Figure 3.

Attenuation of cervical cancer ferroptosis by TAM is involved in downregulation of ALOX15. (A–C) HeLa and SiHa cells were treated with CM from M0 macrophage and TAM for 48 h. Ferroptosis-associated gene was detected by RT-PCR. The ratio of genes expressions in cells treated with TAMs-CM relative to M0-CM were shown in the heatmap (A). Volcano map indicated the relationship between the observed fold change in gene expression and the P value significance of such changes in cells treated with CM. The horizontal dotted lines represented the P = 0.05, and vertical dotted line represented the 2-fold change cut-offs, the red dots represented the selected ALOX15 gene (B, C). (D, E) HeLa cells were treated with CM or exosome (EXO) from PBMC or THP-1-derived M0, M2 macrophage and TAM for 48 h. ALOX15 mRNA levels was detected by RT-PCR. (F) HeLa cells were treated with CM or EXO from PBMC or THP-1-derived M0, M2 macrophage and TAM for 48 h. ALOX15 protein levels was detected by Western blot. The relative quantification of protein was analyzed by scanning densitometry using Image J software and β-actin was used as the loading control. (G) TAM (THP-1) and HeLa in combination or HeLa alone were inoculated subcutaneously in nude mice. And then, the ALOX15 and macrophage marker CD68 expression was detected by immunofluorescence. (H, I) HeLa and SiHa cells were transfected with ALOX15 siRNAs for 48 h. Cell death induced by ERA was detected (H). ALOX15 protein levels were detected by Western blot. The relative quantification of protein was analyzed by scanning densitometry using Image J software and β-actin was used as the loading control (I). (J, K) HeLa and SiHa cells were transfected with ALOX15 siRNAs and treated by TAM-CM for 48 h. Cell death induced by ERA was detected. Data represented the mean ± SEM of at least three times biological replicates. NS, not significant; ∗∗∗P < 0.001, compared to control.