S. A., B. Westernblot analysis on the effect of 50 (A, A549) and 75 (B, H358) lovastatin lactone on COX-2 and PPAR protein expression over a 48-h incubation period. C., D. Concentration-dependent effect of lovastatin lactone on COX-2 protein expression following a 24-h incubation period of A549 (C) and H358 (D) cells. Densitometric evaluations of Western blots are presented as % of vehicle control (100 ) in the charts (A,B; automobile indicated as dashed line) or above the blots (C; D). All densitometric values were normalized to actin. Values are imply SEM of n = 3- 4 (A), n = four – 8 (B) or n = four (C; D) blots. *P 0.05; **P 0.01; ***P 0.001 vs. corresponding automobile manage; Student t test (A; B). www.impactjournals.com/oncotarget 10350 OncotargetFigure 5: Effect of mevalonic acid on modulation of viability, DNA fragmentation and COX-2 expression by lovastatin lactone in A549 and H358 cells. Mevalonic acid at one hundred or 500 was added 1 h prior to addition of lovastatin lactone at 50(A549) or 75 (H358) and incubation was continued for a different 48 h (A549) or 24 h (H358). A., B. Viability (WST-1 test) and DNA fragmentation analyses. C. Western blot analyses of COX-2 expression. Densitometric evaluations of Western blots are presented as percent of car handle (100 ). All densitometric values were normalized to actin. Values are mean SEM of n = 12 (A), n = 4 (B, left; C, left), n = eight (B, suitable) or n = 3 (C, appropriate). *P 0.05; ***P 0.001 vs. corresponding vehicle manage; #P 0.05; ###P 0.001 vs. lovastatin lactone; one-way ANOVA plus Bonferroni test. www.impactjournals.com/oncotarget 10351 OncotargetFigure six: Impact of lovastatin lactone on PG synthesis by A549 and H358 cells.IL-21R Protein custom synthesis A.Animal-Free IFN-gamma Protein manufacturer , B. Cells had been treated with vehicle orlovastatin lactone at 50 (A, A549) or 75 (B, H358) for 24 h inside the presence or absence of NS-398 (1 ) that was added to the cells 1 h before the incubation with lovastatin lactone. PG levels had been determined in cell culture media and have been normalized to total cellular protein amounts. Percent handle represents comparison with vehicle-treated cells (100 ) inside the absence of test substances. Basal, proteinunnormalized PG levels in cell culture media of vehicle-treated cells were as follows: PGE2, 98.27 5.97 pM (A549); PGE2, 39.05 1.33 pM (H358); PGD2, 34.84 9.13 pM (A549); PGD2, 16.60 three.19 pM (H358); 15d-PGJ2, 14.76 four.75 pM (A549); 15d-PGJ2, 18.PMID:23937941 65 2.80 pM (H358). Values are mean SEM of n = four (A, PGE2; B, 15d-PGJ2), n = 8 (A, 15d-PGJ2), n = 7 – 8 (A, PGD2), n = 3 – 4 (B, PGD2) and n = 2 – 4 (B, PGE2 [2 values of your group treated with NS-398 were under the limit of PGE2 detection]). *P 0.05; **P 0.01; ***P 0.001 vs. corresponding automobile control; ##P 0.01; ###P 0.001 vs. lovastatin lactone, one-way ANOVA plus Bonferroni test. www.impactjournals.com/oncotarget 10352 OncotargetImpact of COX-2 and PPAR on lovastatin lactone-induced apoptotic cell deathTo investigate a prospective involvement of COX2 and PPAR in lovastatin lactone-induced apoptotic cell death, experiments working with NS-398 plus the PPAR antagonist GW9662 were performed. As shown in Figure 7, NS-398 and GW9662 inhibited each toxicity (Figure 7A, 7B) and DNA fragmentation (Figure 7C, 7D) by lovastatin lactone in every single cell line. To further substantiate the role of de novo expressed COX-2 in lovastatin lactone-induced apoptotic cell death,transfection experiments were performed utilizing siRNA targeting COX-2. Transfection of cells with COX-2 siRNA was shown t.
