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. 2023 Jan 24;12:e69521. doi: 10.7554/eLife.69521

Figure 5. AURKA and GSK3B regulate c-MYC in PKA-dependent cell models.

(A) Summary data from the FLX1 cell line treated with 352 kinase inhibitors from an advanced clinical compound library at 2 μM for 120 hr. The targets of selected compounds with a z-score ≥2 are highlighted, with the PKA inhibiting tool compound H89 shown. Average of three biological replicates is shown. (B) Impact of doxycycline (dox) induction of 3xFLAG-PRKAR1AG325D on drug sensitivity in FLX1: Cells were incubated with 1 μg/ml dox overnight and compound added on the following day; log2FC vs. median was derived ± dox, and then subtracted to identify those compounds whose activity was altered by 3xFLAG-PRKAR1AG325D. Data are averaged from three biological replicates. Inhibitors with p<0.05 were marked. Selected inhibitors were color coded based on their targets. (C) Kinase pooled siRNA library screen with FLX1 in 384 well plates shows the effect of each target kinase on cell proliferation (average of four biological replicates). Selected non-metabolic kinases that decrease cell proliferation with z-score –1 were marked. (D) FLX1 cells treated with dose curves of multiple AURKA inhibitors for 120 hr. Relative cell viability was measured by CTG assay vs. untreated control samples. Results are the mean ± SEM of triple biological replicates, three technical replicates per biological replicate. Inhibitors are color coded based on their binding mode. (E) FLX1 cells treated with dose curves of multiple PIM inhibitors as in B. (F) Effect of 24 hr treatment with 5 μM of different PIM inhibitors ±4 hr treatment with 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX). (G) Immunoblot showing the change of PKA activity, as indicated by phospho-PKA substrate, and c-MYC and n-MYC levels in Colo741 and FLX1 cells after treatment with DMSO, 1 μM CD532, MLN8237, CX6258, or combination of 1 μM MLN8237 and 1 μM CX6258 for 24 hr.

Figure 5—source data 1. Tables for Figure 5A.
Figure 5—source data 2. Tables for Figure 5B.
Figure 5—source data 3. Tables for Figure 5C.
Figure 5—source data 4. Tables for Figure 5D.
Figure 5—source data 5. Tables for Figure 5E.
Figure 5—source data 6. Images for Figure 5F.

Figure 5.

Figure 5—figure supplement 1. Signaling effects on c-MYC in PKA-driven cells.

Figure 5—figure supplement 1.

(A) Colo741 cells treated with dose curves of multiple AURKA inhibitors for 72 hr. Relative cell viability was measured by CTG assay vs. untreated control samples. Results are the mean ± SEM of three biological replicates and three technical replicates per biological replicate. Inhibitors are color coded based on their binding mode. (B) Immunoblots showing the levels of pAURKA T288 in FLX1 cells either spontaneously cycling or synchronized overnight with nocodazole and treated with 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) for 4 hr. Total AURKA was not clearly detected so is not included. (C) Immunoblots showing the expression of c-MYC FLX1 cells after treating with 1 μM of the GSK3A/B inhibitor CHIR99021 for 24 hr and/or 50 μM FSK/IBMX for 2 hr.
Figure 5—figure supplement 1—source data 1. Tables for Figure 5—figure supplement 1A.
Figure 5—figure supplement 1—source data 2. Images for Figure 5—figure supplement 1B.
Figure 5—figure supplement 1—source data 3. Images for Figure 5—figure supplement 1C.
Figure 5—figure supplement 2. Proteasome-independent PKA effects on c-MYC, (A) immunoblots showing the change of c-MYC protein in FLX1 cells after treatment with 50 μM forskolin (FSK)/3-isobutyl-1-methylxanthine (IBMX) and/or 20 μM MG132 for 2 hr.

Figure 5—figure supplement 2.

(B) Immunoblots showing the change of c-MYC level in engineered FLX1 cells with doxycycline (dox)-inducible 3xFLAG- PRKAR1AG325D after dox for 24 hr and/or 20 μM MG132 for 2 hr. (C) Li-Cor western blot analysis showing the change of c-MYC level in FLX1 during a cycloheximide chase after stimulation with 50 μM FSK/IBMX for 2 hr and 30 min. Degradation curve and half-life of c-MYC as quantified by Li-Cor from individual samples shown below, T1/2 calculated using nonlinear regression in GraphPad Prism.
Figure 5—figure supplement 2—source data 1. Images for Figure 5—figure supplement 2A.
Figure 5—figure supplement 2—source data 2. Images for Figure 5—figure supplement 2B.
Figure 5—figure supplement 2—source data 3. Images and tables for Figure 5—figure supplement 2C.