Fig. 3: Effects of ERO1A inhibition on VEGF secretion and ERO1 redox state in vivo. | Cell Death & Disease

Fig. 3: Effects of ERO1A inhibition on VEGF secretion and ERO1 redox state in vivo.

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

Fig. 3

A FLAG Immunoblot on conditioned media of HeLa cells transfected with FLAG-VEGF121 and treated with vehicle or inhibitors at 20 μM. Below is a bar graph representing the inhibition of VEGF secretion mediated by the different compounds when compared to the control; the ratio between the VEGF secreted from CTRL and the benchmark EN460 was arbitrarily set to 1 and calculated relatively to 1 for all the other compounds (N = 2, mean ± SD, One-Way ANOVA). B FLAG Immunoblot on conditioned media of HeLa cells transfected with FLAG-VEGF121 and treated with vehicle or inhibitors at 20 μM. Below, sensorgrams (time course of SPR signal in Resonance Units, RU) of FLAG-VEGF121 signal obtained by flowing the conditioned media over the immobilized FLAG M2 antibody (10 microgram/mL); these sensorgrams show the specific binding, already subtracted of the signal obtained on the reference surface with no antibody immobilized. C Non-reducing Immunoblot of endogenous ERO1A (henceforth, ERO1) in lysates of vehicle-treated HeLa cells or exposed to DTT, EN460 and the different inhibitors (I), as indicated. ERO1 red. stands for ERO1 reduced, ERO1 ox. for oxidized and * indicates a background band. ERO1 KO HeLa cells were loaded to indicate the background band. Ponceau stain indicated the protein loading control. The experiment was reproduced twice. Below is a bar graph representing reduced levels of ERO1A on total ERO1A (oxidized +reduced), which was arbitrarily set to 1 for the DTT-treated cells (mean ± SEM, One-Way ANOVA). D Non-reducing Immunoblot of endogenous ERO1A in lysates of vehicle-treated HeLa cells or exposed to DTT, and the indicated concentrations of EN460, I2 and I3. Representative experiment reproduced twice. Below is a bar graph representing reduced levels of ERO1A on total ERO1A (oxidized + reduced), which was arbitrarily set to 1 for the DTT-treated cells (mean ± SEM, One-Way ANOVA). Ponceau stain indicated the protein loading control.

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