Fig. 6: TWEAK/Fn14-activated TNBC SE drives NAMPT expression and TNBC progression.

Western blot analysis of NAMPT expression in (a) untreated and TWEAK-treated TNBC, HER2 and ER-positive breast cancer cell lines, and (b) TNBC patient tumours (T) and their matched normal samples (N). c NAMPT locus. Top: H3K27ac HiChIP heatmap of TWEAK-treated MDA-MB-231 cells. Middle: H3K27ac ChIP-seq of untreated and TWEAK-treated MDA-MB-231, Hs578T, SKBR3, MDA-MB-453, MCF7 and CAMA1 cells. Bottom: chromatin interactions in untreated and TWEAK-treated MDA-MB-231 cells, and merged H3K27ac ChIP-seq of Fn14 high TNBC tumours and their matched normal samples (2086, 8370, 2963, 6122, 8850 and 5929). d Western blot analysis of NAMPT and NFκB2 expression in Control and NAMPT enhancer #1, #2 and #3 deleted MDA-MB-231 cells, with and without TWEAK. e Transwell invasion assay performed in Control and NAMPT enhancer #3 deleted MDA-MB-231, cells with and without TWEAK. Scale bars: 400 µm. f Plot depicts the average number of invaded cells/frame (mean ± s.d), across four fields per replicate. g IVIS imaging of mice injected with control and NAMPT enhancer #3 deleted MDA-MB-231 cells overexpressing TWEAK and luciferase. Representative bioluminescent images were taken 1 and 8 weeks after injection (luminescent signal of Control mouse #2 at week 1 was detectable but very low). h Plot depicting the total flux (mean ± s.d) at the metastatic sites of each animal at week 8. (Representative image of Control Mouse #3 was taken at week 7 as the mouse died before week 8). The western blot samples derive from the same experiment and same gel for NAMPT and GAPDH in (a) and (b). The western blot samples derive from the same experiment but different gels for NFKB2 and GAPDH and another for NAMPT were processed in parallel in (d). In vivo assays represent n = 5 biological replicates, other assays represent n = 3 biological replicates. Experiments involving cell lines were performed 3 times independently, each time on different days. Two-sided t test was used for in vivo assay, two-sided two-way ANOVA was used for the other assays where *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file.