Fig 2: EP1 activation protects cardiomyocytes from DOX-induced ferroptosis by mobilizing Ca2+ to activate PKC/Nrf2 signalingA. Effect of FR900359 (1 μM) or PTX (10 μM) on the cell viability of H9C2 cells treated with 17-PT-PGE2 in the presence of erastin. *P < 0.05 as indicated; n = 8.B. Representative fluorescence images showing the intracellular Ca2+ spark in neonatal rat cardiomyocytes treated with 17-PT-PGE2. Scale bars: 10 μmC. The intracellular Ca2+ flux in neonatal rat cardiomyocytes treated with 17-PT-PGE2. The arrow indicates the injection of 17-PT-PGE2.D. PKC kinase activity in H9C2 cells after 17-PT-PGE2 treatment. *P < 0.05 as indicated; n = 5–6.E, F. The effect of U73122 (5 μM) on the cell viability (E) and intracellular ROS production (F) in H9C2 cells treated with 17-PT-PGE2 in the presence of erastin. *P < 0.05 as indicated; E, n = 8; F, n = 5–6.G. Western blot analysis of effect of U73122 on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE2 in the presence of erastin.H. Quantification of relative protein expression levels in (G). *P < 0.05 as indicated; n = 4.I. Western blot analysis of effect of U73122 on the expression of p-Nrf2, Nrf2, GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE2 in the presence of DOX.J. Quantification of relative protein expression levels in (I). *P < 0.05 as indicated; n = 4.K. Schematic illustration of EP1-mediated myocardial protection in DOX-induced cardiomyocytes ferroptosis through Ca2+/PKC/Nrf2 signaling pathway.

Fig 3: EP1 activation reduces DOX-induced ferroptosis in cardiomyocytes through evoking Nrf2 activityA. Effect of GPX4 inhibitor RSL3 on erastin-induced ferroptosis of H9C2 cells after17-PT-PGE2 treatment. *P < 0.05 vs indicated; n = 8.B. Western blot analysis of p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE2 or SC-51322 in the presence of erastin.C. Quantification of relative protein expression levels in (B). *P < 0.05 as indicated; n = 4.D. Western blot analysis of p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE2 or SC-51322 in the presence of DOX.E. Quantification of relative protein expression levels in (D). *P < 0.05 as indicated; n = 4.F. Western blot analysis of nuclear p-Nrf2 and Nrf2 expression in H9C2 cells treated with17-PT-PGE2 or SC-51322 in the presence of erastin.G. Quantification of relative protein expression levels in (F). *P < 0.05 as indicated; n = 4.H. Western blot analysis of nuclear p-Nrf2 and Nrf2 expression in H9C2 cells treated with 17-PT-PGE2 or SC-51322 in the presence of DOX.I. Quantification of relative protein expression levels in (H). *P < 0.05 as indicated; n = 4.J, K. Effect of ML-385 (5 μM) on cell viability (J) and intracellular ROS production (K) in H9C2 cells treated with 17-PT-PGE2 in the presence of erastin. *P < 0.05 as indicated; (J), n = 8; (K), n = 6.L. Western blot analysis of effect of ML-385 on the expression of GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE2 in the presence of erastin.M. Quantification of relative protein expression levels in (L). *P < 0.05 as indicated; n = 4.N. Western blot analysis of effect of ML-385 on the expression of GPX4 and SLC7A11 in H9C2 cells treated with 17-PT-PGE2 in the presence of DOX.O. Quantification of relative protein expression levels in (N). *P < 0.05 as indicated; n = 4.

Fig 4: Expression of ferroptotic genes upon E2F1 overexpression.Adenovirus (100 MOI) expressing E2F1 or CMV control viruses were transduced for two days into (A) HT1080 or (B) NARF2 cells. Then, qPCR was performed to measure the mRNA levels of SLC7A11, NFE2L2, GPX4, ATF4, and CCNE1 as a reference of E2F1 target using SYBR green. (n=3 and * p < 0.05)

Fig 5: EP1 activation prevents DOX-induced ferroptosis in human cardiomyocytesA. Cell viability of AC16 cells treated with 17-PT-PGE2 (2.5 μM) or SC-51322 (10 μM) in the presence of erastin (5 μM). *P < 0.05 vs indicated; n = 8.B. Representative flow cytometric plots showing the intracellular ROS levels in AC16 cells treated with 17-PT-PGE2 or SC-51322 in the presence of erastin.C. Quantification of ROS production in (B). *P < 0.05 vs indicated; n = 5–8.D. Western blot analysis of p-Nrf2 and total Nrf2 expression in AC16 cells treated with 17-PT-PGE2 or SC-51322in the presence of erastin.E. Quantification of relative protein expression levels in (D). *P < 0.05 as indicated; n = 4.F. Cell viability of AC16 cells treated with 17-PT-PGE2 or SC-51322 in the presence of DOX. *P < 0.05 vs indicated; n = 8.G. Representative flow cytometric plots showing the intracellular ROS levels in AC16 cells treated with 17-PT-PGE2 or SC-51322 in the presence of DOX.H. Quantification of ROS production in (G). *P < 0.05 vs indicated; n = 6.I. Western blot analysis of p-Nrf2 and total Nrf2 expression in AC16 cells treated with 17-PT-PGE2 or SC-51322 in the presence of DOX.J. Quantification of relative protein expression levels in (I). *P < 0.05 as indicated; n = 4.