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. 2020 Aug 18;117(35):21420–21431. doi: 10.1073/pnas.2010275117

Fig. 7.

Fig. 7.

GPX8-KO cellular effect is mediated by IL-6/JAK/STAT3 signaling pathway. (A) GPX8 loss leads to a reduction in the gene expression pattern for “hallmark IL-6/JAK/STAT3 signaling.” MDA-MB-231 WT cells and GPX8-KO-1 were subjected to RNA-Seq analysis. The expression ratio of all genes was calculated and ranked based on the relative expression in GPX8 WT and KO. The samples were subjected to GSEA. The FDR q-value was computed by GSEA. (B) STAT3 signaling is reduced by the addition of neutralizing IL-6 antibodies to the conditioned media. Conditioned media from the WT cultured for 3 d were added to GPX8-KO-1 cells in the absence or presence of neutralizing IL-6 antibodies (800 ng/mL) for 1 h. The cells were lysed and subjected to immunoblot using the indicated antibodies. (C) GPX8 WT and KO cells respond to soluble IL6 receptor and Hyper IL-6 but not to IL-6 treatment. GPX8-WT and GPX8-KO-1 cells were starved with 0.1% FBS medium for 24 h and treated with 20 ng/mL IL-6, 300 ng/mL soluble IL-6 receptor, and 50 ng/mL Hyper IL-6 for 1 h. Cells were subjected to immunoblot using the indicated Abs. (D) Hyper-IL6 induces EMT markers expression in GPX8-KO cells. WT and GPX8 KO cells were stimulated with Hyper IL-6 (50 ng/mL) for 3 d. The cells were then lysed and subjected to immunoblot using the indicated antibodies. (E) The soluble IL-6 receptor level is affected by the expression level of GPX8 in cell growth media. Cell growth media were collected from each of the indicated samples after 24 h starvation. The level of sIL6R was determined using a specific ELISA kit (n = 4). The P value was determined by Student t test. (F) Ectopic expression of full-length IL-6 receptor activates the STAT3 signaling pathway only in WT cells. The soluble IL6R and the full-length IL6R were overexpressed (SIL6R-OE, IL6R-OE, respectively, both FLAG-tag) in both GPX8-WT and GPX8-KO-1 cells, were lysed after 24 h, and subjected to immunoblot using the indicated antibodies. (G) A scheme representing the role of GPX8 in IL-6/JAK/STAT3 activation. Green arrows represent IL-6 secretion, and dark blue arrows represent intracellular IL-6 signaling. Dashed arrows and reduced font size represent impaired secretion and intracellular signaling. In WT cells, IL-6 and IL6R are processed in the ER; gradient color represents proteins before maturation and solid color after maturation.