Fig. 3: ERX-41 induces ER stress in TNBC.
a,b, Volcano plots showing relative effect after 4 h of treatment by 1 μM ERX-41 vehicle (Veh) in MDA-MB-231 (a) and BT-549 (b) (n = 2 replicates). c, Heatmap of top ER stress/UPR genes in TNBC cells after 2 h and 4 h of 1 μM ERX-41. Fold change values in heatmap were calculated by normalization of FPKM values to those for vehicle. d–f, Effect over time of 1 μM ERX-41 on mRNA levels of ER stress genes sXBP1 (d), DDIT3 (e) and HSPA5 (f) in MDA-MB-231, BT-549 and HMEC cells. Data presented as mean ± s.e.m.; n = 3 biologically independent samples. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Adjusted P values of last time points are shown. g,h, TEM of MDA-MB-231 cells showing effect of vehicle (g) and 1 μM ERX-41 (h) on subcellular structures at 4 h; ER denoted by yellow arrowheads. i–l, Airyscan imaging showing effect on SUM-159 cells stably expressing the ER membrane marker mCherry-RAMP4 before (i) and 2 h after 1 μM ERX-41 (j); right, zoomed-in images. Representative graph showing distribution of normalized tubule intensity (white lines in i,j) from vehicle and ERX-41-treated samples (k). Dilated ER is represented by two peaks, with signal between them indicating continuously dilated ER tubules. Histogram comparing distribution of ER tubule width (in μm) between untreated and treated samples (l). Data presented as mean ± s.d.; n = 102 measurements for vehicle, n = 104 measurements for ERX-41. Significance was determined by unpaired two-tailed Student’s t-test. m, Immunoblotting showing effect over time of 1 μM ERX-41 on UPR protein expression in SUM-159 cells. n, Immunoblot showing effect of 1 μM ERX-41 on IRE1-α expression in SUM-159 cells. Phos-tag gel (top) distinguishes between hypophosphorylated (0) and hyperphosphorylated (p) forms of IRE1-α. Total IRE1-α levels are shown (middle) with GAPDH (lower) control. o, RT–PCR showing effect over time of 1 μM ERX-41 on expression of unspliced (XBP1u) and spliced (XBP1s) XBP1 and GAPDH transcripts in SUM-159 cells. Effect of positive control (100 nM thapsigargin) is also shown. Experiments in g,h,j,l–o were repeated twice independently, with similar results. Numerical source data for d–f,k,I and uncropped blots for m–o are provided. bp, base pairs.
