Figure 1

UVB dose-dependently stabilised FOXM1B protein expression through inhibition of proteolysis. (A) Fluorescence and phase-contrast microscopy of EGFP or EGFP-FOXM1B transduced cells 24 hours following UVB exposure. (B) Digital densitometry of fluorescence micrographs in (A) as mean ± s.e.m. (n = 3) fold fluorescence activation over control un-irradiated cells. Statistical significance levels: *(P < 0.05), **(P < 0.01) and ***(P < 0.001). (C) Time-lapse fluorescence microscopy of EGFP-FOXM1-transduced N/TERT cells following no-exposure (controls) or UVB irradiation. Each point represents mean ± s.e.m. (n = 6) fold fluorescence activation over control un-irradiated cells at time 0 hour. (D) qPCR showing no change in FOXM1B mRNA during the first 6 hours following UVB exposure. Control non-irradiated cells showed significant increase in FOXM1B mRNA, corresponding cell cycle phases were verified by FACS analyses. (E) Immunoblots showing increased in EGFP-FOXM1 protein levels (using GFP antibody) at 24 and 48 hours following UVB exposure. EGFP-expressing N/TERT showed no change in EGFP protein levels at all time points. GAPDH showed sample loading density in each blot. (F) Proteasomal proteolysis inhibition by MG132 prevented protein degradation leading to stabilisation of FOXM1B proteins. N/TERT cells transduced with EGFP-FOXM1B were treated with either vehicle (0.001% DMSO) or MG132 (1 μM; 24 hours). Fluorescence densitometry showed over 95% ***(P < 0.001) re-activation of EGFP-FOXM1B following MG132 treatment. (G) Immunoblots showing FOXM1 protein stabilisation by UVB and MG132.