Fig 2: miR-10b inhibition reduces FLC cell metabolic activity and proliferation.(A) Protein expression of DNAJB1-PRKACA (DP) is detected with a protein kinase A catalytic a subunit (PKA) antibody. WT PKAc, DP major, and DP minor are identified. Lane 1, FLC-H cell line; lane 2, nonmalignant liver; lane 3, FLC patient sample; lane 4, FLC PDX sample. Vinculin expression for loading control is shown in the lower panel. Uncropped immunoblot shown in Supplemental Figure 4, A and B. (B) qPCR showing the RQV of miR-10b in FLC-H cells 6 days after 500 nM treatment with miR-10b LNA or scrambled sequence compared with mock (n = 4 each condition). (C) Luciferase signal (RLU) in FLC-H cells after 6 days of 500 nM miR-10b LNA treatment is shown as RQV compared with the scrambled negative control (6 trials, n = 6 each condition). (D) qPCR showing the RQV of FANCC, KLF11, SEC14L2, SIRT5, SUN2, and TRIM35 in FLC-H cells after 6 days of 500 nM miR-10b LNA treatment compared with the negative control (n = 5–7 trials with 3 replicates for each condition, SIRT5 n = 2 trials). (E) Soft agar colony formation of FLC-H cells 35 days after 500 nM miR-10b LNA compared with the negative control shown as RQV. (F) Representative nitro blue tetrazolium–stained images shown (2 trials, n = 8 each condition). (G) EdU incorporation in FLC-H cells 6 days after 500 nM treatment with miR-10b LNA compared with the negative control shown as RQV (2 trials, n = 6 each condition). (H) Representative DAPI- and EdU-stained images show total and proliferative cells, respectively. Scale bars: 100 µm. In all assays, each dot represents the average signal across technical replicates for a single biological replicate. P values are calculated by 2-tailed Student’s t test. P values reported in B and D were adjusted for multiple testing correction post hoc by the Benjamini-Hochberg method.

Fig 3: PKA activates DLK via phosphorylation of its activation loop.(A-B) Changes in endogenous DLK abundance in response to treatment with forskolin (30 µM) (A) or the PKA inhibitor H-89 (5 µM) (B) for 6 hr in cultured rat embryonic cortical neurons. Quantification shows relative DLK/Tubulin levels in western blots. (C) Alignment of activation loop sequences in different species. (D) The anti-pDLKS302 antibodies recognize transfected Flag-DLKWT, but not activation loop mutation Flag-DLKS301A,S305A. Both proteins were transiently expressed in HEK293 cells. Western blots were probed with anti-pDLKS302 antibody, anti-Flag antibody to detect the total DLK expression levels, and anti-Tubulin (which remains similar in all manipulations) for normalization. (E) PKACA stimulates phosphorylation of DLK S302 in HEK293 cells. HEK293 cells were co-transfected with Flag-tagged DLKWT and an empty control plasmid or PKACA. Cell lysates were probed with anti-DLK antibody, anti-pDLKS302 antibody, anti-PKA C antibody and anti-tubulin antibody. (F) PKACA stimulates an increase in DLK molecular weight. Flag-DLK protein was immunoprecipitated from HEK293 cells co-transfected with DLK and either Flag-tagged DLKWT and an empty control plasmid or PKACA. The immunoprecipitated Flag-DLK was then incubated with either glycerol (control) or lambda protein phosphatase (?PP). PKACA induced a upward shift in DLK molecular weight, which was lost upon phosphatase treatment. (G) The activation loop is required for axonal regeneration in Drosophila neurons. Single axons in Drosophila third instar larva are labeled by mCD8RFP using eve-Gal4 driver. 24 hr after injury, these neurons in animals heterozygyous for wnd (wnd3/+) show robust axonal sprouting. However, sprouting fails to occur in wnd3/wnd3 animals. Expression of UAS-Wnd (WT) can restore axonal regeneration in wnd mutant background (UAS-Wnd, wnd3; wnd3,eve-Gal4, UAS-mCD8RFP). However, expression of activation loop mutant UAS-WndS301A,S305A failed to rescue the sprouting defect in wnd mutant animals (UAS-WndS301A,S305A, wnd3; wnd3, eve-Gal4,m12-mCD8RFP). Quantification of the volume of axonal membrane within 100 µm of the distal ending of the proximal stump. n> 50 axons for each genotype. Data are presented as mean ± SEM for 3 independent experiments; ***p<0.001; scale bar, 100 µm.DOI: http://dx.doi.org/10.7554/eLife.14048.005

