Fig 2: Biophysical and structural characterization of VH298, a new potent VHL inhibitor.(a) Chemical structure of VH032 and VH298 inhibitors. (b) Crystal structure of VBC in complex with VH298 (orange carbons, PDB code 5LLI). VHL is shown as a purple surface and the VHL residues forming the binding pocket as grey stick representations. The bound ligand is shown as sticks representation with orange carbons, nitrogen atoms in blue, oxygen in red and sulfur in dark yellow. The inset panel shows the Fo-Fc omit map contoured at 3 s around the ligand. (c) ITC titrations of 300 µM VH298 (blue) and 300 µM cisVH298 (green) into 30 µM VBC protein. (d) Competitive fluorescence polarization binding assays of VH298 and VH032 displacing a 20-mer FAM-labelled HIF-1a peptide binding to VBC (Kd=3 nM), confirming the trend in relative potency observed by ITC (see full data in Table 1). (e) SPR sensogram of VH298 binding into surface-immobilized VBC. Used ligand concentrations are reported in the inset legend. Cellular thermal shift assays to monitor cellular target engagement of (f) VHL and (g) PHD2. VH298 and FG-4592 (100 µM) were incubated in HeLa cell lysates for 15–30 min. Representative western blots are shown. Data are presented as means±s.e.m. from three independent experiments.

Fig 3: LIGHT, a non-canonical NF-?B inducer, induces HIF expression and activity. (A) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Whole cell lysates were prepared and analysed by western blot for the indicated non-canonical NF-?B pathway regulators, subunits and target genes. ß-Actin was used as loading control. (B) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior mRNA extraction and RT-qPCR analysis for RANTES transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated sample. The graphs depict mean and SEM determined from at least three independent experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, *** p = 0.001. (C) HeLa cells, stably transfected with HRE luciferase reporter, were treated with 100 ng/mL LIGHT for 8 and 24 h prior to luciferase measurements. All of the values were normalised to the untreated sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. One way Anova analysis was performed and significance determined as follows: ns = not significant, *** p = 0.001. (D) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior collection of whole cell lysates and western blot analysis for the depicted proteins. ß-Actin was used as loading control. (E) HeLa cells were treated with 10 ng/mL TNF-a and 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for the depicted proteins. ß-Actin was used as loading control. (F) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for a subset of HIF-a specific targets. ß-Actin was used as loading control. (G) HeLa cells and (H) A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h, prior mRNA extraction and RT-qPCR analysis for PHD2 gene transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated samples. The graph depicts mean and SEM determined from at least three independent biological experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, * p = 0.05, ** p = 0.01, *** p = 0.001. (I) Analysis of canonical NF-?B signalling following NIK expression vectors for 48 h prior to luciferase measurements. All the values were normalised to the control sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. Student t-test analysis was performed and significance determined as follows: ns = not significant, ** p = 0.01. (J) HeLa cells were transfected with control and NIK expression vectors for 48 h prior to cell lysis. Western blot analysis was performed using the indicating antibodies. ß-Actin was used as loading control.