Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell–cell interactions in vivo
Version of Record online: 4 OCT 2010
© 2010 John Wiley & Sons A/S
Pigment Cell & Melanoma Research
Volume 23, Issue 6, pages 795–808, December 2010
How to Cite
DiVito, K. A., Trabosh, V. A., Chen, Y.-S., Chen, Y., Albanese, C., Javelaud, D., Mauviel, A., Simbulan-Rosenthal, C. M. and Rosenthal, D. S. (2010), Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell–cell interactions in vivo. Pigment Cell & Melanoma Research, 23: 795–808. doi: 10.1111/j.1755-148X.2010.00758.x
- Issue online: 13 OCT 2010
- Version of Record online: 4 OCT 2010
- Accepted manuscript online: 25 AUG 2010 01:53AM EST
- PUBLICATION DATA Received 2 June 2010, revised and accepted for publication 18 August 2010, published online 25 August 2010
Figure S1. Expressing Smad7 in 1205Lu Metastatic Melanoma Cells. Top panel, stable over-expression of Smad7 in 1205Lu (right); 1205Lu-Vc showed undetectable levels (left). Middle panel, phosphorylated-Smad3, a known marker of TGF-β activation, was undetectable following Smad7 expression; expression remained in 1205Lu-Vc. GAPDH was used as a loading control.
Figure S2. Representative Spectral Plots. Left and right panels compare HFK-GFP versus 1205Lu-DsRed target signal (top panel), followed by autofluorescence intensity (middle panel). Correction following spectra-unmixing shows enhanced, true signal devoid of autofluorescence (lower panel). Target signal emission following spectral correction was plotted in dashed lines along with the corrected autofluorescence (dashed white curve).
Figure S3. 1205Lu-Vc Grafts are Proliferative. Matched 1205Lu-DsRed grafts were stained for the proliferation marker Ki-67. S3A Top right, 1205Lu-Vc cells positive for Ki-67. Lower right, 1205Lu-Smad7 cells were predominantly Ki-67 negative. S3B Quantitating Ki-67 expression. Results indicated approximately 13–22% of 1205Lu-Vc cells express Ki-67, compared to only 3–6% of 1205Lu-Smad7 cells 3C, Assaying Apoptosis. Caspase-3 cleavage was addressed by immunofluorescence (DAPI is shown in blue; caspase-3 in green) and western blot. An average of 4.2% of 1205Lu-Vc cells showed activated caspase-3 compared to an average of 2.6% of 1205-Smad7 cells. As positive control, cells were treated with 1 μM Staurosporine for 4 h, then assayed for caspase-3 cleavage. No statistical difference (P > 0.19) in caspase-3 activity was found when comparing 1205-Lu-Vc versus 1205Lu-Smad7 cells; this was confirmed by western blot. (D) Activated caspase-3 in vivo (20X). Tissue from matched day 12 grafts also showed no difference (P > 0.93) in caspase-3 activation, quantification is shown on the right. Five high-power fields representing >500 cells were used to assess both Ki-67 and caspase-3 activation.
Figure S4. Grafted 1205Lu-Smad7 Cells Remain Near the Dermal-Epidermal Junction at Day 25. Tissue from matched day 25 tissue sections of 1205Lu-Smad7 grafts show Smad7-expressing cells remain just below the dermal-epidermal junction and do not invade, in contrast to the large tumor masses found in vector control. Overlaid images are shown far right. Epidermis and dermis are depicted in the right-hand margin.
Figure S5. Pharmacological Inhibition of TGF-β Does Not Affect N-cadherin Expression. 1205Lu cells were grown in serum free media for 24hrs then treated with 10 μM TGF-β receptor kinase inhibitor SB3431542 (Sigma-Aldrich, St. Louis, MO, USA) for 24 and 48 h. 30 μg of total cell lysate was separated on 8% SDS-PAGE and then probed for N-cadherin (clone-32, BD Biosciences). Loss of phospho-Smad3 was used to show TGF-β inhibition. Neither 24 nor 48 h of treatment effected N-cadherin resulted in appreciable changes in N-cadherin.
|PCMR_758_sm_figsS1-S5_re.zip||7636K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.