Figure 1. Spontaneous mutation of leucine residues in the bLZip domain of Zhangfei in D-17 cells stably expressing the protein in the presence of tetracycline. (A) Doxycycline induces the expression of Zhangfei protein in D-17 cell clones. Twenty-four hours after induction with 1 µg/ml doxycycline, Zhangfei protein in D-17 clones was detected by immunofluorescence and immunoblotting. (B) Growth of D-17 cells after induction of Zhangfei. Six D-17 clones were treated with 1µg/ml doxycycline to induce Zhangfei expression. Cell growth characteristics were monitored at different time points using WST-1 assay. Growth characteristics of the original D-17 cells, mock-infected (MI) or infected with adenovirus vectors expressing either ZF (Adeno-ZF) or β-galactosidase (Adeno-LacZ) are shown for comparison. (C) Mutations in the leucine zipper of Zhangfei-expressing D-17 clones. Amino acid sequence of the LZip region for Zhangfei recovered by PCR from the vector used to develop the clones and the doxycycline-inducible Zhangfei expressing clones. Dashes (−) indicate no changes. Leucine residues (L) comprising the zipper are shown and mutations are in bold. (D) Induction of mutant Zhangfei has no effect on the UPR. Clones expressing ZF with mutations in the leucine zipper did not inhibit the UPR. Original D-17 cells and clones 4 and 15 were treated with 1 µg/ml doxycycline for 24 h, followed by 4 h of treatment of thapsigargin. Then cells were harvested and UPR transcripts were estimated by qRT-PCR. The original D-17 cells, mock-infected or infected with Adeno-ZF were also analyzed for comparison. The values represent fold changes in levels of transcripts between Adeno-ZF infected cells with mock-infected cells (in original D-17 cells) or between doxycycline treated with untreated cells (in Tet-on clones). Error bars indicated standard deviations from means of 3 individual experiments. Dashed lines indicate fold changes of less or more than 2-fold and changes exceeding these limits were arbitrarily regarded as significant. Bar = 100 µm.