Human genes repressed by IE1 are IL6- and OSM-responsive pSTAT3 target genes.

Abstract

<p>(A) TetR (w/o) and TetR-IE1 (IE1) cells were treated with dox for 72 h. Relative mRNA levels were determined by RT-qPCR with primers specific for the C4A, CHL1, CXCL12, IFI16, RASL11A or SOCS3 genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to TetR cells (set to 1). (B) TetR cells were treated with solvent or IL6 plus IL6R (IL6/Rα) for 6 h or 24 h. Relative mRNA levels in comparison to solvent-treated cells (set to 1) were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown. (C) TetR cells were treated with solvent or OSM for 24 h. Relative mRNA levels in comparison to solvent-treated cells (set to 1) were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown. (D) TetR cells were transfected with the indicated siRNA duplexes. Two and five days post transfection (p.t.), whole cell protein extracts were prepared and subjected to immunoblotting for STAT3α and GAPDH (left panel). Five days post transfection, relative mRNA levels were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to control siRNA-transfected cells (set to 1) (right panel). (E) Whole cell protein extracts from TetR cells without (w/o) or with stable expression of the indicated STAT3α-Myc proteins were subjected to immunoblotting for STAT3 (Myc tag), TUBA, pSTAT3 (Y705) and pSTAT3 (S727) (left panel). Total RNA samples from TetR cells overexpressing either wild-type STAT3α-Myc or STAT3α_Y705F-Myc were subjected to RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to cells expressing wild-type STAT3α-Myc (set to 1) (right panel).</p