Figure 5.
DCAF13-deficient T cells showed impaired new protein synthesis caused by abnormal ribosomal maturation. (A) Confocal z-stack three-dimensional reconstruction of nucleoli from CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells to compare nucleolar sizes. Representative images (left) and corresponding quantitation of the nucleolar volume (right) from three independent experiments (naïve T cells, n = 60; stimulated T cells, n = 50). DAPI (blue) and NPM1 (cyan) are shown. Scale bar, 2 μm. (B) Quantitation of the nucleolar number in naïve or stimulated CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells from three independent experiments (naïve T cells, n = 60; stimulated T cells, n = 50). (C) FSAs of CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. (D) Confocal z-stack three-dimensional reconstruction of stimulated T cells isolated from CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) cells to compare their ratios of cytoplasm and nuclei. Representative images (left) and corresponding quantitation from three independent experiments (n = 45) are shown. DAPI (blue) and CD4 (green). Scale bar, 2 μm. (E) Flow cytometry analysis of HPG signals in CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. (F) Immunofluorescence staining of HPG signals in CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. Representative pictures (left) and quantification of the HPG intensity (right; WT, n = 6; cKO, n = 3) are shown. Scale bar, 10 μm. (G) Polysome profile analysis of stimulated T cells with anti-CD3/CD28 treatment for 72 h. These cells were isolated from ERT2 Cre+Dcaf13fl/fl T cells and treated with tamoxifen (regarded as DCAF13 KO) or without tamoxifen (regarded as WT). Cells were loaded in equal amounts. Representative pictures of the A254 absorbance profile in three independent experiments (left) and quantification of the 40S peaks (right, n = 3) are shown. (H) Ribosomal RNA processing products in stimulated T cells were detected by northern blotting with OTSI and OTSII oligos. Loaded RNA was purified from the same number of cells. The gray values of bands were quantified with ImageJ. WT cells were normalized to 1.0. The fold changes of 45S, 30S, and 18S-E rRNA were summarized from three independent experiments (n = 3). Data are presented as the mean ± SD. A two-tailed Student’s t test was used. ns is not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F5.

DCAF13-deficient T cells showed impaired new protein synthesis caused by abnormal ribosomal maturation. (A) Confocal z-stack three-dimensional reconstruction of nucleoli from CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells to compare nucleolar sizes. Representative images (left) and corresponding quantitation of the nucleolar volume (right) from three independent experiments (naïve T cells, n = 60; stimulated T cells, n = 50). DAPI (blue) and NPM1 (cyan) are shown. Scale bar, 2 μm. (B) Quantitation of the nucleolar number in naïve or stimulated CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells from three independent experiments (naïve T cells, n = 60; stimulated T cells, n = 50). (C) FSAs of CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. (D) Confocal z-stack three-dimensional reconstruction of stimulated T cells isolated from CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) cells to compare their ratios of cytoplasm and nuclei. Representative images (left) and corresponding quantitation from three independent experiments (n = 45) are shown. DAPI (blue) and CD4 (green). Scale bar, 2 μm. (E) Flow cytometry analysis of HPG signals in CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. (F) Immunofluorescence staining of HPG signals in CD4 Cre−Dcaf13fl/fl (WT) and CD4 Cre+Dcaf13fl/fl (cKO) T cells stimulated with anti-CD3/CD28 for 24 h. Representative pictures (left) and quantification of the HPG intensity (right; WT, n = 6; cKO, n = 3) are shown. Scale bar, 10 μm. (G) Polysome profile analysis of stimulated T cells with anti-CD3/CD28 treatment for 72 h. These cells were isolated from ERT2 Cre+Dcaf13fl/fl T cells and treated with tamoxifen (regarded as DCAF13 KO) or without tamoxifen (regarded as WT). Cells were loaded in equal amounts. Representative pictures of the A254 absorbance profile in three independent experiments (left) and quantification of the 40S peaks (right, n = 3) are shown. (H) Ribosomal RNA processing products in stimulated T cells were detected by northern blotting with OTSI and OTSII oligos. Loaded RNA was purified from the same number of cells. The gray values of bands were quantified with ImageJ. WT cells were normalized to 1.0. The fold changes of 45S, 30S, and 18S-E rRNA were summarized from three independent experiments (n = 3). Data are presented as the mean ± SD. A two-tailed Student’s t test was used. ns is not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F5.

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