Fig. 6: ERO1A inhibition triggers cytotoxicity and restrains VEGF in an ERO1A-dependent manner in TNBC cells. | Cell Death & Disease

Fig. 6: ERO1A inhibition triggers cytotoxicity and restrains VEGF in an ERO1A-dependent manner in TNBC cells.

From: Small molecule-mediated inhibition of the oxidoreductase ERO1A restrains aggressive breast cancer by impairing VEGF and PD-L1 in the tumor microenvironment

Fig. 6

A MTS assay of MDAMB231 cells treated with different inhibitors (I). The value of the cells challenged with the vehicle alone was arbitrarily set to 100 and the value of the treated cells calculated as percentages (N = 5, mean ± SEM, Two-way ANOVA). B EN460 and I2 intracellular signal revealed by MALDI imaging in MDAMB231 cells from EN460 and I2-treated MDAMB231 cells, compared with the levels of the standard EN460 and I2. Left-over reports for the signal of the residual compound in the media, i.e., that was not taken up (N = 4). C Non-reducing Immunoblot of endogenous ERO1 in lysates of vehicle-treated MDAMB231 cells or exposed to DTT, EN460 and the different inhibitors (I). ERO1 red. stands for ERO1 reduced, ERO1 ox. for oxidized and * indicates a background band. Representative experiment reproduced twice. On the right, bar graph representing reduced levels of ERO1 on total ERO1 (oxidized + reduced), that was arbitrarily set to 1 for the DTT-treated cells (mean ± SEM, One-Way ANOVA). D MTS assay of MDAMB231 cells and ERO1A KO MDAMB231 cells treated with EN460 and I2 as indicated (N = 5, mean ± SEM, Two-way ANOVA). E Analysis of apoptotic cells. WT and ERO1 KO MDAMB231 were treated with EN460 or I2 for 6 (T1) and 14 hours (T2) (shown in the picture) and later analyzed for Annexin V and Propidium Iodide staining by flow cytometry. Based on the positivity to the stainings, cells were divided as live, necrotic, early and late apoptotic. Below, bar graph indicating the percentages of live, necrotic, early and late apoptotic and the significantly higher level of late apoptotic cells in EN460-treated cells (N = 3, t-test). F Dot plots representing the migrated MDAMB231 subjected to a Boyden Chamber motility assay (N = 3, mean ± SEM, One-way ANOVA). G Bar Graph representing VEGF secreted from MDAMB231cells by VEGF ELISA (N = 4, mean ± SEM, One-way ANOVA). H MTS assay of E0771 cells treated with EN460 and I2 (N = 5, mean ± SEM, Two-way ANOVA). I Dot plots representing migrated E0771 cells subjected to a Boyden chamber motility assay (N = 3, mean ± SEM, One-way ANOVA). L Bar graph representing VEGF secreted from E0771 cells by VEGF ELISA (N = 4, mean ± SEM, One-way ANOVA).

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