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. 2023 Mar 7;16(1):133–159. doi: 10.1016/j.jcmgh.2023.03.001

Figure 4.

Figure 4

FSP1 knockdown induced ferroptotic HCC cell death. (A) Stable FSP1-knockdown subclones (shFSP1) were established using shRNAs. RT-qPCR and Western blotting confirmed the knockdown efficiencies at mRNA and protein levels, respectively. (B) LC-MS demonstrated decrease in relative CoQ10H2/CoQ10 ratio in MHCC97L-shFSP1 cells. (C) BODIPY 581/591 C11 staining in MHCC97L-shFSP1 cells revealed a significant increase in lipid peroxidation. (D) Cell proliferation rates of MHCC97L-NTC and -shFSP1 cells by cell counting daily revealed that FSP1 knockdown significantly suppressed HCC cell growth. Suppressed growth rates were rescued using ferroptosis inhibitor Fer-1. (E) Orthotopic implantation of luciferase-labeled MHCC97L-NTC and MHCC97L-shFSP1-23, and MHCC97L-shFSP1-24 cells into the livers of nude mice (n = 5 mice per experimental group) revealed that FSP1 knockdown suppressed HCC tumor formation in vivo. Top, left: Representative picture of orthotopic xenografts and quantification of tumor volume. Top, right: Bioluminescent images of mice implanted with different stable subclones. Bottom: Bioluminescent images of mice lung tissues with corresponding incidence of lung metastases. (F) Quantitative measurements of natural lipid peroxidation by-products Left: 4-HNE and Right: MDA in MHCC97L-NTC and MHCC97L-shFSP1 cells further suggest increased lipid peroxidation upon FSP1 knockdown. (G) Quantification of relative LDH release in MHCC97L-NTC and MHCC97L-shFSP1 cells demonstrated that intracellular lipid peroxidation induced by FSP1 knockdown significantly increased cell death. (H) SYTOX Green (SG+) staining in MHCC97L-NTC and MHCC97L-shFSP1 cells demonstrated increased cell death from FSP1 knockdown were rescued with Fer-1. (I) Cell proliferation rates of MHCC97L-NTC and MHCC97L-shFSP1 cells determined using BrdU assay indicated that Fer-1, not inhibitors of other modes of RCD GSK’872 (necroptosis), Z-VAD-FMK (apoptosis), and MRT68921 (autophagy), can rescue proliferation rates suppressed by FSP1 knockdown. Error bars indicate mean ± SD. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 vs NTC or Fer-1 as indicated. Student t test. E: scale bar = 1 cm