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. 2022 Sep 2;13:930614. doi: 10.3389/fphar.2022.930614

FIGURE 2.

FIGURE 2

Ferroptosis induced by Ferric ammonium citrate (FAC) could aggravate fibrosis. (A–B) Visualization of HEcESCs viability in the different dose of FAC treated groups (A) over time and CCK8 assay (B) in different treated groups over time. (C–D) Visualization of HEcESCs viability in the different dose of erastin treated groups (C) over time and CCK8 assay (D) in different treated groups over time. With the increasing in drug concentration, all cells in the erastin group tended to be sensitive to ferroptosis, but the cells in the FAC group didn’t exist the same phenomenon after the FAC reached 50 mg/L. (E–F) Visualization of HEcESCs viability in FAC and ferroptosis inhibitor Fer-1 (E) and CCK8 assay (F) in different treated groups over time. (G–H) Visualization of HEcESCs viability in erastin and ferroptosis inhibitor Fer-1 (G) and CCK8 assay (H) in different treated groups over time.The induction of both reagents can be blocked by ferroptosis inhibitor Fer-1. Scar bars: 50 μm. (I) The ROS level of HEcESCs treated with FAC and/or Fer-1. (J) The MDA level of HEcESCs treated with FAC and/or Fer-1. (K) The ROS level of HEcESCs treated with Erastin and/or Fer-1. (L) The MDA level of HEcESCs treated with Erastin and/or Fer-1. (M–N) Typical mitochondrial picture of ferroptosis under a transmission electron microscope in different treated groups (M) and statistical results of typical mitochondrial ratio (N). Only a part of cell mitochondria changed in the FAC-treated group. Scale bars: 500 nm. (O–P). Representative images (O) and quantitative analyses (P) of the fibrosis-related protein Collagen1 and a-SMA expression levels of HEcESCs in FAC-treated groups. (Q–R). Representative images (Q) and quantitative analyses (R) of the fibrosis-related protein Collagen1 and a-SMA expression levels of HEcESCs in erastin-treated groups. FAC treatment promoted fibrosis-related protein expressions, while erastin exerted opposite effects. Summary data are presented as the mean ± SEM. Significance in (F,H–L,N) was calculated using a one-way analysis of variance; Significance in P and R was calculated using a two-way analysis of variance; *p < 0.05, **p < 0.01, ***p < 0.001.