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. 2012 Apr 27;7(4):e35467. doi: 10.1371/journal.pone.0035467

Figure 5. Physiological cell models confirm the dominant-negative effect of new ASM splice variants.

Figure 5

A. PMA induces mRNA expression of ASM isoforms in THP-1 cells. Upon PMA treatment, ASM-1 mRNA expression was time-dependently induced. Expression levels of ASM-5 to -7 rose disproportionately strong. THP-1 cells were treated with 160 nM PMA. After 24, 36 and 48 h, quantitative RT-PCR analyses were conducted. All ASM RT-qPCR values were measured in duplicates, normalised against values from the non-regulated reference gene HPRT and included primer efficiency calculations. DMSO served as a negative control. The displayed data represent mean values of triplicates, error bars denote SD. The results were replicated three times. B. ASM activity levels remain constant upon PMA treatment of THP-1 cells. Upon 24, 36 and 48 h of PMA treatment (160 nM), cell lysates were subjected to an in vitro enzyme activity assay. DMSO served as a negative control. The displayed data represent mean values of triplicates, error bars denote SD. Results were replicated three times. C. High density of H4 cells induces mRNA expression of full-length ASM-1. Depending on increasing cell density, ASM-1 mRNA expression was significantly induced (p<0.001), in contrast to the expression of splice variants ASM-5, -6 and –7. H4 cells were grown to varying levels of confluence. After 72 h, quantitative RT-PCR analyses were conducted. All ASM RT-qPCR values were measured in duplicates and normalised against values from the non-regulated reference gene HPRT. The displayed data represent the mean values of three independent experiments, error bars denote SD. Statistical significance was calculated using one-way ANOVA including a post-hoc test for linear trend (*** p<0.001). D. High density of H4 cells increases ASM activity. The increase in cellular density significantly increased cellular ASM activity (p<0.001). H4 cells were grown to varying levels of confluence. Upon 72 h cell lysates were subjected to an in vitro enzyme activity assay. The displayed data represent the mean values of three independent experiments, error bars denote SD. Statistical significance was calculated using one-way ANOVA including a post-hoc test for linear trend (*** p<0.001).