Figure 4.

The CaMK inhibitor KN-93 reduces RBL1/p107 levels in A549 and MSTO-211H cells. (A). A549 and MSTO211H cells were treated for 16 h with the CaMK inhibitor KN-93 (20 µM) or the CaMKK inhibitor ST-609 (20 µM) and RBL1/p107 levels assessed by immunoblot. (B). A549 and MSTO-211H cells were treated with 0.2 µM Abemaciclib or 20 µM KN-93 for 16 h. Levels of total and phosphorylated RBL1/p107 (Ser975) were analyzed by immunoblot. (C). mRNA levels of RBL1/p107 relative to β-actin were assessed by qPCR in A549 and MSTO-211H treated with 20 µM KN-93 or control for 16 h. Mean ± SD. (D). Protein levels of RBL1/p107 and RBL2/p130 were analyzed in A549 and MSTO211H cells treated with the CaMK inhibitor KN-93 (20 µM) or with the inactive derivative of KN-93, KN-92 (20 µM), for 16 h. * Indicates an unspecific band recognized by the anti-RBL2/p130 antibody. (E). A549 and MSTO-211H cells were transiently transfected with plasmids encoding wild type (WT) CaMKI, CaMKI kinase-inactive mutant K49A, WT CaMKII, CaMKII kinase-inactive mutant K42R, CaMKII constitutively active mutant T286D, WT CaMKIV, CaMKIV kinase-inactive mutant K75M, or an empty vector used as control. 6 h post transfection, cells were transferred in serum free-media and incubated for additional 24 h. Cells were then serum-stimulated for 16 h. RBL1/p107 levels were analyzed from cell lysates by immunoblot. CamKII was detected using an anti-CaMKII antibody, while CamKI and CaMKIV were assessed using an anti-c-myc tag antibody. (F). RBL1/p107 levels were analyzed in cytoplasmic (C) and nuclear (N) fractions of A549 and MSTO-211H cells pre-incubated with the calpain inhibitor ALLN (100 µM) or a solvent control for 1 h and then incubated for 16 h with 100 µM ALLN, 12.5 µM AKTi VIII, 20 µM KN-93 alone or the indicated combination. Quantification was performed by densitometric analysis using the ImageJ program and expressed as % of control. All experiments were performed three times.