Isoform B of myosin II heavy chain mediates actomyosin contractility during TNF-induced apoptosis

JCS022640 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Validation of the immunofluorescence microscopy studies on activation of caspase 3 in adherent MHCIIB^−/− embryonic fibroblasts treated with TNFα and CHX (TNFα+CHX). The cells were incubated with culture medium containing TNFα and CHX for 16 hours. Next, they were processed for fluorescence microscopy with Rhodamine-phalloidin and antibody against active caspase 3. A treated MHCIIB^−/− cell that had remained attached stained positive for active-caspase 3. Another treated-MHCIIB^−/− fibroblast (arrow) that had remained flat showed no evidence of caspase 3 activation (arrows). Bar: 50 µM.

• Supplemental Figure S2 -

  Fig. S2. Effect of PKCζ inhibition on caspase-3 cleavage. MHCIIB^−/− embryonic fibroblasts were incubated either with culture medium alone (control) or with culture medium containing TNFα and CHX both in the absence or presence of the specific PKCζ inhibitor myr-PKCζ pseudosubstrate (PKCζ-in; at 10 µM, final concentration) for 16 hours. Next, total cell lysates were prepared and then subjected to SDS-PAGE followed by immunoblotting with antibody against active caspase 3. PKCζ inhibition neither induced the cleavage of caspase 3 nor affected TNFα-induced caspase-3 cleavage.