Extended Data Fig. 4: p95HER2 induces the expression of several immune-related cytokines independently of activating senescence.
(a) p95HER2 acutely induces IL-6 protein secretion. ELISAs were performed to measure IL-6 protein level in the media from PB2 cells expressing either p95HER2 or FL-HER2 under control of doxycycline (0.5 µg/mL for 48 h). Similarly, p95HER2 induces IL-6 secretion from MDA-MB-453 cells, which is abolished by neratinib (50 nM for 24 h). N = 3 independent biologic samples per condition. Data represent mean ± SEM. Statistical analyses by ordinary one-way ANOVA with Tukey’s correction for multiple comparisons. P values are indicated within the plots. (b) The same cytokine array as depicted in Fig. 4i, presented with different exposure levels. Cytokines that are induced by p95HER2 are annotated in red. (c) Short-term expression of p95HER2 fails to induce cellular senescence in both MCF-7 and PB2 cells. Cells were treated with doxycycline for 48 h to induce either FL-HER2 or p95HER2, then fixed and evaluated with cellular senescence assay kit (β-galactosidase). As a positive control, cells were treated with etoposide for 24 h and allowed to recover prior to assaying senescence. (d) Expression of p95HER2 fails to induce p21 expression in PB2 cells. Cells were treated with increasing doses of doxycycline for 48 h to induce either FL-HER2 or p95HER2, then harvested for Western analysis to measure expression of proteins as indicated. (e) Cellular senescence signatures are downregulated in FinHER specimens with a high p95HER2/HER2 ratio. GSVA enrichment score calculations for cellular senescence signatures in HER2+ tumors within the FinHER dataset (median-split categorical analysis. p95HER2/HER2 ratio: low n = 90, high n = 90 for each plot). Boxes span the upper to lower quartiles with a median center line. Whiskers extend from the minimum to the maximum values in the dataset. Statistical analyses by unpaired, two-tailed Student’s t test. P values are indicated within the plots.
