the total cell number in the counted fields. Lower panel: A total of 200 cells from each of the indicated treatment samples were examined for SAHF formation. The percentage of SAHF-positive cells was calculated relative to the total cell number in the counted fields. (B) Effect of AOA on senescence-inducing regulators. After treatment for 6 days, cell lysates were prepared and analyzed by immunoblotting and densitometry analysis for p53, p21 CIP1 , Rb, P-Rb-S807/811 and p16 INK4A , β-actin served as a loading control. The arrowheads show the indicated antibody recognized specific signals. All quantitative data are expressed as the mean±s.e.m. ( n =3) of three independent experiments. Different uppercase letters indicate significant difference of the same treatment group at different time-points ( P <0.05). Asterisk (*) designates a significant difference compared with the respective vehicle control at the same time-point ( P <0.05). Different lowercase letters indicate significant difference among treatment groups ( P <0.05).

Article Snippet: The WI38 normal human embryonic lung fibroblast cell line and the U2OS human osteosarcoma cell line (ATCC, Manassas, USA) were grown in minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotic (100 U ml −1 penicillin and 100 μg ml −1 streptomycin) at 37°C in 5% CO 2 .

Techniques: Staining, Western Blot, Control

Journal: Biology Open

Article Title: Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response

doi: 10.1242/bio.038257

Figure Lengend Snippet: AOA treatment leads to inhibition of proliferation, increase of p16 antibody-reactive p12 and cellular senescence in p16 INK4A -knockdown WI38 cells. Cells were first transfected with p16 INK4A siRNA or control siRNA for 24 h, and then treated with vehicle or 3 mM AOA in fresh medium for the indicated time-periods with medium changed at 2-day intervals. (A) Effect of 6-day AOA treatment and 3-day AOA removal on cell proliferation. The relative cell numbers were calculated by normalizing against the value of day 0. After 6 days of AOA treatment, cells were refreshed with AOA-free growth medium and incubated for an additional 3 days. (B) Effect of AOA on cellular senescence. The senescent cells were assessed using the SA-β-gal staining assay. SA-β-gal positive cells were counted in at least five microscopic fields each of the triplicate cultures of all treatment groups. The percentage of SA-β-gal positive cells was calculated relative to the total cell number (DAPI-stained positive cells) in the counted fields. (C) Effect of AOA on the senescence-inducing regulator p16 INK4A −Rb pathway. At the end of 6-day AOA treatment, cell lysates were prepared for immunoblotting and densitometry analysis of p16 INK4A , Rb and P-Rb-S807/811 with β-actin served as a loading control. All quantitative data are expressed as the mean±s.e.m. ( n =5) of two independent experiments. The arrowheads show the indicated antibody recognized specific signals. Different lowercase letters indicate significant difference among treatment groups at the same time-point ( P <0.05). Different uppercase letters indicate significant difference of the same treatment group at different time-points ( P <0.05).

Article Snippet: The WI38 normal human embryonic lung fibroblast cell line and the U2OS human osteosarcoma cell line (ATCC, Manassas, USA) were grown in minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotic (100 U ml −1 penicillin and 100 μg ml −1 streptomycin) at 37°C in 5% CO 2 .

Techniques: Inhibition, Knockdown, Transfection, Control, Incubation, Staining, Western Blot

Journal: Biology Open

Article Title: Blockage of glutamine-dependent anaplerosis affects mTORC1/2 activity and ultimately leads to cellular senescence-like response

doi: 10.1242/bio.038257

Figure Lengend Snippet: AOA-induced cell growth arrest and senescence of WI38 cells was not mediated by ROS signaling. (A) Effect of AOA on cellular ROS level. WI38 cells were treated with 3 mM AOA for 2 and 4 days ( n =9). As a positive control, cells were exposed to 0.4 mM H 2 O 2 for 2 h on the first 2 days. At the indicated time-period, cellular content of ROS was determined using DCFH-DA staining as detailed in the Materials and Methods. (B) Effect of ROS scavenger NAC on cell proliferation. Cells were pre-treated with vehicle or NAC for 1 h and then treated with or without 3 mM AOA for the indicated time-periods with medium changed at 2-day intervals. Cell numbers were assessed and calculated as described in Fig. 1 A. (C) Effect of NAC on AOA-induced SA-β-gal activity. Cells were pre-treated with vehicle or 0.5 mM NAC for 1 h prior to treatment with AOA or H 2 O 2 for the indicated time-period. As a positive control, WI38 cells were exposed to 0.4 mM H 2 O 2 for 2 h on the first 2 days in the absence or presence of NAC. Senescent-like cells were evaluated by measuring SA β-gal activity as described in Fig. 3 A. (D) Effect of chronic AOA treatment on cell morphology of WI38 cells. The cells were grown in the absence (control) or presence (+AOA, 6 day) of 3 mM AOA for 6 days, or in the presence of AOA for 6 days followed by removal of the AOA for an additional day (Removal of AOA). The H 2 O 2 -induced senescent WI38 cells (+H 2 O 2 , 6 day) serving as a reference became enlarged and flattened, which is a documented typical senescent morphology. All quantitative data are expressed as the mean±s.e.m. ( n =3) of three independent experiments. Different lowercase letters indicate significant difference among treatment groups at the same time-point ( P <0.05). Different uppercase letters indicate significant difference of the same treatment group at different time-points ( P <0.05). Asterisk (*) designates a significant difference compared with the respective vehicle control at the same time-point ( P <0.05).

Article Snippet: The WI38 normal human embryonic lung fibroblast cell line and the U2OS human osteosarcoma cell line (ATCC, Manassas, USA) were grown in minimum essential medium (MEM; Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate and antibiotic (100 U ml −1 penicillin and 100 μg ml −1 streptomycin) at 37°C in 5% CO 2 .

Techniques: Positive Control, Staining, Activity Assay, Control