Figure 2

14-3-3ε modulates Bcl-x splicing and its response to DNA damage. (A) A plasmid programmed to express HA-14-3-3ε was co-transfected with minigene X2 or ∆SB1. Expression of HA-14-3-3ε was confirmed by immunoblots shown on top. Detection of actin was used as a loading control. The impact on Bcl-x splicing was monitored by RT-PCR assays using a minigene-specific pair of primers; radiolabeled RT-PCR products are shown for one experiment with the positions of the Bcl-xS and Bcl-xL products indicated. Histograms represent the average production of Bcl-xS in percentage from triplicates with standard deviations. The CTRL samples were transfected with the Bcl-x minigenes only, and pCDNA3.1 is an empty expression plasmid. (B) Cells were treated with si14-3-3ε, and an immunoblot was performed with anti-14-3-3ε antibodies (top). Detection of tubulin was used as a loading control. The middle panel shows one representative RT-PCR analysis of Bcl-x splicing, while the bottom panel displays the production of Bcl-xS from triplicate experiments. (C) Same as in (B) except that difopein (eYFP-D) was used to inactivate 14-3-3 proteins. (D) Cells treated or not with si14-3-3ε were transfected with HA-SRSF10 and minigene X2. Immunoblots on top display the depletion of 14-3-3ε and the expression of HA-SRSF10. (E) Same as in (D) except that eYFP-D was used to inactivate 14-3-3 proteins. F, SRSF10 contributes to the activity of 14-3-3ε. Minigene X2 was used to follow the impact of depleting SRSF10 with siSRSF10 when HA-14-3-3ε is ectopically expressed. (G) Immunoprecipitation assays using 293 cells treated or not with oxaliplatin and transfected or not with HA-14-3-3ε and FLAG-SRSF10. The material recovered was fractionated on gel and transferred on nitrocellulose decorated with anti-FLAG and anti-HA antibodies. (H) RT-PCR assays on Bcl-x after cells were transfected with Bcl-x minigenes, X2, ∆B2 and ∆B2G (the position of the B2 and B2G elements is shown in Fig. 1A) along with the HA-14-3-3ε expression plasmid. Expression of HA-14-3-3ε was confirmed by immunoblotting (shown on top). (I) Cells treated with the siRNA against 14-3-3ε were incubated or not with oxaliplatin and a RT-PCR assay was carried out to determine the impact of these treatments on the production of endogenous Bcl-xS. Immunoblots confirming the depletion are shown on top. (J) Impact of depleting 14-3-3ε on SRSF10 binding to the Bcl-x transcript. Immunoprecipitations with anti-Flag antibody were carried out on cells treated or not with si14-3-3ε and transfected with Flag-SRSF10. RNA was quantitated for Bcl-x pre-mRNA using primers shown in Fig. 3F and procedures described in the legend of Fig. 3F. (K) Impact of depleting 14-3-3ε on the interaction between hnRNP F and SRSF10. Immunoprecipitations using cells transfected with Flag-SRSF10 and with or without si14-3-3ε were carried out with anti-hnRNP F antibody and recovery of Flag-SRSF10 was monitored by immunoblotting. Oxaliplatin treatment was used as a control. (L) Interaction of mutated SRSF10 with 14-3-3ε. Cells transfected with Flag-14-3-3ε and wild-type (HA-SS), alanine-substituted (HA-AA) or serine-deleted (HA-∆∆) HA-SRSF10 at positions 131 and 133 were used in immunoprecipitation assays with the anti-Flag antibody. Recovered proteins were detected by immunoblot with the anti-HA antibody. In all cases, error bars indicate SD, and asterisks represent significant P values (two-tailed Student’s t test) comparing the means between samples and their respective controls. *P < 0.05, **P < 0.01 and ***P < 0.001; n.s. = not significant. In immunoblots, “xx” and “x” respectively indicate the large and small immunoglobulin subunits that react with the secondary antibody. Gels and blots cropped from different parts of the same gels/blots are indicated by white space between sections in panels A-I and K.