Fig. 1

Characterization of the molecular responses of H₂O₂-stimulated cells used in the discovery screening. a-b Time-course profile for H₂O₂-induce ERK 1/2 and Akt activation in HeLa cells. Cells were treated with H₂O₂ for the indicated periods, and the activation profiles of ERK1/2 (a), and Akt (b) were studied by western blot using phospho-specific and anti-total ERK1/2 and Akt antibodies. Anti-GAPDH antibody was used to check protein loading. For each pathway, it is shown a representative western blot (bottom panels) and the profile of activation (top panels) determined by the relative phosphorylation to control conditions (time 0 min). The line indicates the mean, and shadow indicates the SEM of four replicates. Red arrowheads indicate the time-point selected for the differential secretome analysis. c-f Biochemical evaluation of HeLa cells response to the proposed oxidative stress condition (1 mM H₂O₂ for 40 min). LDH release (c), cell toxicity (d), ATP levels (e), and reactive oxygen species (ROS) levels (f) were measured as an indication of the cell integrity, metabolic activity, and oxidative status, respectively. LDH release and cell toxicity (using the CellTox™ Green probe) were measured 24 h after the acute stimuli. LDH cytotoxicity (c) represents the relative levels of the released LDH over the total levels of LDH (dots), while the total levels of LDH (triangles) were used as an indication of changes in the number of cells between conditions. ATP and reactive oxygen species levels (measured using the CellROX™ Orange probe) were determined immediately after the 40 min stimulation (dots) and after 24 h (triangles). Bar and error bars correspond to the mean and interquartile range of four experiments, respectively. n.s. and * indicate a p ≥ 0.05 and p < 0.05, respectively, for statistically significant differences between control (Ctrl, in blue) and oxidative stress (Stress, in red) conditions using the Wilcoxon Signed Rank Test