Figure 4.

NONO directly mediates STAT3 function in TNBC cells (A) Alignment of the STAT3 locus sequence. (B) RNA-IP was performed with anti-Myc ab or endogenous NONO ab in Myc-NONO-overexpressing MDA-MB-231 cells or MDA-MB-231 cells. After RNA-IP, the cells were analyzed by qRT-PCR with the indicated probes. (C) A dual-luciferase assay in HEK293T cells, which harbored a luciferase reporter vector containing the wild or mutant-type sequence of STAT3 locus. Luciferase activities were measured after transfecting the indicated constructs. (D) HEK293T cells were transfected with Flag-STAT3 or Myc-NONO alone or in combination. The cells were then lysed and co-immunoprecipitated with Myc ab, and western blotting was performed with Myc and Flag antibodies (left panel). MDA-MB-231 cell lysates were immunoprecipitated with IgG and NONO (center panel) and STAT3 (right panel) antibodies, and western blotting was performed with STAT3 and NONO antibodies. (E) Protein interaction amplitudes based on the correlation functions obtained in the cells co-expressing GFP and RFP (F) Computational docking model for human NONO (cyan) and STAT3 (olive) predicted using ClusPro ¹⁷ (see Materials and Methods). (G) Cell localization of NONO and STAT3 in MDA-MB-231 cells. The cells were immunostained with the indicated antibodies and visualized using microscopy. (H) Schematic of the CCND1 promoter region. ChIP assays were performed in MDA-MB-231 cells using a STAT3 or NONO antibody. Recruitment of NONO to the CCDN1 promoter via STAT3 was analyzed using primers specific to this promoter. IgG was used as an internal control. (I) Dual‐luciferase reporter gene assay to determine the STAT3 activity level following transfection of NONO, STAT3, and a STAT3-reporter into HEK293T cells. (J) The STAT3-reporter was transfected into shNONO (or shGFP) infected MDA-MB-231 cells and rescued by STAT3 re-introduction. The cells were then used to measure luciferase activity. (K and L) MDA-MB-231 cells were stably transfected with shNONO or shGFP. After transfection, the cells were treated with DMSO, cycloheximide (CHX; 50μg/ml), or actinomycin D (Act D; 1μM) and harvested at the indicated time points. Total proteins or RNAs were extracted from the indicated cells and analyzed by western blotting with the indicated antibodies or qRT-PCR, respectively. Western blot bands were quantified using the software ImageJ. STAT3 protein levels were normalized to those of β-actin. The relative STAT3 protein or RNA level was designated as the protein or RNA half-life. All results are shown as means plus standard deviations from three-independent replicates (*p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001).