Ation drastically upregulated MMPs RNA expression, 16 5 of As shown inwhereas AATP treatment efficiently decreased the levels of MMP1, two, 3, 9, 13 under PMA stimulation. In zymography analysis and western blotting evaluation, we found that AATP therapy stimulation. In zymography evaluation and western blotting analysis, we found that AATP remedy drastically suppressed PMAinduced the activities and protein expressions of MMP2 and MMP9 drastically suppressed PMAinduced the activities and protein expressions of MMP2 and MMP9 in in HT1080 cells in a dosedependent manner (Figure 3b and c). HT1080 cells within a dosedependent manner (Figure 3b,c).(a)(b)Figure three. Cont.Mar. Drugs 2019, 17, 244 FOR PEER Overview Mar. Drugs 2019, 17, x6 of 6 of 16Mar. Drugs 2019, 17, x FOR PEER REVIEW6 of(c)Figure Impact of of AATP therapy on expression of matrix metalloproteinase (MMPs) (a) treated Figure three. three. Impact AATP remedy on expression of matrix metalloproteinase (MMPs) (a) CellsCells treated with 1 h were 1 h have been in PMA in ng/mL). ng/mL). h, the expression of MMPs MMPs with AATP for AATP for incubatedincubated(10 PMA (10 After 24 Just after 24 h, the expression ofRNA was RNA was quantitative genuine time PCR. actin was used as a loading handle. (b) Gelatin zymography analyzed by analyzed by quantitative real time PCR. actin was employed as a loading control. (b) Gelatin zymography for the determination of MMP2 and(c) MMP9 activities in AATPtreated HT1080 cells. for the determination of MMP2 and MMP9 activities in AATPtreated HT1080 cells. HT1080 cells HT1080Figurewere treated withtreatment on 50, and 100 h matrixstimulated by PMA (ten ng/mL) for 72 h. cells three. Effect of AATP AATP 100 ) for 1of and metalloproteinase (MMPs)PMA (ten ng/mL) (20, expression M) for 1 h and stimulated by (a) Cells have been treated with AATP (20, 50, and treated with AATP for 1 h had been incubated in PMA (ten ng/mL). conditioned media have been detected by for 72 h. Gelatinolytic MMP2 of MMP9 in conditioned Immediately after 24 h, the expression of MMPs Gelatinolytic activities of activitiesand MMP2 and MMP9 in media were detected by electrophoresis RNA was analyzed by quantitative real time as a (b) Gelatin electrophoresis of soluble protein on a gelatin PCR. actin was usedgel. loading handle. Untreated handle a of solublezymography for the determination of MMP2containing 10 5 lipoxygenase Inhibitors targets polyacrylamide gel. protein on a gelatin containing 10 polyacrylamide in AATPtreated controlcells. utilised as Untreated HT1080 was and MMP9 activities was used as a (c) Expression (c) MMP2 and MMP9 and Lysates cell detected making use of western blot detected loading manage.loading manage.withExpression of MMP2in for MMP9 in wasLysates was ng/mL) working with HT1080 cells have been treated of AATP (20, 50, and 100 M) cell h and stimulated by PMA (ten 1 Ilaprazole web westernfor 72 evaluation. actin was usedMMP2 and MMP9 in conditioned media have been with AATP (20, 50, blot h. Gelatinolytic activities of as a loading handle. HT1080 cells treated detected by evaluation. actin was employed as a loading handle. HT1080 cells treated with AATP (20, 50, and 100 ) and 100electrophoresisand stimulated by PMA (10 ng/mL) for 24 h. The relative amounts of MMP2 and M) for 1 h of soluble protein for 1 h and stimulated by PMA (ten on a gelatin containingThe relative amounts of MMP2 and MMP9 ng/mL) for 24 h. ten polyacrylamide gel. Untreated manage MMP9was used as a loading control. (c) Expression of MMP2 and MMP9 in cellLysates vs. untreated control, p were quantified by densitometry measur.