Figure 2
Figure 2. TNF-α gene expression is elevated in FA-C lymphoblasts. (A) Mutant FA-C lymphoblasts (HSC536N and PD149) produce more TNF-α than isogenic complemented FA-C/C cells. Secreted TNF-α was measured in culture media by the use of ELISA assays. Shown are results from 1 of 3 independent experiments. All error bars in this figure represent mean (± SD). (B) TNF-α RNA is elevated in FA-C lymphoblasts. TNF-α mRNA is increased in FA-C cells. TNF-α mRNA was analyzed by real-time RT-PCR. Results shown are from 1 of 3 identical experiments. (C) TNF-α mRNA decay rates are equivalent in mutant and complemented cells. To rule out differential TNF-α mRNA decay as an explanation for the increase in mutant cells, mRNA stability was measured by real-time RT-PCR after treatment of FA-C lymphoblasts (HSC536) with the transcription inhibitor actinomycin-D. The mRNA decay rates were identical, demonstrating that the differences in mRNA levels between FA-C and FA-C/C cells are attributable to differences in mRNA production rather than differences in mRNA stability. (D) TLR8 siRNA decreases TLR8 and TNF-α mRNA in FA-C cells. By using real-time RT-PCR for both TLR8 and TNF-α mRNA, we found that both were reduced in FA-C cells treated with siRNA targeting TLR8, demonstrating that TNF-α production in these cells is TLR8-dependent. (E) Ground-state activation of NF-κB was demonstrated by use of the electromobility shift assay in 2 different FA-C lymphoblastoid cell lines in the 2 lanes to the far left, HSC536N and HSC536N/FANCC (complemented with FANCC cDNA) and in the 6 lanes on the right, PD149 and PD149/FANCC. The top right panel is a short exposure of the same gel exposed longer shown in the bottom right panel. NF-κB signal is reduced in the both complemented cells. Supershift experiments were performed by the use of nuclear lysates of PD149 and PD149/FANCC cells and revealed that both p50 and p65 (best seen on the longer exposure) were contained within the NF-κB band. Shown are results of 1 representative of 4 identical experiments. Autoradiographs of both full gels are shown in supplemental Figure 7. This figure also shows that in mutant FA-C lymphoblasts, NF-κB activation is high in the ground state and does not require exposure to interferon-γ or TNF-α. Again, this is distinctly different from primary mononuclear phagocytes and the THP-1 shFANCC cells, which require exposure to a TLR-agonist to reveal overexpression of TNF-α.

TNF-α gene expression is elevated in FA-C lymphoblasts. (A) Mutant FA-C lymphoblasts (HSC536N and PD149) produce more TNF-α than isogenic complemented FA-C/C cells. Secreted TNF-α was measured in culture media by the use of ELISA assays. Shown are results from 1 of 3 independent experiments. All error bars in this figure represent mean (± SD). (B) TNF-α RNA is elevated in FA-C lymphoblasts. TNF-α mRNA is increased in FA-C cells. TNF-α mRNA was analyzed by real-time RT-PCR. Results shown are from 1 of 3 identical experiments. (C) TNF-α mRNA decay rates are equivalent in mutant and complemented cells. To rule out differential TNF-α mRNA decay as an explanation for the increase in mutant cells, mRNA stability was measured by real-time RT-PCR after treatment of FA-C lymphoblasts (HSC536) with the transcription inhibitor actinomycin-D. The mRNA decay rates were identical, demonstrating that the differences in mRNA levels between FA-C and FA-C/C cells are attributable to differences in mRNA production rather than differences in mRNA stability. (D) TLR8 siRNA decreases TLR8 and TNF-α mRNA in FA-C cells. By using real-time RT-PCR for both TLR8 and TNF-α mRNA, we found that both were reduced in FA-C cells treated with siRNA targeting TLR8, demonstrating that TNF-α production in these cells is TLR8-dependent. (E) Ground-state activation of NF-κB was demonstrated by use of the electromobility shift assay in 2 different FA-C lymphoblastoid cell lines in the 2 lanes to the far left, HSC536N and HSC536N/FANCC (complemented with FANCC cDNA) and in the 6 lanes on the right, PD149 and PD149/FANCC. The top right panel is a short exposure of the same gel exposed longer shown in the bottom right panel. NF-κB signal is reduced in the both complemented cells. Supershift experiments were performed by the use of nuclear lysates of PD149 and PD149/FANCC cells and revealed that both p50 and p65 (best seen on the longer exposure) were contained within the NF-κB band. Shown are results of 1 representative of 4 identical experiments. Autoradiographs of both full gels are shown in supplemental Figure 7. This figure also shows that in mutant FA-C lymphoblasts, NF-κB activation is high in the ground state and does not require exposure to interferon-γ or TNF-α. Again, this is distinctly different from primary mononuclear phagocytes and the THP-1 shFANCC cells, which require exposure to a TLR-agonist to reveal overexpression of TNF-α.

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