Fig 2: The AKT inhibitor AKTi VIII reduces RBL1/p107 levels in A549 and MSTO-211H cells. (A). A549 and MSTO-211H cells were treated with the indicated concentrations of the AKT inhibitor AKTi VIII for 16 h. RBL1/p107 levels were analyzed in cell lysates by immunoblot. (B). A549 and MSTO-211H cells were treated with 0.2 µM Abemaciclib or 12.5 µM AKTi VIII for 16 h. Levels of total and phosphorylated p107 (ser975) were analyzed by immunoblot. Arrows indicate the two major bands corresponding to hyper- and hypo-phosphorylated RBL1/p107. (C). RBL1/p107 levels were analyzed in cell lysates of parental and A549 cells with RBL1/p107 knock-out by CRISPR/Cas9. MM: molecular mass. (D). RBL1/p107 mRNA levels were determined by RT-PCR in A549 and MSTO-211H cells treated with 12.5 µM AKTi VIII for the indicated time points. (E). RBL1/p107 levels were determined by immunoblot in A549 and MSTO-211H cells preincubated with the MEK1/2 inhibitor U0126 or the p38 inhibitor SB202190, at the concentration of 10 and 20 µM, respectively, for 2 h and then treated with the same concentrations of U0126 or SB202190 alone or in combination with 12.5 µM AKTi VIII for 16 h (F). A549 cells were treated with 50 ng/mL EGF for the indicated time and RBL1/p107 protein levels analyzed by immunoblot. (G). RBL1/p107 levels were analyzed in cell lysates of A549 cells treated for the indicated time with 50 ng/mL EGF or 10 nM IGF-2. Western blot quantification was performed using the ImageJ program, normalized to ß-actin, a-tubulin or GAPDH expression and reported as % of control (CTR). All experiments were performed three times.

Fig 3: 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 ß-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.

Fig 4: KN-93 effect on cell cycle of A549 and MSTO-211H cells. (A). The cell cycle was analyzed by FACS in A549 and MSTO-211H cells treated with 20 µM KN-93 or 20 µM KN-92 for 16 h. (B). Protein levels of cyclin D1 (CycD1) and cyclin D3 (CycD3) were assessed in A549 and MSTO-211H cells treated for 16 h with 20 µM KN-93. (C). A549 and MSTO-211H cells were co-transfected with plasmids coding for cyclin D1, CDK6-HA, CDK6-DN-HA or an empty vector as control and then treated with 12.5 µM AKTi VIII or 20 µM KN-93 for 16 h. RBL1/p107 levels were assessed by immunoblot. CDK6 was detected using an anti-HA antibody. (D). RBL1/p107 protein levels in A549 and MSTO-211H cells transduced with a lentivirus expressing CDK6 S178P or control lentivirus and transfected with plasmids coding for cyclin D3 or empty vector and then treated with 12.5 µM AKTi VIII or 20 µM KN-93 for 16 h. The expression of exogenous CDK6 S178P was monitored by detecting enhanced green fluorescent protein (EGFP), encoded by the bicistronic IRES pHAGE vector. Quantification was performed by densitometric analysis using the ImageJ program and expressed as % of CTR (B) or, for each treatment condition, as % empty vector (e. v.)-transfected respective control cells. All experiments were performed three times.

Fig 5: The modulation of AKT expression does not significantly affect RBL1/p107 levels. (A). A549 cells were treated with 12.5 µM AKTi VIII, 20 µM LY294002 or 1 µM Wortmannin for 1 h (left panel) or 16 h (right panel). RBL1/p107 levels were analyzed by immunoblot. (B). A549 and MSTO-211H cells were transfected with siRNA targeting AKT1 or non-targeting control siRNA and the levels of RBL1/p107 assessed by immunoblot. (C). RBL1/p107 levels as determined in A549 and MSTO-211H cells expressing myristoylated AKT1, AKT2, the combination of myristoylated AKT1 and AKT2 or an empty plasmid. AKT1 and AKT2 were detected using an anti-pan AKT antibody recognizing both AKT1 and AKT2. Quantification was performed by densitometric analysis using the ImageJ program and expressed as % of control. All experiments were performed three times.