Zfx loss induces integrated stress response in BCR-activated B cells. (A) Differential gene expression in BCR-stimulated B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. Sorted FO B cells were stimulated with anti-IgM for 0, 2, or 12 hours and analyzed by microarray expression profiling. Shown is pair-wise comparison of control (Ctrl) and CKO cells at each time point; the scatter plots represent normalized log intensities of individual microarray probe signals. The probes increased or decreased greater than 5-fold in CKO cells are indicated in green and red, respectively, with the number of each probe set indicated. Probes for previously identified Zfx targets 6720467C03Rik and Dis3l are highlighted in yellow and blue circles, respectively. The probes for ISR target genes induced in CKO cells are indicated. (B) Principal component analysis (PCA) of global gene expression in BCR-stimulated FO B cells. Shown are the top principal components in the dataset, with gray lines representing individual probes and red lines representing the average probe intensity in the probe set. The average intensities at the zero time point are indicated by dashed lines for each principal component. (C) The expression of ISR genes in BCR-stimulated FO B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. FO B cells were stimulated as in Figure 6A. Shown are normalized expression levels of indicated genes relative to the unstimulated control sample, as determined by qPCR (mean ± SD of triplicate reactions). (D) Expression of ATF4 protein in BCR-stimulated B cells from control and CD19-Cre⁺Zfx^flox/y CKO mice. Resting B cells were stimulated with anti-IgM for the indicated time points. Whole cell lysates were analyzed by Western blotting for ATF4 and β-tubulin. Data are representative of 3 independent experiments.