Extended Data Fig. 3: ERX-41 induces ER stress in TNBC.
a-d: GSEA analysis of the RNA-sequencing data evaluating the treatment-enriched gene sets in three TNBC cell lines, MDA-MB-231 (a), BT-549 (b) and SUM-159 (c) following 4 h exposure to 1μM ERX-41. The top 10 gene sets (total ~15k C5 ontology gene sets) are shown for each cell line. ER- related gene sets are highlighted in blue. For all shown gene sets, the adjusted P value is <0.001 for all gene sets. GSEA showing correlation between ERX-41 treatment and induction of GO intrinsic apoptotic signaling pathway in response to ER stress in each of these three TNBC cell lines (d). e-g: Western blotting shows time course of effect of 1μM ERX-41 or 1μM ERX-11 on expression of UPR proteins in MDA-MB-231 (e), BT-549 (f) and HMEC cells (g). h: RT-PCR shows effect of 1μM ERX-41 on expression of unspliced (XBP1u) and spliced (XBP1s) XBP1 and GAPDH transcripts in MDA-MB-231, BT-549 and HMEC cells. i-k: Immunohistochemical evaluation of the effect of a single dose 10 mg/kg oral ERX-41 on the expression of ER stress markers in MDA-MB-231 TNBC tumor xenografts at 24 h after treatment (i), with quantitation of p-eIF2α (j) and p-PERK (k) in tumor tissue. Data are presented as mean ± SEM. n = 5 fields per group. Significance was determined by unpaired two tailed Student’s t-test l: Time course of effect of 1μM ERX-41 on global de novo protein synthesis in two TNBC (MDA-MB-231 and BT-549) and HMEC cells shown by western blots for puromycin labeled nascent proteins. The experiments in e, f, g, h and l were done twice independently with similar results. Numerical source data for a, b, c, j, k and uncropped images for e, f, g, h, i and l are provided.
