Journal: Biology of Reproduction
Article Title: Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes †
doi: 10.1093/biolre/ioy099
Figure Lengend Snippet: Cellular sensitivity to pyolysin. (A) Bovine endometrial stromal cells were incubated with culture medium containing vehicle, or molecules that target MAPK (5 μM ERK inhibitor, 10 μM JNK II inhibitor, 10 μM p38 inhibitor), cell cycle (50 μM PNU112455A, 80 μM roscovitine, 3 μM butyrolactone I), metabolic signaling pathways (10 μM AICAR, 100 μM compound C, 2 μM Torin 1, 4 μM rapamycin, 40 μM AKT inhibitor IV, 2.8 μM LY294002), or the cholesterol synthesis pathway (1 μM atorvastatin, 100 μM etidronate, 10 μM alendronate, 10 μM zaragozic acid), with 0.5 mM methyl-β-cyclodextrin as a positive control. Cells were then challenged with control medium, black bar ( ) or medium containing 100 HU/ml pyolysin red bars ( ), and cell viability was determined by MTT assay. Data are from at least three animals per treatment, and expressed as percent cell viability compared with cells in control medium. Data are presented as mean (SEM), and were analyzed by one-way ANOVA and Dunnett multiple comparison post hoc test; values differ from vehicle, * P < 0.05, *** P < 0.001. (B, C) Bovine endometrial stromal cells were incubated for 2 h with control medium black bar ( ) or medium containing the indicated concentrations of pyolysin red bars ( ). Cell viability was evaluated by MTT assay (B) and leakage of LDH into cell supernatants (C). Data are presented as mean (SEM); n = 4 animals. Data were analyzed by ANOVA with Dunnett multiple comparison test; values differ from control *** P < 0.001. (Please see the online version for the color figure.)
Article Snippet: To screen for molecules and pathways that might increase cell survival when cells were challenged with pyolysin, stromal cells were incubated for 24 h in serum-free medium containing vehicle, or molecules that target: MAPK (5 μM ERK Activation Inhibitor Peptide I (Calbiochem 328000) to inhibit MAPK3/1, 10 μM JNK inhibitor II (Calbiochem 420128) to inhibit MAPK8, and 10 μM SB 203580 (Calbiochem 559398) to inhibit MAPK14, as used previously [ ]); cell cycle (50 μM PNU112455A (Sigma SML0498) to inhibit cyclin dependent kinase (CDK)2 and CDK5, 80 μM roscovitine (Sigma R7772) to inhibit CDK1, CDK2, CDK5, and CDK7, and 3 μM butyrolactone I (Sigma B7930) to inhibit CDK1, CDK2, and CDK5 [ , ]); metabolic signaling pathways (10 μM AICAR (Cell Signaling 9944) to activate AMPK, 100 μM compound C (Calbiochem 171261) to inhibit AMPK, 2 μM Torin 1 (Cell Signaling 14379) to inhibit mTOR, 4 μM rapamycin (Cell Signaling 9904) to inhibit mTOR, 40 μM AKT inhibitor IV (Calbiochem 124038), and 2.8 μM LY294002 (Cell Signaling 9901) to inhibit phosphoinositide 3-kinases [ , , ]); or 48 h incubation with inhibitors for the cholesterol synthesis pathway (1 μM atorvastatin (Sigma PZ0001) to inhibit HMGCR [ ]; 100 μM etidronate (Sigma P5248) and 10 μM alendronate (Sigma A4978), which inhibit human FDPS with IC 50 of 80 and 0.5 μM, respectively [ ]; 10 μM zaragozic acid (Sigma Z2626), which inhibits FDFT1 [ , ]).
Techniques: Incubation, Protein-Protein interactions, Positive Control, Control, MTT Assay, Comparison
