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

Figure 3. PKA activity correlates with c-MYC and n-MYC protein levels.

(A) Immunoblots showing the change of PKA activity, as indicated by phospho-PKA substrate, and c-MYC and n-MYC expression in Colo741 and FLX1 cells after treatment with 50 μM forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) for 30 min, 2 hr, or 4 hr. n-MYC was not detected in Colo741 so is not shown. (B) Impact of 0–4 hr treatment with FSK/IBMX on MYC mRNA levels in Colo741 and FLX1; ± SD. Technical replicates from a representative experiment shown. (C) Immunoblots showing the change of PKA activity and c-MYC and n-MYC levels in engineered FLX1 cells with doxycycline (dox)-inducible 3xFLAG- PRKAR1AG325D with or without dox for 72 hr. (D) Immunoblots showing the basal level of PKA activity with phosphorylated PKA substrate and c-MYC expression in AML12 wild type (WT; left) and AML12DNAJ-PKAc cells (right). (E) Effect of 4 hr treatment with PKA-inhibiting tool compound H89 over a dose range from 1.25 to 20 μM on PKA substrate phosphorylation and c-MYC level. (F) Immunoblot showing the presence of DNAJ-PKAc and different level of c-MYC and n-MYC in fibrolamellar carcinoma (FLC) tumor samples (FLC) vs adjacent liver (N) from four FLC patients. (G) Summary gene set enrichment analysis (GSEA) of PRKACA amplified/mutant and PRKAR1A inactivated adrenocortical carcinoma or ovarian serous carcinoma vs. WT from TCGA. All significant ‘Hallmark’ gene sets are shown.

Figure 3—source data 1. Images for Figure 3A.
Figure 3—source data 2. Tables for Figure 3B.
Figure 3—source data 3. Images for Figure 3C.
Figure 3—source data 4. Images for Figure 3D.
Figure 3—source data 5. Images for Figure 3E.
Figure 3—source data 6. Images for Figure 3F.
Figure 3—source data 7. Tables for Figure 3G.

Figure 3.

Figure 3—figure supplement 1. Effects of PKA inhibition on FLX1 cell proliferation.

Figure 3—figure supplement 1.

(A) Impact of individual PRKACA-targeting siRNAs on relative cell confluence in FLX1. Confirmation of knockdown shown on right. Experiment was done in duplicate, the representative results shown with mean ± SD, n=6 wells for each siRNA. (B) Relative cell confluence of FLX1 cells with doxycycline (dox)-inducible 3xFLAG- PRKAR1AG325D in 96 well plates after treatment with or without 1 μg/ml dox. Experiment was done in duplicate, the representative results shown with mean ± SD, n=15 wells for each condition.
Figure 3—figure supplement 1—source data 1. Tables for Figure 3—figure supplement 1A.
Figure 3—figure supplement 1—source data 2. Tables for Figure 3—figure supplement 1B.