Targeted disruption of the Klf13 gene. A, Schematic representation of the wild-type Klf13 allele, the targeting vector, and the predicted targeted allele are shown. In the targeted Klf13 allele, Neo replaces exon 1 plus 0.7 kb upstream and 0.6 kb downstream of the Klf13 gene. B, BamHI; H, HindIII; S, SacII; N, NotI; TK, thymidine kinase; Neo, neomycin resistance. B, Southern blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. After BamHI digestion, the wild-type allele (Klf13^WT) is detected as a 3.6-kb fragment, whereas the targeted allele (Klf13^Neo) is detected as a 4.5-kb fragment using probe as indicated in Fig. 1A. C, Western blot of KLF13 expression in thymocytes from two littermate pairs of wild-type (+/+) and mutant (−/−) mice, with TUBA1 used as an internal control. D, Western blot of KLF13 expression in splenocytes, with JAB1 used as an internal control. Splenocytes from Klf13^+/+ and Klf13^−/− littermates were left untreated (Day 0) or activated with anti-CD3 and anti-CD28 Abs for 5 days (Day 5). E, KLF13 positively regulates RANTES expression in T lymphocytes. Splenic CD3⁺ T lymphocytes isolated from Klf13^+/+ and Klf13^−/− littermates were stimulated with anti-CD3 and anti-CD28 Abs. Supernatants were removed on day 1 and day 5 and RANTES was measured by ELISA. Data presented are the mean ± SD from three pairs of littermates (**, p < 0.01).

Phenotypic comparison of thymus and spleen from Klf13^+/+, Klf13^+/−, and Klf13^+/− littermates. A, Increased mass of lymphoid organs and increased numbers of thymocytes and splenocytes in Klf13^−/− mice. Three sets of littermates were subjected to pathological examination and all other parameters examined were normal. B, Enlarged spleens of Klf13^−/− mice from three pairs of littermates. C, Increased numbers of thymocytes in Klf13^−/− mice at different ages as indicated. All values are the mean ± SD from three pairs of littermates (*, p < 0.05 and ***, p < 0.001). D, Increased numbers of splenocytes in Klf13^−/− mice at different ages as indicated. All values are the mean ± SD from three pairs of littermates (**, p < 0.01 and ***, p < 0.001).

Normal proliferation and cell division in cells from Klf13^−/− mice. A, Incorporation of BrdU into bone marrow cells (BM, top panels) and thymocytes (T, bottom panels) from Klf13^+/+ and Klf13^−/− mice. B, Cell division in activated Klf13^+/+ and Klf13^−/− CD4⁺ or CD8⁺ splenocytes. CFSE-labeled cells were cultured in vitro with anti-CD3 and anti-CD28 Abs and analyzed by flow cytometry at the indicated times. For both experiments, three littermate pairs were tested and data from one littermate pair are presented.

Thymocytes in Klf13^−/− mice exhibit defective cell death. A, TUNEL assay. Fold decrease in apoptotic cells of thymocytes in Klf13^−/− mice in comparison to Klf13^+/+ mice. Data are the mean ± SD from three pairs of littermates (***, p < 0.001). B, Klf13^−/− thymocytes are more resistant to spontaneous cell death when cultured in vitro. Data are the mean ± SD from three pairs of littermates. Differences between Klf13^+/+ and Klf13^−/− mice were analyzed by ANOVA and shown to be significant (p < 0.0001). C, Klf13^−/− thymocytes are more resistant to activation-induced cell death than Klf13^+/+ thymocytes when cultured in vitro with Con A (10 µg/ml). Data are the mean ± SD from three pairs of littermates. Differences between Klf13^+/+ and Klf13^−/− mice were analyzed by ANOVA and shown to be significant (p < 0.0017). D, Cells from Klf13^−/− mice are more resistant to staurosporine-induced apoptosis compared with Klf13^+/+ mice. Three littermate pairs were tested in triplicate and data from one littermate pair are presented as the mean ± SD (**, p < 0.01 and ***, p < 0.001).

Increased expression of BCL-X_L in thymocytes and splenocytes from Klf13^−/− mice. A, rt-qPCR analysis. Fold increase in Bcl-xL expression in thymocytes and splenocytes of Klf13^−/− and Klf13^+/− mice compared with Klf13^+/+ mice was measured by rt-qPCR and normalized by Gusb. Values are the mean ± SD for three sets of littermates (*, p < 0.05 and **, p < 0.01). B, Western blot analysis. Fold increase in BCL-X_L expression in thymocytes and splenocytes of Klf13^−/− and Klf13^+/− mice compared with Klf13^+/+ mice was detected in whole cell lysates and normalized to JAB1. Quantitation was performed by densitometry and values are graphed as the mean ± SD from three sets of littermates (*, p < 0.05 and ***, p < 0.001). Blots shown are representative of one littermate set. C, rt-qPCR analysis of CD4⁺CD8⁻ thymocytes. Fold increase in Bcl-xL expression in CD4⁺CD8⁻ cells of Klf13^−/− and Klf13^+/− mice compared with Klf13^+/+ mice was measured by rt-qPCR and normalized by Gusb. Values are the mean ± SD for three sets of littermates (*, p < 0.05 and **, p < 0.01).

KLF13 is a negative regulator of Bcl-xL expression. A, Schematic representation of the Bcl-xL promoter showing KLF13 consensus binding sites (❙). Numbers are relative to the +1 Bcl-xL translational start site. B, EMSA of recombinant GST-KLF13 binding to the Bcl-xL promoter was performed using probes corresponding to sequential 300-bp regions of the Bcl-xL promoter from −1,800 to −1 bp relative to the translational start site. Purified GST from empty pGEX4T-1 vector was used as a negative control. Supershift using anti-KLF13 Ab or a negative control Ab (p53 Ab) is indicated. C, KLF13 represses Bcl-xL promoter activity. NIH3T3 cells were cotransfected with a series of luciferase reporter constructs containing nested deletions or sequential 300-bp regions of the Bcl-xL promoter from −1,800 to −1 bp relative to the translational start site as indicated, and either pcDNA3.1(+) or mouse KLF13 expression plasmid. Luciferase activity was measured in cell lysates from transfected cells and normalized by total cellular protein concentration. Values are the mean ± SD for three independent transfections (*, p < 0.05, **, p < 0.01, and ***, p < 0.001).