Legend to Supplemental Material
Suppl. Table I
Primary antibodies, concentrations and suppliers.
Suppl. Figure 1
Inhibition of MMP-2 activity using an anti-MMP-2 monoclonal antibody and was confirmed by
gelatin zymography. Note that gelatinolytic activity at the exected MW is present in CM from
H357 CAFs only.
Suppl. Figure 2
Western blot analysis of DSG3. Treatment of H357 keratinocytes with CM from senescent
fibroblasts (GU-OSCC/H357F) generated a 75kDa fragment of DSG3; inhibition of MMP-2
activity reduced the cleavage of DSG3. CM from H357 keratinocytes treated with normal
fibroblasts (NHOF1) CM or serum-free medium (SFM) were used as negative controls.
Suppl. Figure 3
Keratinocytes (H357) were incubated with conditioned media from normal fibroblasts (NHOF1),
CAF from GS-OSCC (BICR66 and BICR18) and from GU-OSCC (BICR31 and BICR63).
Dispase-based assay was undertaken as specified in Material and Methods. In some experiments
following the shaking step the fragments and single cells were split into two parts. One half was
fixed in 10% buffered formalin to prevent further disaggregation and gently centrifuged for 5
mintes at 800 rpm. The single cells present in this half were counted following resuspension in
PBS. The other half was centrifuged in a similar manner and the cells incubated in trypsin/EDTA
for 10-15 minutes before counting the total number of cells present in each sheet. The results
were expressed as the fraction of single cells in each sheet as a percentage of the total.