Fig 1: Gomisin B promoted ferroptosis in NB cells. Different concentrations of Gomisin B were used to treat NB cell lines, SH-SY5Y (0, 25, 50, 100, 200 μM), SK-N-SH (0, 25, 50, 100, 200 μM), IMR-32 (0, 12.5, 25, 50, 100 μM). (A) CCK-8 was employed to measure cell viability. (B) ESR1 mRNA level was measured by RT-qPCR. (C) DMSO or Gomisin B (GB) or Ferrostatin 1 (Fer-1) or Necrostatin-1 (Nec-1) or Ac-DMPD (Ac-D) or Z-VAD-FMK (Z-VAD) or 3-Methyladenine (3-MA) was applied to treat SH-SY5Y and IMR-32 cells, and cell viabilities were analyzed by CCK-8. SH-SY5Y and IMR-32 cells were treated with RSL3 and GB. (D) The commercial kits were implemented to determine Fe2+, GSH, MDA and LPO levels. (E) C-11 BODIPY 581/591 fluorescent probe was adopted to assess lipid ROS levels (100 μm). (F) SCD1, GPX4, and ACSL4 protein levels were visualized by western blot. (G) Transmission electron microscopy was performed to examine mitochondrial morphological alterations in cells (500 nm). N = 3, *p < 0.05, **p < 0.01, ***p < 0.001
Fig 2: Graphical abstract: Gomisin B inhibits the transcriptional activation of USP7 by downregulating ESR1 to suppress the deubiquitination of SREBF1, thereby enhancing NB ferroptosis
Fig 3: Gomisin B promoted ferroptosis in NB cells by downregulating ESR1. SH-SY5Y and IMR-32 cells were transfected with ESR1 overexpressing lentivirus and treated with Gomisin B and RSL3. (A) RT-qPCR was utilized to check ESR1 expression. (B) Cell viability was estimated by CCK-8. (C) GSH, MDA and LPO levels were detected by their commercial kits. (D) Lipid ROS levels were measured using C-11 BODIPY 581/591 fluorescent probe (100 μm). (E) Western blot was conducted to detect the protein abundance of ESR1, SREBF1 and SCD1. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001
Fig 4: ESR1 inhibited ferroptosis in NB cells by transcriptionally activating USP7. (A) JASPAR (http://jaspar.genereg.net) was applied to predict the binding site between ESR1 and USP7 promoter. (B) The binding relationship between ESR1 and promoter of USP7 was verified using a dual-luciferase reporter. (C) ChIP was utilized to confirm the binding relationship between ESR1 and the upstream promoter region of USP7. SH-SY5Y and IMR-32 cells were transfected with sh-ESR1 or OE-USP7, and RSL3 was used to induce ferroptosis in NB cells. (D) RT-qPCR was carried out to measure ESR1 and USP7 levels. (E) CCK-8 was implemented to examine cell viability. (F) GSH, MDA and LPO levels were tested via commercial kits. (G) C-11 BODIPY 581/591 fluorescent probe was deployed to assess lipid ROS level (100 μm). (H) ESR1, USP7, SREBF1 and SCD1 expressions were calculated by western blot. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001
Fig 5: Gomisin B promoted ferroptosis and inhibited tumor growth in NB tumor-bearing nude mice. NB cells were injected subcutaneously into BALB/c nude mice to construct NB tumor-bearing nude mice, and then Gomisin B or RSL3 was injected intraperitoneally every other day. The experimental groups were: Control, DMSO, RSL3, RSL3 + GB. (A) Representative pictures of different tumor tissues were taken. (B) Tumor growth curves were plotted, and the tumor weight was recorded. (C) The commercial kits were utilized to test tumor tissue GSH, MDA and LPO levels. (D) C-11 BODIPY 581/591 fluorescent probe was performed to estimate tissue lipid ROS level (50 μm). (E) ESR1, USP7, SREBF1 and SCD1 expressions were examined using western blot. N = 5, *p < 0.05, **p < 0.01, ***p < 0.001
from Cell Signaling Technology for SimpleChIP ® Human ESR1 Promoter Primers