Fig 2: PFKFB4 hinders the ubiquitylation of HIF-1α by binding to the E3 ubiquitin ligase FBXO28.A FBXO28-Halo and HIF-1α were overexpressed in HEK293T cells, and an FBXO28 pulldown was performed using Halo resin. FBXO28 and associated proteins were eluted by TEV-cleavage. Immunoblot was performed with FBXO28 and HIF-1α antibodies. B Upper panel: HIF-1α IP from lysate of NCH421k cells stably expressing HA-ubiquitin, in the presence of the proteasome inhibitor MG132 (500 nM, 6 hours) and with and without FBXO28 silencing (3 days). The immunoblot (IB) shows HA-ubiquitin and HIF-1α levels. The bracket indicate poly-ubiquitinated HIF-1α and the arrow shows the expected migration of unmodified HIF-1α (also applies to 5D and 5E). Lower panel: HIF-1α and FBXO28 protein levels in the input lysates used in the IP above. β-actin is displayed as a loading control in all panels. C Scheme of the HIF-1α protein domains including the ubiquitinylated lysines which were mutated to alanine (K532, K538, K547) (Adapted from ref. [54]). D Upper panel: HIF-1α IP from lysate of NCH421k cells stably expressing HA-ubiquitin and silenced (3 days) for endogenous HIF1A, in the presence of MG132 (500 nM, 6 hours), with and without PFKFB4 silencing and overexpression of wild-type or lysine-mutated HIF-1α. The IB shows HA-ubiquitin and HIF-1α levels. Lower panel: PFKFB4 protein levels in the input lysates used in the IP above. E Upper panel: HIF-1α immunoprecipitated (IP) from lysate of HEK293T cells in the absence and presence of MG132 (500 nM, 6 hours) and PFKFB4 overexpression (3 days). HIF-1α was overexpressed in all conditions. The IB shows ubiquitin (Ub) and HIF-1α levels. Lower panel: PFKFB4 protein levels in the input lysates used for the IP above.

Fig 3: FBXO28 is a novel interaction partner of PFKFB4.A Interaction partners of PFKFB4 in NCH421k GSCs as determined by mass spectrometry of immunoprecipitated PFKFB4 from NCH421k lysate. Proteins were only considered as identified if more than one unique peptide had an individual ion score exceeding the MASCOT identity threshold and if three or more unique peptide sequences were identified in all replicates and not in the controls. The experiment was performed in biological triplicate, with data from one biological replicate displayed here; further replicates shown in Table S2. B Immunoprecipitation of FBXO28 or PFKFB4 from NCH421k lysate, with IgG as a control. PFKFB4 and FBXO28 are shown on the immunoblot. C Immunofluorescent staining of PFKFB4 and FBXO28 in NCH421k GSCs. Scale bar = 10 µm. D Split NanoLuc® Luciferase assay to determine the best combination of split fragments with the large (LgBiT) or small (SmBiT) BiT cloned onto the C- or N-terminal of either PFKFB4 or FBXO28. Data are normalized to the negative control, which is a combination of the halo tag labelled with the small BiT with either PFKFB4 or FBXO28 tagged with the large BiT. A luminescent signal that is at least 10 times the negative control was considered as positive. Data are represented as mean of biological triplicates ± SD. E Verification of the specificity of the interaction of SmBiT-tagged FBXO28 (N-term) to LgBiT-tagged PFKFB4 (C-term) using the Split NanoLuc® Luciferase system. Increasing amounts of untagged PFKFB4-overexpressing pLVX plasmid were added to displace the tagged protein, with empty pLVX plasmid as a negative control. Data are depicted as percent inhibition compared with the pLVX only control. (n = 3, mean ± SD, two sided t-test, *p value < 0.05, ***p value< 0.001). F ADP-Glo™ kinase assay measuring the percentage ATP to ADP conversion when increasing amounts of recombinant PFKFB4 was incubated with fructose-6-phophate, recombinant FBXO28 or no substrate. G FBXO28 mRNA in normal brain (n = 8) compared with GBM patients (n = 159). Data are represented as mean ± the minimum and maximum. The whiskers are drawn down to the 25th percentile and up to the 75th percentiles. H Survival of GBM patients with high (top tertile, n = 53; red) and low (bottom tertile, n = 53; blue) FBXO28 expression.