Figure 6.

DIPY suppresses HMOX1 through the deactivation of CREB1. (A) Schematic illustration for screening TFs for the human HMOX1 gene. (B) A549 and HUVEC cells were treated with RSL3, DIPY, and RSL3 with DIPY for 8 hours as shown in Figure 1. Cell lysates were collected and analyzed with the indicated antibodies (n = 3 per group). (C) hAOs were treated with LPS, DIPY, and LPS with DIPY as shown in Figure S3 (n = 5 per group). The lysates were subjected to immunoblotting with the indicated antibodies. (D-E) Western blot (D) and IHC assays (E) examined the expression of p-CREB1 in LPS-induced ALI mouse models with or without DIPY treatment as shown in Figure 2 (n = 6 per group). Scale bar, 50 μm. (F-K) CREB1 knockout A549 cell lines (sgCREB1) and the control cells (sgCtrl) were treated with RSL3 (5 μM) for 8 hours (n = 3 per group). Confirmation of CREB1 knockout via sequencing (F). Cell lysates were subjected to immunoblotting with the indicated antibodies (G). Cell viability assessment (H). Representative phase-contrast images of cells (I). Scale bar, 100 μm. L-ROS levels measurement (J). MDA contents and Fe²⁺ levels (K). (L-N) sgCREB1 A549 cells or the control cells were infected with lentiviruses carrying Lv-HMOX1 or Lv. The cells were treated with RSL3 (5 μM) for 8 hours (n = 3 per group). Cell lysates were subjected to immunoblotting with the indicated antibodies (L). L-ROS levels (M). MDA contents and Fe²⁺ levels (N). Results are shown as mean ± SD of 3-6 independent experiments. Statistical significance is indicated as **P < 0.01; ***P < 0.001; ****P < 0.0001.