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. 2015 Feb;21(2):262–278. doi: 10.1261/rna.048090.114

FIGURE 3.

FIGURE 3.

PROMPTs are accumulating by anisomycin- and UV-C light-induced p38MAPK-activation in HeLa cells. (A) Treatment of HeLa cells with 10 µg/mL anisomycin for 2 h or irradiation with 100 J/m2 UV-C light results in the strong activation of p38MAPK as shown by Western blot for phospho-T180 and phospho-Y182 in p38 and the activation of its downstream kinase MK2 as monitored by T334 phosphorylation. Inhibition of p38MAPK-activity by the inhibitor BIRB796 (BIRB) prevents p38- and MK2-phosphorylation. Total p38 and MK2 protein remained unchanged throughout the experiments. The translation elongation factor eEF2 served as a loading control and RBM7 and ZCCHC8 showed similar band patterns in response to stress like in previous experiments (see Figs. 1E–G, 4A,C). (B) Using the anisomycin stimulation experimental setup, expression of four well-characterized PROMPTs (proRBM39, proEXT1, proIFNAR1, and proDNAJB4) was analyzed at different time points (0, 60, 120, and 240 min anisomycin stimulation) by qRT-PCR. (C) As a control the anisomycin-induced downstream expression of RBM39 was analyzed. (D) Like the anisomycin-induced up-regulation of PROMPTs the UV-C light-mediated accumulation can be abolished by p38MAPK-inhibition using BIRB796. All results are shown as x-fold induction relative to the nonstimulated expression that was set to 1. All graphs are displayed with standard deviations. All experiments were performed independently a minimum of two times and showed similar effects. (E) Overexpression of GFP-RBM7S136A leads to diminished PROMPTs accumulation upon anisomycin stimulation indicating that stress targets the exosomal degradation of PROMPTs via RBM7. (F,G) HeLa cells were treated with either a Control siRNA or hRRP40- and RBM7-specific siRNAs and 48 h post-transfection treated as indicated. Levels of proRBM39 and proDNAJB4 were determined by qRT-PCR and accumulation was calculated relative to untreated cells for each siRNA knockdown. Knockdown efficiency of hRRP40 and RBM7 was assayed by Western blot (Supplemental Fig. S7B).