Journal: PLoS ONE

Article Title: IPF Fibroblasts Are Desensitized to Type I Collagen Matrix-Induced Cell Death by Suppressing Low Autophagy via Aberrant Akt/mTOR Kinases

doi: 10.1371/journal.pone.0094616

Figure Lengend Snippet: A) 3×10 4 control or IPF fibroblasts were cultured on polymerized collagen for 24 h in serum free medium in the presence of various doses of 3-methyladenine (3MA) or chloroquine (CQ), and cell viability was measured as described in the Materials and Methods. Shown is the % decrease in control and IPF fibroblasts in the presence of autophagic inhibitors on polymerized collagen (the viable IPF and control fibroblasts on collagen matrix in the absence of inhibitors (0 µM) are considered as 100% viable cells). B) 3×10 4 control or IPF fibroblasts were cultured on 96 well tissue culture plates in the absence of polymerized collagen in serum free medium, and viable cells were measured in the presence of various doses of 3-methyl adenine (3MA, left panel) or chloroquine (CQ, right panel) for 24 h. Shown is the % decrease in viability in control and IPF fibroblasts in the presence of 3MA or CQ. All assays were performed in triplicate. C) 3×10 4 control or IPF fibroblasts were cultured on polymerized collagen in serum free medium, and fibroblast proliferation was measured in the presence of 3MA (left panel) or CQ (right panel) for 24 h as described in the Materials and Methods. Shown is the % decrease in proliferation in control and IPF fibroblasts in the presence of 3MA or CQ. All assays were performed in three separate experiments to measure fibroblast viability or their proliferation using IPF cells in lane 5 and control fibroblasts in lane 3 in an upper panel in .

Article Snippet: For the autophagy inhibition assay, 3-methyladenine (3MA), and chloroquine (CQ) were purchased from Calbiochem (Rockland, MA) and InvivoGen (San Diego, CA) respectively.

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: IPF Fibroblasts Are Desensitized to Type I Collagen Matrix-Induced Cell Death by Suppressing Low Autophagy via Aberrant Akt/mTOR Kinases

doi: 10.1371/journal.pone.0094616

Figure Lengend Snippet: A) 3×10 4 IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) were cultured on polymerized collagen in the presence of 100 µM of 3MA or DMSO in serum free medium and viable cells were measured at 24 h. p <0.01 versus GFP, p <0.02 versus GFP. B) IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) were cultured on polymerized collagen in the presence of 100 µM of CQ or water in serum free medium and viable cells were measured at 24 h. p <0.01 versus GFP, p <0.02 versus GFP. C) 3x10 4 control or IPF fibroblasts grown on 96 well plates coated with polymerized collagen were treated with 100 µM of 3 methyl adenine (MA) and chloroquine (CQ) together, and viable cells were measured at 24 h in serum free medium. Shown is the % change in viable control or IPF fibroblasts treated with 3MA and CQ on collagen matrix. p <0.01 versus control fibroblasts. D) IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP), dominant negative Akt (DA) or empty vector (GFP) cultured on polymerized collagen matrix were treated with both 100 µM of 3MA and CQ together (3MA+CQ), and viable cells were measured at 24 h. p <0.01 versus GFP, p <0.03 versus GFP, p <0.03 versus DMSO/water as indicated. All assays were performed in triplicate using IPF cells shown in lane 5 or control fibroblasts in lane 3 in an upper panel in .

Article Snippet: For the autophagy inhibition assay, 3-methyladenine (3MA), and chloroquine (CQ) were purchased from Calbiochem (Rockland, MA) and InvivoGen (San Diego, CA) respectively.

Techniques: Infection, Expressing, Dominant Negative Mutation, Plasmid Preparation, Cell Culture