Fig 2: TGFΒ treatment destabilizes ΔNp63α in a manner that is dependent upon the kinase activity of ALK5. A.TGFΒ increases the rate of ΔNp63α turnover. IMECs were treated with vehicle or cycloheximide followed by treatment with vehicle, TGFΒ or A8301. B. TGFΒ-stimulation selectively increases the rate of turnover of phospho-ΔNp63α. IMECs were treated with vehicle, TGFΒ or A83-01 followed by vehicle or cycloheximide. C. Phospho-p63 is stabilized by the 26S proteosome inhibitor MG132. H1299 cells were transfected with GFP, ΔNp63α WT and ΔNp63α AA and treated for 2 hours with 1 µM MG-132.

Fig 3: ALK5 mediates phosphorylation of ΔNp63α in response to ultraviolet irradiation. A. IMEC cells were plated and treated with the indicated amounts of A83-01 or vehicle for 12 hrs after which cells were exposed to 50 J/m2 UV radiation. Whole cell lysates were collected after 1 hr of UV treatment and analyzed by immunoblotting for phospho-P63, total-P63, phospho-SMAD2 and β-actin levels. Phospho-SMAD1/5/8 was used as a control to show that there were no off-target effects for A83-01 at the concentrations used. B. H1299 cells were transfected with GFP, ΔNp63α-WT or ΔNp63α-AA expression vectors and cells were treated with 2 µM A83-01 or vehicle control. Twenty-four hours later cells were exposed to 50 J/m2 UV radiation and whole cell extracts were collected after 1 hr to analyze the levels of phospho-P63, total-P63 and β-actin by immunoblotting.

Fig 4: TGFΒ is anti-proliferative and suppresses ALDH1 and ΔNp63α protein levels activity in a mammary stem cell model. A. Cell counts plotted for IMEC cells treated with control, TGFβ1 and A83-01 for the indicated time points. Data are representative of multiple B. Cell cycle analysis was performed by PI staining on IMEC cells after treatment with control, TGFβ1 or A83-01 for the indicated time points. Dashed lines flank the S-phase region. C. IMEC cells were sorted based on the ALDH staining, ΔNp63α mRNA expression levels were analyzed using quantitative PCR. D. IMEC cells expressing ALDH high and low were analyzed after treating IMEC cells with vehicle control, TGFβ1 and A83-01 at the indicated time points. E. IMEC cells were treated with control or cycloheximide (CHX) for 2 hrs and then treated with TGFβ1 or A83-01 for 2 hrs. Whole cell lysates were analyzed for ΔNp63α and β-actin. F. IMEC cells were treated with control, TGFβ1 and A83-01 for 1 hr, after which cells were treated with control and CHX for 4 hrs. Whole cell lysates were analyzed for Phospho-p63 and β-actin.

Fig 5: Nuclear accumulation of the intracellular kinase domain of ALK5 (ALK5IKD) in response to TGFβ. A. Three different siRNAs targeted against ALK5 were transfected into IMEC cells, 48 hrs later cells were transfected with GFP, ΔNp63α-WT and ΔNp63α-AA expression vectors, whole cell lysates were harvested after 24 hrs and analyzed for phospho-p63, total p63, TGFβR1 and β-actin. B. H1299 cells were transfected with an ALK5-WT-Flag expression vector, whole cell lysates were harvested and analyzed for full length and cleaved fragments with anti-Flag antibody. C. Analysis of ALK5 distribution in IMECs indicates that the 34 kDa C-terminal ALK5 fragment is present only in the nucleus. D. H1299 cells were transfected with ALK5-GFP expression vector, 6 hrs after transfection cells were treated with vehicle control or TGFβ1 for 8 hrs and imaged for subcellular distribution of GFP. E. H1299 cells were serum starved for 12 hr and then induced with vehicle control or TGFβ1 for 1hour. Cells were stained with anti- TGFβR1 (V-22) antibody. Depletion of TGFβR1in H1299cells by siRNA is shown as a control for the specificity of the antibody.