Fig 4: PKA modulates the levels of Wnd protein and downstream signaling in Drosophila neurons.(A) The puc-lacZ transcriptional reporter for Wnd/JNK signaling indicates that activated PKA stimulates Wnd signaling. A pan-neuronal driver (BG380-Gal4) is used to express UAS-dnc-RNAi, UAS-PKACA or UAS-PKACA together with UAS-wnd-RNAi or a control-RNAi. Example images are shown of cell bodies in the dorsal midline of the ventral nerve cord; (all but two of these neurons are motoneurons). Quantification (described in methods) was carried out for 10 animals per genotype. (B) Endogenous Wnd protein levels are increased in PKACA expressing neurons. Ventral nerve cords were dissected from third instar larvae (BG380-Gal4 [WT control] and BG380-Gal4; UAS-PKACA/+) and processed for Western blotting with anti-Wnd and anti-tubulin antibodies. The quantification shows Wnd/tubulin ratios (normalized to WT control) averaged from 3 independent experiments (25 nerve cords per experiment). (C) PKA increases DLK levels via a posttranscriptional mechanism. GFP-tagged kinase dead Wnd (GFP-WndKD) was ectopically expressed using m12-Gal4 driver (WT control) or co-expressed with UAS-PKACA and imaged directly after fixation. Example images and quantification of GFP-WndKD intensity in cell bodies and axons within segmental nerves. n>10 animals for each condition. (D) PKA-C1 is required for induction of Wnd protein after axonal injury. Example images and quantification of GFP-WndKD in nerve cords and segmental nerves before and after (8 hr) injury. UAS-GFP-WndKD was expressed in motoneurons by OK6-Gal4. WT or together with UAS-pka-c1-RNAi or UAS-moody-RNAi (control) and imaged similarly to Figure 2C. The quantification method for GFP intensity is described in materials and methods. n>10 animals for each condition. All data are represented as mean ± SEM; ***p<0.001, **p<0.01, *p<0.05, ‘n.s.’ indicates non-significant; scale bars, 10 µm.DOI: http://dx.doi.org/10.7554/eLife.14048.004

Fig 5: pVHL protects TFAM from LONP1 degradation.a,b, Immunoblot analysis of primary Egln3+/+ and Egln3-/- MEFs (a) and 786-O cells (b) treated with 1 µM LONP1 inhibitor bortezomib (BTZ) for 16 h. c, Immunoblot analysis of HEK293 cells transfected with transposon vectors pB-TRE-TFAM-WT-Luc2, pB-TRE-TFAM-mut-Luc2 and transposase vector pCAG-hyPBase. d, Immunoblot analysis of PKA activity assay using biotinylated TFAM peptides with double hydroxyl-prolines 53 and 66. e,f, Immunoblot analysis of 786-O cells treated with 20 µM PKA activator forskolin (24 h) (e) or 5 µM PKA inhibitor H89 (24 h) (f). g, Immunoblot analysis of TFAM degradation assay by LONP1 using purified His-TFAM, GST-VHL, LONP1 and IVT-synthesized Flag-EGLN3 WT or Flag-EGLN3 catalytic mutant. In a–g, n = 3 biological independent experiments. h,i, Kaplan–Meier overall survival curve for individuals with high (blue) and low (red) expression of PKA catalytic subunit (PRKACA) (h) and LONP1 (i) using the Kidney Renal Clear Cell Carcinoma dataset from The Cancer Genome Atlas which contains 533 tumour samples (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi; minimal patient group size of 50 in the iterations). The overall survival probability was estimated with the KaplanScanner tool, using a Bonferroni-corrected logrank test between the two groups of patients. The graph depicts the best P value corrected for multiple testing (Bonferroni method). j, Crystal violet staining of 786-O cells pretreated with BTZ (10 nM) for 24 h and then treated for 48 h with sorafenib (Sora; 20 µM), BTZ (10 nM) or a combination (combo) of these two drugs as indicated. k, Cell apoptosis rate was detected by Annexin V-FITC/propidium iodide (PI) staining using flow cytometry. Data are presented as mean values ± s.d. Two-way ANOVA Tukey’s multiple-comparison test. ****P < 0.0001. n = 3 biological independent experiments. l, Female athymic NCrnu/nu mice were implanted subcutaneously with 786-O cells. Sorafenib (n = 4) or vehicle control (DMSO, n = 5) was administered orally, once a day at the dose of 15 mg per kg body weight. BTZ (n = 5) was administered by intraperitoneal injection, twice per week at a dose of 1 mg per kg body weight. Combined treatment: 1 mg per kg body weight BTZ + 15 mg per kg body weight sorafenib (n = 5). Mean (±s.e.m.) tumour volume data are shown. *P < 0.01, **P < 0.01, ***P < 0.001. m, Representative images of tumours after dissection and quantification of tumour weight of each treatment group. n, Representative H&E (scale bar indicates 50 µm, ×100), TFAM and MTCO2 immunofluorescence stainings (scale bar indicates 50 µm, ×400) of tumour tissues including quantification. NS, not significant.Source data