JCS031443 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Movie S1. Enhanced visualization of PH_CRAC-GFP by subtraction of cytosol signal. Cells expressing both PH_CRAC-GFP and cytosolic mRFP chemotaxing towards a cAMP-filled microtip (tip is outside field of view towards upper-left corner) were recorded with a confocal laser-scanning microscope (frame interval is 8 seconds). GFP- and mRFP-signals were recorded separately. The cell of interest is marked with a white arrow in the first frame. Panel A contains the original PH_CRAC-GFP signal. After background subtraction, the red signal was normalized to the mean cytosolic green fluorescence of the cell of interest; panel B shows the normalized mRFP signal. This normalized signal was subtracted from the green channel, yielding the cytosol-corrected PH_CRAC signal (panel C). Note that the cytosol correction must be performed separately for each individual cell. The movie was used for quantification presented in Fig. 4.

• Supplemental Figure S2 -

  Fig. S2. Overexpression of GFP-tagged lap2β or HB-EGF-V5-C at the INM neither induces heterochromatin formation nor global transcriptional repression. (A) HeLa cells transiently expressing GFP-tagged lap2β or HB-EGF-V5-C were incubated with or without TPA and then stained with indicated antibodies and Hoechst 33342. (B,C) 5 mM Br-U was added with or without TPA to the culture medium of HeLa cells transiently expressing GFP-tagged lap2β (B) or HB-EGF-V5-C (C), followed by a 30 minute incubation. Cells were fixed and immunostained with anti-BrdU mAb, and Hoechst 33342. Scale bars: 5 µm.

• Supplemental Figure S3 -

  Fig. S3. Lamin A/C-knockdown cells did not repress global transcriptional. HeLa cells were transfected twice: first with 20 nM lamin A/C-targeting duplexes siRNA (si-lamin A/C) or control siRNA (si-control); and, after 48 hours with wild-type AREG plasmid. After 24 hours, 5 mM Br-U was added in the presence or absence of TPA to the culture medium and cells were incubated for 30 minutes, and then fixed. Cells were stained with anti-AR-C pAb, anti-BrdU mAb and Hoechst 33342. Scale bars: 5 µm