Figure 4.
Figure 4. Effect of A20 preservation on mitochondrial integrity in PAECs after TNF treatment. A20 expression in PAECs preserves mitochondrial integrity following TNF treatment by inhibiting proteolytic cleavage of Bid, maintaining the Δψm, and preventing cytochrome c release and activation of caspase 9. (A) Western blot analysis of BID cleavage in NI PAECs and PAECs infected with rAdA20 or rAdβ-gal 6 hours following treatment with CHX/TNF. A20 expression in PAECs inhibited TNF-induced cleavage of BID (24 kD). Data shown are representative of 3 independent experiments. (B) NI PAECs and PAECs infected with rAdA20 or rAdβ-gal were treated with CHX/TNF for 4 hours. PAECs were then recovered and labeled with the RH 123 dye, and the Δψm was analyzed by FACS analysis in fluorescence channel 1 (FL-1) for green fluorescence. Cells with normal Δψm exhibit strong rhodamine fluorescence. Cells with low fluorescence reflect loss of the Δψm (gated area). Expression of A20 in PAECs significantly decreased the loss of the Δψm. Data shown are representative of 3 independent experiments. (C) NI PAECs and PAECs infected with rAdA20 or rAdβ-gal were treated with CHX or CHX/TNF for 6 hours. Cytoplasmic cell extracts were recovered and evaluated by Western blot analysis for cytochrome c expression. Expression of A20 inhibited TNF-mediated release of cytochrome c in the cytoplasm. Data shown are representative of 3 independent experiments. (D) NI PAECs and PAECs infected with rAd.β-gal or rAdA20 were treated with CHX alone or with CHX/TNF for 6 hours. Caspase 9 activity was analyzed in PAEC lysates by means of a colorimetric assay based on spectrophotometric detection of the chromophore pNA after cleavage from the caspase-specific–labeled substrate LEHD-pNA. Expression of A20 significantly inhibited TNF-induced activation of caspase 9. Western blot analysis of procaspase 9 demonstrated that A20 inhibited TNF-induced proteolytic cleavage of caspase 9. The molecular mass of the inactive proform of the procaspase 9 (48 kD) is indicated (arrows). Data are expressed as mean ± SD of triplicate values. Results shown are representative of 4 independent experiments.

Effect of A20 preservation on mitochondrial integrity in PAECs after TNF treatment. A20 expression in PAECs preserves mitochondrial integrity following TNF treatment by inhibiting proteolytic cleavage of Bid, maintaining the Δψm, and preventing cytochrome c release and activation of caspase 9. (A) Western blot analysis of BID cleavage in NI PAECs and PAECs infected with rAdA20 or rAdβ-gal 6 hours following treatment with CHX/TNF. A20 expression in PAECs inhibited TNF-induced cleavage of BID (24 kD). Data shown are representative of 3 independent experiments. (B) NI PAECs and PAECs infected with rAdA20 or rAdβ-gal were treated with CHX/TNF for 4 hours. PAECs were then recovered and labeled with the RH 123 dye, and the Δψm was analyzed by FACS analysis in fluorescence channel 1 (FL-1) for green fluorescence. Cells with normal Δψm exhibit strong rhodamine fluorescence. Cells with low fluorescence reflect loss of the Δψm (gated area). Expression of A20 in PAECs significantly decreased the loss of the Δψm. Data shown are representative of 3 independent experiments. (C) NI PAECs and PAECs infected with rAdA20 or rAdβ-gal were treated with CHX or CHX/TNF for 6 hours. Cytoplasmic cell extracts were recovered and evaluated by Western blot analysis for cytochrome c expression. Expression of A20 inhibited TNF-mediated release of cytochrome c in the cytoplasm. Data shown are representative of 3 independent experiments. (D) NI PAECs and PAECs infected with rAd.β-gal or rAdA20 were treated with CHX alone or with CHX/TNF for 6 hours. Caspase 9 activity was analyzed in PAEC lysates by means of a colorimetric assay based on spectrophotometric detection of the chromophore pNA after cleavage from the caspase-specific–labeled substrate LEHD-pNA. Expression of A20 significantly inhibited TNF-induced activation of caspase 9. Western blot analysis of procaspase 9 demonstrated that A20 inhibited TNF-induced proteolytic cleavage of caspase 9. The molecular mass of the inactive proform of the procaspase 9 (48 kD) is indicated (arrows). Data are expressed as mean ± SD of triplicate values. Results shown are representative of 4 independent experiments.

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