Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: Gene targeting strategy and analysis of genomic DNA. A. Gene-targeting strategy used to generate the null alleles at the RGC-32 locus. Diagrams representing the wild type (top) and targeted (bottom) RGC-32 alleles. Tick marks represent 1 kb distances, except for contracted longer stretches marked with SS marks. Green and white boxes represent coding regions and UTRs respectively, and splice junctions are indicated by angled black lines. Homologous recombination targeting arms (5′ – 4.2 kb and 3′ – 2.7 kb) are indicated by black lines, and the 3′ southern blot probe as a black box under the locus. FRT sites (blue circles) flank the PGK-Neo Cassette. Genotyping primers for the wild type (thin black arrowheads − 167 bp) are spanning exon 1 and encompass the ATG. Genotyping primers for the KO (thin white arrow heads − 600 bp) are placed in the PGK-Neo cassette and the 3′ arm respectively. Eco RV (R) digestion of genomic DNA and hybridization with the indicated 3′ probe result in 8.5 kb (wild type) and 3.5 kb (knock-out) fragments. In the KO allele, exons 1 and 2, including the ATG are removed. B. Southern blot analysis of genomic DNA. Southern blot analysis of genomic DNA extracted from ES clones. The DNA was digested with EcoRV and hybridized with probe. The sizes of the wild type (WT) and disrupted (KO) alleles are shown. The genotype of the clones is shown above each lane. C. Southern blot analysis of genomic DNA extracted from mouse tails using a DNA Easy tissue kit (Qiagen). The sizes of the WT and KO alleles are shown. The genotypes of the animals are presented above the lanes. D. PCR amplification of DNA extracted from mouse tails. The sizes of the PCR fragments used to identify the WT and KO alleles are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Homologous Recombination, Southern Blot, Hybridization, Knock-Out, Clone Assay, Amplification

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: RGC-32 expression in mouse tissues. A. RGC-32 mRNA expression analyzed by Northern blotting. Tissue expression of RGC-32 was analyzed by Northern blotting in WT and RGC-32 KO mice. RGC-32 mRNA expression was detected in tissues obtained from the WT mice but not detected in the RGC-32 KO mice. β-Actin was used as a housekeeping gene. B. RGC-32 protein expression analyzed by Western blotting. Tissue expression of RGC-32 protein was analyzed by Western blotting. RGC-32 protein expression was detected in tissue obtained from the WT mice. No RGC-32 protein expression was detected in the RGC-32 KO mice.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Expressing, Northern Blot, Western Blot

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: Immunohistochemical localization of RGC-32. RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32−/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector). A significant amount of RGC-32 protein expression was found in WT mice in Purkinje cells (A), lung bronchiolar epithelium (C), thymus (E), renal tubular cells (G), and spleen, where the stained cells were in the periarteriolar lymphoid sheets and germinal center follicular cells (I). RGC-32 expression was absent from the tissues obtained from the RGC-32−/− mice (B, D, F, H, and J). Original magnification: ×400.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Immunohistochemical staining, Expressing, Staining, Plasmid Preparation

