Fig. 1: Cytotoxic therapy-induced release of PGE₂ is exclusive to COX-2 expressing tumor cells and requires transcriptional upregulation of Ptgs2 via endogenous promoter activity.

a 4T1 tumor cells were treated with cisplatin (50 µM) or 5-FU (100 µM) and PGE₂ release into the cell culture medium was measured over time. Mean ±SEM of triplicate wells, representative plot shown of n = 2 independent experiments. b COX-2 protein levels in 4T1 cells following treatment with cisplatin (50 µM) or 5-FU (100 µM). β-Tubulin ran as a loading control on the same membrane. Data representative of n = 2 independent experiments. c PGE₂ release from 4T1 cells treated with cisplatin (20 µM) or 5-FU (100 µM) for 24 h in the presence or absence of the selective COX-2 inhibitor celecoxib (CXB, 5 µM). Mean ±SEM of n = 2 independent experiments with triplicate wells. d PGE₂ release (top panel) and Ptgs2 expression relative to Hprt (bottom panel) in multiple murine tumor cell lines following 24 h 5-FU (100 µM) treatment. ND = not detected. Mean ±SEM of n = 3 (CT26, 4T1, TB32047), 2 (5555, 3LL, 4434, E0771, YUMM1.1) independent experiments with duplicate wells, or 1 (B16F10, Renca) with duplicate wells. e Log10 normalized values of COX-2 transcript expression relative to Hprt in tumor cell lines with or without 24 h 5-FU (100 µM) treatment. Spearman’s rank correlation coefficient and p value are shown. Murine tumor lines are from multiple origins, including: melanoma (5555, 4434, YUMM1.1, B16F10), colorectal (CT26, MC38), breast (4T1, E0771), lung (3LL), renal (Renca) and pancreatic (TB32043, TB32047, TB32908). n = 1 (B16F10, Renca, TB32043, MC38), 2 (5555, 3LL, 4434, E0771, YUMM1.1, TB32908) or 3 (CT26, 4T1, TB32047) independent experiments with duplicate wells. f Histograms showing the expression of Ptgs2 in COX-2^KO 4T1 cells and in DMSO- or 5-FU- (100 µM) treated COX-2^WT 4T1 cells at 24 h determined using PrimeFlow by gating on live, Gapdh⁺ cells. Geometric mean fluorescence intensity per group is shown. g Schematic depicting promoter control of Ptgs2 expression in COX-2^WT, COX-2^KO and COX-2^REST cells. h COX-2 protein levels in 4T1 cells following 48 h treatment with 5-FU (100 µM). β-Tubulin ran as a loading control on the same membrane. Data representative of n = 2 independent experiments. i PGE₂ release from 4T1 and CT26 cells treated with 5-FU (100 µM) for 24 h. Mean ±SEM of n = 3 independent experiments with duplicate wells. ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by one-way ANOVA with Tukey’s multiple comparisons test (a, i) or unpaired two-tailed t-test (d). Source data and exact p values are provided as a Source Data file.