Supplemental Material

This article contains the following supporting material:

• Corrected version of Figure 5
• Figure S1 - Transfection of siRNA for RAGE activated assembly of GWB. SiRNA for RAGE were transfected into HeLa cells to examine its effects on GWB formation. Mock transfected cells or cells transfected with siRNA for luciferase served as controls. Cells were fixed on day 3 after transfection and stained with human anti-GWB serum (green) and counterstained with DAPI (blue). The number and size of GWB increased only in cells transfected with siRNA for RAGE (ii). Scale bar, 10µm.
• Figure S2 - siRNA for GFP efficiently knocked down the expression of target. GFP3T3 cells were either mock transfected (iii, iv) or transfected with 100nM siRNA for GFP (i, ii), and then fixed 3 days later. Nuclei were counterstained with DAPI (blue). Scale bar, 10µm.
• Figure S3 - The increase of GWB induced by lamin A/C siRNA was prominent on day 3. HeLa cells were either mock transfected (i) or transfected with 100nM siRNA for lamin A/C (ii) or luciferase (iii), and then fixed on day 3 after transfection. Cells were counterstained with index human anti-GWB serum (green) to monitor GWB and DAPI for nuclei (blue).
  (A) A majority of the cells transfected with siRNA for lamin A/C had larger and greater numbers of foci. Scale bar, 10µm.
  (B) Quantitative analysis of the number of foci per cell showed that both the percentage of cells with foci and the average number of foci per cell increased in cell treated with lamin A/C siRNA. The number of foci per cells was quantitated as described in Figure 3B and methods. The median with the interquartile range is indicated for each data group. The lamin A/C siRNA group, as indicated by bracket, was significantly higher than the other two groups (Dunn's multiple comparison test, P<0.001); ns, no significance (Dunn's multiple comparison test, P>0.05).
• Figure S4 - Lamin A/C siRNA induced assembly of GWB and recruited residual rck/p54 to the reassembled GWB in rck/p54-knockdown cells. HeLa cells were either mock transfected (i-iii) or transfected with 100nM siRNA for rck/p54 (iv-vi) for 3 days. In the sequential transfection, 100nM lamin A/C siRNA were transfected 24 hours after the initial transfection of siRNA for rck/p54 and the cells were fixed 2 days after that 2^nd transfection (vii-ix). The transfected cells were stained with human anti-GWB serum (green), rabbit anti-rck/p54 (red), mouse anti-lamin A/C (magenta) and DAPI (blue). Merge image of anti-rck/p54 and anti-GWB are shown in the right column. Insets are enlarged about 1.5-fold and the rck/p54 signal in rck/p54-knockdown cells (vii, ix) is enhanced to show the localization of rck/p54 to GWB. Arrowheads indicate co-localization. Scale bar, 10µm.