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Figure 3


Fig. 3. TNF{alpha} treatment decreased MHCIIA levels but did not affect MHCIIB levels. (A) TtT/GF cells were treated with culture medium alone or with medium containing 20 ng/ml TNF{alpha} both in the absence or in the presence of 5 µM Z-VAD-fmk for 96 hours. Next, non-cytoskeleton (N) and cytoskeleton (C) fractions were obtained and subjected to electrophoresis and western blotting with antibodies against MHCIIA. The membrane was then stripped and reprobed with anti-MHCIIB. The actin immunoreactive bands correspond to the loading control. Z-VAD-fmk slightly decreased MHCIIA levels in the cytoskeleton fraction. TNF{alpha} reduced MHCIIA levels in both subcellular fractions. Inhibition of caspase activity restored control non-cytoskeleton MHCIIA levels but the recovery was not complete in the cytoskeleton fraction. MHCIIB levels in the cytoskeleton fraction were slightly decreased by Z-VAD-fmk but were not affected by TNF{alpha}. (B) TtT/GF and NIH 3T3 cells were treated with different combinations of TNF{alpha} (20 ng/ml), cycloheximide (CHX, 5 µg/ml) and Z-VAD-fmk (5 µM). Following a 24-hour treatment, MHCIIA and MHCIIB expression levels were analysed by western blotting in whole-cell lysates. Short-term incubation of the two cell lines with TNF{alpha} in combination with CHX drastically reduced MHCIIA, whereas MHCIIB expression remained almost unchanged in TtT/GF cells and was slightly reduced in NIH 3T3 fibroblasts. The TNF{alpha}+CHX-induced drop of MHCIIA levels was abolished by the caspase inhibitor Z-VAD-fmk in both cell lines. (C) TtT/GF cells were incubated with the protein synthesis inhibitor cycloheximide (CHX, 50 µg/ml) for increasing periods of time. Following treatments, total cell proteins were subjected to SDS-PAGE followed by immunoblotting with anti-MHCIIA. Next, the membrane was stripped and incubated with anti-MHCIIB. The actin immunoreactive bands correspond to the loading control. The figure shows representative immunoblots. The bands were scanned and the mean intensity values for each myosin isoform were plotted. Values shown are the mean ± s.e.m. of three independent experiments. (D) TtT/GF cells were incubated with the translation inhibitor actinomycin D (ActD, 1 µg/ml) for increasing periods of time. Blockade of protein or mRNA synthesis affected MHCIIA more rapidly than MHCIIB.