Hyal-2 induces WOX1 expression in L929 cells. (A) L929 cells were electroporated with nothing (null), GFP, GFP-Hyal-1 or GFP-Hyal-2 construct (30 g DNA / 3 × 106 cells) and cultured for 48 hr. Transient expression of GFP-Hyal-1 and GFP-Hyal-2 in L929 cells induced WOX1 expression (46 and 31 kDa), whereas no WOX1 expression was observed in non-transfected cells or control GFP-expressing cells. The 46-kDa band is full-length WOX1, and the 31-kDa band is a degraded WOX1 . No detectable p53 expression was observed in these cells. α-Tubulin was examined to confirm equal protein loading onto each lane of the gel. (B) In comparison, the GFP-Hyal-2 stable transfectant constitutively expressed cytosolic WOX1 (46 kDa) and TNF (50 ng/ml) reduced the WOX1 expression. The reduction is probably due to TNF-mediated WOX1 nuclear translocation from the mitochondria . In contrast, WOX1 was not expressed in the stable GFP-Hyal-1 cells, and TNF induced the expression of WOX1 in a time-related manner. TNF could not induce WOX1 expression in the control GFP-expressing cells. (C) L929 cells were transfected with GFP-Hyal-2 and/or WOX1 constructs by electroporation. GFP-Hyal-2 enhanced WOX1-mediated L929 cell death, as determined 48 hr post-transfection using crystal violet staining (two representative rows of cell stains shown at left and the analyzed data shown at right; mean ± standard deviation, n = 4). cell death. In controls, GFP-Hyal-1 or GFP-Hyal-2 alone failed to induce cell death.