Fig. 5.
RGS11-dependent regulation of ATF3 requires CaMKII. (A) Co-immunoprecipitation of ATF3 with RGS11or CaMKII in AC-16 cells. (B) Control or RGS11 (n = 3) CRISPR KO AC-16 cells were transfected with control plasmid (vector) ± CaMKII (HA-tagged) or phosphorylation (T287A) or oxidation (M281/282 V) deficient CaMKII (HA-tagged) where indicated. (C–E) Control (scramble shRNA) or ATF3 KD VCM were treated with doxorubicin (3 μM, 16 h) ± pre-treatment with CaMKII inhibitor KN-93 (50 μM, 1 h). (C) NRG1 concentration in cell culture media (n = 5). (D) CM-H2-DCFDA fluorescence (total ROS; n = 5). (E) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). Immunoblots are accompanied by a densitometric quantification wherein expression is normalized to the corresponding control group. β-Actin serves as a loading control for western blots. Data were analyzed by student's t-test or one- or two-way ANOVA with Sidak's post-hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns = not significant. Data are presented as mean ± SEM.