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. 2012 Jun 25;1(6):e18. doi: 10.1038/oncsis.2012.18

Figure 4.

Figure 4

Forskolin and downstream effectors PKA and JunD affect RASSF10 expression. (a) Stability of RASSF10 mRNA was determined after 10 μℳ α-amanitin treatment from 1 to 12 h. 28S rRNA was used as control. (b) Cells were treated with 20 μℳ forskolin and 500 μℳ IBMX (F+I) or DMSO for 12 h with or without 10 μℳ α-amanitin. RASSF10 expression was analyzed by qRT–PCR and normalized to 28S rRNA. DMSO alone was set 1. (c) H89 (10 μℳ) inhibition of RASSF10 induction by F+I is shown. F+I is depicted relative to DMSO and F+I+H89 is shown relative DMSO+H89. Cells were transfected with PKACα or empty vector and (d) RASSF10 and (e) JunD expression were analyzed after 24 h. Expression was normalized to ACTB. (f) Cells were transfected with AP-1 members or empty vector. RASSF10 expression was analyzed after 24 h by qRT–PCR (normalized to ACTB). (g) Cells were transfected with siControl, siJunD and siFra2. After 72 h cells were trypsinized, seeded at 10% density and upon attachment serum-starved overnight. Then cells were stimulated with F+I or DMSO for 12 h. RASSF10 expression was normalized to ACTB. (h) JunD and (i) Fra2 knockdown were verified for assay described in (g). All experiments were performed in A549 cells at least in triplicates and mean as well as respective SD are shown. P values were calculated using two tailed t-test. DMSO treatment, vector alone or siControl are set 1.