Fig. 4

Effect of PA5 on melanoma spheroids invasion and survival. Melanoma spheroids were transferred onto a type I collagen matrix. At 24 h later, spheroids were treated with PA5. (a) Pictures of treated or NT spheroids were taken every 24 h up to 72 h. (b) Histograms representing the % Frag. of PA5-treated or -NT spheroids at each time point. (c) Graphs representing the changes of the Inv. areas of PA5-treated spheroids versus that of NT spheroids evaluated by the ImageJ software. (d) Histograms representing the Inv. Dist. reached by cells in treated versus those reached by NT spheroids. Data represent the means of at least three spheroids ± SD of triplicate biological experiments. Student’s t-test was used for statistical analyses of PA5-treated samples compared with NT. (e) SKMEL-28‒ and (f) M121224-derived spheroids were treated with PA5 (250 nM) for 24 h, and the expression (RQ) of BIRC5, CDKN1A, and CXCL8 mRNAs was evaluated by real-time qPCR. β-Actin was used as a reference gene. Data represent the mean ± SD of triplicate determinations. Student’s t-test was used for statistical analysis. (g) BIM and Survivin protein levels in SKMEL-28 spheroids after PA5 treatment. (h) Protein levels (RQ) represented as mean of protein/β-actin ratio ± SD from three biological replicates. NT data were set as 1. Student’s t-test was applied for statistical analysis. ∗∗∗P < 0.001; ∗∗0.001 < P < 0.01; ∗0.01 < P < 0.05. % Frag., percentage of fragmentation; Dist., distance; h, hour; Inv., invasion; NT, not treated; PA, protein kinase G activator; RQ, relative quantity.