Fig. 2

HDAC7 facilitates FOXP1 protein stabilization. a The expression of FOXP1 protein increased as the dosage of HDAC7 increased. Different levels (0, 1 μg and 2 μg) of HDAC7 and FOXP1 (1 μg) expression plasmids were transfected into 293 T cells and then analyzed 24 h later. β-actin levels served as loading control here and in subsequent sub-figures. b The expression of FOXP1 increased following harvesting from 293 T cells post-transfection at the indicated times (12, 24, 36, 48 h) with the indicated plasmids (2 μg). c FOXP1 expression assessed in 293 T cells that were treated for 12 h with either Cycloheximide (CHX, an inhibitor of protein synthesis, 10 μg/ml), or Chloroquine (an inhibitor of lysosomal activity, 50 μM), or Bafilomycin A1 (an inhibitor of autophagy, 100 μM) and with MG132 (a proteasome inhibitor, 10 μM). d Stabilization of FOXP1 by HDAC7 is sensitive to HDAC inhibitor TSA. 293 T cells transfected with the indicated plasmids were treated with 40 nM TSA for 48 h, or 10 μM MG132 for 24 h prior harvesting of FOXP1 via its His tag. FOXP1 acetylation was assessed simultaneously (as described in the legend for Fig. 1). e HDAC7 reduces FOXP1 ubiquitination levels in 293 T cells transfected with the plasmids indicated on the figure. f HDAC7 facilitates FOXP1 accumulation both within the nucleus and the cytoplasm of 293 T cells transfected with the indicated plasmids. g Luciferase reporter assays demonstrate that FOXP1 transcriptional repression of p16 is enhanced by HDAC7. C3H/10T1/2 cells were co-transfected with p16-Luc, FOXP1-His, and/or HDAC7-Flag. Reporter activity is presented as relative luciferase units (RLU). Data shown are representative of 3 independent experiments). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, non-significant