However, the levels were reverted by either reconstituted expression of Sec-containing TrxR1 (480±94 nM H2O2) or by addition of catalase (41±16 nM H2O2), but not by reconstitution of the Cys variant of the enzyme (1473±204 nM H2O2, Figure 6c), thereby correlating well with the protective effects of Sec-containing TrxR1 reconstitution or catalase supplementation.
For stable reconstituted expression of Bcl-xL, we cloned its full-length coding region with an N-terminal Flag-tag in pLenti6.3 vector (Invitrogen).
Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/WTBRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar.
Strikingly, in IKKγΔ-reconstituted cells, the expression of all three type I IFNs was significantly less, and for IFNα1, indistinguishable from that of IKKγ−/− MEFs (Figure 2A).
However, expression of a dominant negative form of p73β in p65 reconstituted cells successfully blocked expression of Noxa in these cells and prevented Noxa induction by etoposide treatment.
B: KHC-2 cells with reconstituted USP18 expression maintained expression for the duration of growth in mice.
Immunoblot analysis of the reconstituted MEFs verified that expression of the Stat1 mutants was equal to that of Stat1 WT (Fig. 2A, panel a).
Interestingly, although the induction of Noxa in the p65-reconstituted cells was completely ablated by DN-p73β, the ectopic expression of a transactivation-competent version of p73β failed to activate expression of Noxa in the p65-null cells.
It is also important to note that this approach does not involve supraphysiologic overexpression of LXRs, as the level of reconstituted protein expression in these cells was within the physiologic range and restored target gene expression to physiologic levels.
For example, Olaso et al. have demonstrated that skin fibroblasts from DDR2 knockout mice present impaired proliferation and migration through a reconstituted basement membrane concomitantly with the expression of MMP-2 [ 7].
