Fig. 3

HMGA1 regulates FOXM1 at a post-transcriptional level via the proteasome pathway. a qRT-PCR of FOXM1 mRNA level in MDA-MB-231 cells after 24, 48 and 72 h after HMGA1 silencing. GAPDH was used for normalization. The data are compared to the control condition and are presented as the mean ± SD (n ≥ 3), **p < 0.01; two-tailed Student’s t-test. b Western blot analysis of FOXM1 protein level in MDA-MB-231 cells upon 24, 48 and 72 h of HMGA1 silencing. β-actin was used as a loading control. c Western blot analysis of FOXM1 protein levels in MDA-MB-231 control cells and cells silenced for HMGA1, treated with cycloheximide (CHX) for the indicated times. The FOXM1 band intensity was normalized to total protein lysate stained with Red Ponceau, based on ImageJ quantification. The protein levels of FOXM1 relative to the time point 0 of each group were calculated and are shown in the graph on the right, n = 3, *p < 0.05; two-tailed Student’s t-test. d Western blot analysis of FOXM1 protein level in MDA-MB-231 cells silenced for HMGA1 and treated with the proteasome inhibitor MG-132. e Representative immunofluorescence images of the translocation of FOXM1 (green) and its colocalization with the Subunit β2 of the proteasome (red) in MDA-MB-231 control cells versus cells silenced for HMGA1. Images were taken at 60X magnification