Figure 2.

CREB knockdown inhibits cell growth via stimulating apoptosis- and ferroptosis-like cell death concurrently. (A) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) (scale bar, 50 µm). (B) Cell viability, 3D cell with SYTOX Green staining and MDA were measured in cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB (scale bar, 50 µm). (C) Fe²⁺ and lipid ROS generation were measured in CREB-knockdown cells with additional treatment using Nec-1 (20 µM), ZVAD-FMK (20 µM) or Fer-1 (1 µM) or cells treated with erastin (10 µM) or apoptozole (10 µM) with or without ectopically expressed CREB. The data are presented as the mean ± SD from three biological replicates. *P<0.05, **P<0.01 indicates statistical significance. Data from A, B and C were analyzed using a one-way ANOVA followed by Bonferroni's post hoc test. CREB, cAMP response element-binding protein; MDA, malondialdehyde; NEC-1, necrostatin-1; Fer-1, ferrostatin-1; ROS, reactive oxygen species; sh, short hairpin.