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. Author manuscript; available in PMC: 2019 Nov 26.
Published in final edited form as: Sci Transl Med. 2019 Mar 13;11(483):eaaq1238. doi: 10.1126/scitranslmed.aaq1238

Fig. 3. ZEB1 regulates MAPK signaling by suppressing the scaffold protein IL17RD.

Fig. 3.

(A) Western blot of MAPK signaling molecules in H157 cells +/− miR-200 expression after 24 hours of serum-free starvation, followed by stimulation with complete serum medium, serum-free medium, or EGF for 4 hours.

(B) QPCR analysis for relative expression of SPRY1, KSR2, 14-3-3σ/SFN, CNK2, SHOC2, and IL17RD in H157 cells with inducible miR-200 expression. *p<0.05.

(C) Cluster plot analysis of Spearman’s rank correlation between IL17RD gene expression and EMT scores of 77 human lung cancer cell lines.

(D and E) Western blots of IL17RD in (D) a panel of human epithelial and mesenchymal KRAS mutant lung cancer cell lines and (E) H157 or H441 cells with induced miR-200 or ZEB1 expression, respectively.

(F) Western blot of IL17RD and MAPK signaling molecules after transient shRNA knockdown of IL17RD in H157 cells with induced miR-200 expression for 72 hours.

(G and H) Western blots of IL17RD and MAPK signaling molecules after 48-hour constitutive expression of IL17RD in (G) H157 and (H) A549 cells.

(I) Schematic of human IL17RD promoter region containing predicted ZEB1 binding sites represented by color-coded ellipses. Black arrows indicate location of genomic region used for qPCR amplification, containing potential ZEB1 binding sites in the IL17RD promoter after ZEB1 chromatin immunoprecipitation (ChIP). The IL17RD promoter was cloned 1,066 base pairs upstream of the transcriptional start site and inserted into a luciferase reporter vector. Mutations of potential ZEB1 binding sites indicated with yellow X.

(J) Fold enrichment by qPCR analysis of IL17RD promoter segments containing potential ZEB1 binding sites after endogenous ZEB1 ChIP in H157 cells with inducible vector control or miR-200 expression, using ZEB1 antibody or IgG control antibody. Promoter regions analyzed by qPCR labeled with black arrows in (I).

(K) Relative luciferase activity of IL17RD promoter reporter constructs in (I) transfected into epithelial H441 cells with induced GFP control or ZEB1 expression. Relative luciferin signal was normalized to promoter-less vector control signal.