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: RGC-32 suppresses cell proliferation in T cells. A. Splenocytes from WT or KO (RGC-32−/−) mice were stimulated with the indicated concentrations of plate-coated anti-CD3 (5 μg/ml) plus soluble anti-CD28 (2.5 μg/ml) mAb for 48 h. [3H]thymidine was added for the final 18 h of culture, and proliferation was measured. Significantly higher levels of [3H]thymidine incorporation were found in RGC-32−/− mice when compared to WT mice. Results (mean ± SEM from three separate experiments) are shown as stimulation index (p < 0.03). B. Purified CD4+ T cells from WT or RGC-32−/− mice were stimulated with anti-CD3 (5 μg/ml) plus soluble anti-CD28 (2.5 μg/ml) for 48 h. [3H]thymidine was added for the final 18 h of culture, and proliferation was measured by tritium uptake. Significantly higher levels of [3H]thymidine incorporation were found in CD4+ T cells isolated from spleens of RGC-32−/− mice when compared to WT mice (p < 0.01). C. Ki-67 uptake after stimulation with anti-CD3 (5 μg/ml) plus soluble anti-CD28 (2.5 μg/ml) for 48 h was measured in CD4 and CD8 cells isolated from RGC-32−/− and WT mice by FACS analysis. A significant increase in the percentage of Ki-67-positive CD4 (p < 0.007) and CD8 (p < 0.05) cells was found after stimulation with CD3/CD28 in RGC-32−/− mice when compared to WT mice. D. Effect of LY294002 on cell proliferation. Purified CD4+ T cells from WT or RGC-32−/− mice were stimulated with anti-CD3 (5 μg/ml) plus soluble anti-CD28 (2.5 μg/ml) for 48 h in the presence or absence of LY294002 (10 μM). [3H]thymidine was added for the final 18 h of culture, and proliferation was measured. LY294002 pretreatment significantly reduced the increase in cell proliferation induced after anti-CD3/CD28 stimulation in both WT and RGC-32−/− mice. Results of three separate experiments are expressed as mean ± SEM.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Purification, Isolation

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: RGC-32 regulates Akt phosphorylation in CD4+ T cells. Purified CD4+ T cells from WT or RGC-32−/− mice were stimulated with anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml) for the indicated periods of time. Cells were lysed, and Akt phosphorylation was assessed by Western blotting with an anti-Akt IgG specific for Akt phosphorylated at Ser 473. The absence of RGC-32 from CD4+ T cells resulted in a significant increase in Akt phosphorylation 1 h after the initiation of co-stimulation (p < 0.05). Results of three separate experiments are expressed as mean ± SEM.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Purification, Western Blot

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: RGC-32 regulates FOXO1 phosphorylation in CD4 T cells. Purified CD4+ T cells from WT or RGC-32−/− mice were stimulated with anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml) for the indicated periods of time. Cells were lysed with RIPA buffer, and FOXO1 phosphorylation was assessed by Western blotting with antibodies specific for FOXO1 phosphorylated at Ser 256 and anti-FOXO1 IgG. In the absence of RGC-32 in CD4+ T cells, a significant increase in FOXO1 phosphorylation was seen at 30 min and 1 h after initiation of co-stimulation (p < 0.01) when compared to unstimulated cells. Results of three separate experiments are expressed as mean ± SEM.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Purification, Western Blot

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: RGC-32 regulates IL-2 expression and release. Purified CD4+ T cells from WT or RGC-32−/− mice were stimulated with anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml) for the indicated periods of time. A. IL-2 mRNA expression was significantly increased after 24 h of stimulation (p < 0.05). B. A similar effect was seen in the IL-2 levels released into the culture supernatant after stimulation. IL-2 protein levels were determined by ELISA. Results of three separate experiments are expressed as mean ± SEM.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Expressing, Purification, Enzyme-linked Immunosorbent Assay

Journal: Experimental and molecular pathology

Article Title: RGC-32 is a novel regulator of the T-lymphocyte cell cycle

doi: 10.1016/j.yexmp.2015.03.011

Figure Lengend Snippet: Role of RGC-32 in T-cell receptor signaling. Signaling through the T-cell receptor (TCR) is known to involve phosphoinositide 3-kinase (PI3K). The co-stimulatory receptors CD28 contain a consensus PI3K-binding motifs (YXXM), which might also contribute to TCR-dependent PI3K signaling. AKT connects PI3K to signaling pathways that promote cytokine transcription, survival, and cell-cycle activation. RGC-32 acts on Akt which phosphorylates FOXO1 protein, induces IL-2 transcription and cell cycle activation contributing to TCR mediated signaling. Secreted IL-2 has an autocrine effect on T-cells and might be implicated in further activation of PI3K.

Article Snippet: RGC-32 expression was localized by immunohistochemical staining in tissues from WT and RGC-32 −/− mice using a rabbit IgG anti-RGC-32 antibody and a RTU Vectastain universal kit (Vector).

Techniques: Binding Assay, Activation Assay