Luciferase reporter assay

To determine the effect of QKI‐5 on the luciferase activity of TGFβR1 3′ UTR, a series of constructs containing the wild‐type and mutated (Mut) 3′ UTR of TGFβR1 fused to the 3′‐end of the luciferase cDNA was generated using pGL3‐Luc reporter vector (Promega, Madison, WI, USA). Briefly, different fragments (Appendix Table S4) were directly synthesized (GENEWIZ Inc.) and subcloned into pGL3‐basic vector to create various constructs (pGL3‐Luc‐TGFβR1 3′ UTR wild type, pGL3‐Luc‐TGFβR1 3′ UTR‐Mut1, pGL3‐Luc‐TGFβR1 3′ UTR‐Mut2, and pGL3‐Luc‐TGFβR1 3′ UTR‐Mut1*2). Then, the above‐mentioned constructs and pRL‐TK plasmids were co‐transfected into A549 and H1299 stable cell lines with vector or QKI‐5 overexpression using Lipofectamine 3000 (Invitrogen).

For analyzing transcriptional regulation of KLF6 on QKI‐5, various QKI‐5 promoter fragments (Appendix Table S4) were synthesized and subcloned into pGL3‐basic vector to generate the constructs (pGL3‐QKI‐5 promoter‐wild‐type‐Luc, pGL3‐QKI‐5 promoter‐mutant 1‐Luc, pGL3‐QKI‐5 promoter‐mutant 2‐Luc, and pGL3‐QKI‐5 promoter‐mutant 1*2‐Luc). Subsequently, the above‐mentioned constructs and pRL‐TK plasmids were co‐transfected into A549 and H1299 cells with si‐NC or si‐KLF6. At 48 h post‐transfection, cells were harvested and luciferase activity was assessed using a Dual‐Luciferase^® Reporter Assay System (Promega). Results are presented as relative luciferase activities (firefly/Renilla), and each luciferase reporter analysis was performed in triplicate.