Fig 2: Knockdown of IL6ST could decrease viability and increase ferroptosis phenotype and related genes. (A-C) Effective knockdown of IL6ST in SW480 CRC cells is detected using RT-PCR. (B, C) Effective knockdown of IL6ST in SW480 CRC cells is detected using western blot. (D) Knockdown of IL6ST could significantly decrease the viability of SW480 CRC cells compared to nonmalignant human colonic epithelial cell lines NCM-460 and vector controls via CCK-8 assays (P = .007 and P = .004, respectively). (E) IL6ST knockdown SW480 cells exhibit shrunken and damaged mitochondria (3200×). (F) Knockdown of IL6ST up-regulated the levels of iron in SW480 cells compared to the control group (P = .007). (G) Knockdown of IL6ST increased the levels of ROS in SW480 cells compared to the control group (P = .005). (H-L) The expressions of PTGS2, NOX1, and ACSL4 are upregulated at the RNA levels, while FTH1 and GPX4 are downregulated in IL6ST knockdown SW480 cells (P < .01). (M-R) The expressions of PTGS2, NOX1, and ACSL4 are upregulated at protein levels in IL6ST knockdown SW480 cells, while FTH1 and GPX4 are upregulated in IL6ST overexpression SW620 cells (P < .01).

Fig 3: Correlation between IL6ST and immune cell subtype infiltration. (A) The expression level of IL6ST is positively correlated with the infiltration of T helper cells, macrophages, Tcm cells, Tgd cells, Mast cells, DC cells, Th1 cells, TFH cells, Th2 cells, neutrophils and T cells (P < .05). (B) The enrichment score of T cells is significantly higher in IL6ST high-expressed group than in the low-expressed group.

Fig 4: Upregulated IL6ST in CRC tissues and cells was associated with ferroptosis-related genes. (A) IL6ST expression is upregulated in CRC tissues compared with unpaired controls based on TCGA-COAD database (P = .000). (B) IL6ST expression is upregulated in CRC tissues compared with paired adjacent controls based on TCGA-COAD database (P = .000). (C) Higher expression of IL6ST in CRC tissues is found compared to adjacent controls via IHC assay (P = .0001) (200×). (D) IL6ST is upregulated in SW480, HCT116, HT-29, and SW620 cells than nonmalignant human colonic epithelial cell lines NCM-460. (E) Demonstrated the specificity and sensitivity of IL6ST in predicting CRC. (F) The expression level of IL6ST has a significant association with FTH1, GPX4, NCOA4, PTGS2, SLC7A11, and TFRC expression levels.