Figures in the lower left quadrant indicate the percentage of live cells at the time of analysis. of the floxed region is also sketched. (B) DNA from your targeted CJ7 ES cells was digested with the indicated restriction enzymes and subjected to southern blot analysis with either a probe realizing the short arm (SA) or long arm (LA). The short arm of the targeted allele yields a 5kb fragment upon EcoRI digestion. Similarly, the long arm of the targeted allele yields a 17kb fragment upon EcoRV and NotI digestion.(TIFF) pone.0131071.s001.tiff (3.3M) GUID:?6188C477-C152-4B85-A9DE-2DDEA63DCF17 S2 Fig: PCR Genotyping strategy for mice. A schematic of lengths of the expected PCR products (A) are shown. Tail DNA from mice with the indicated genotypes that were either untreated or tamoxifen-treated, and representative PCR results are shown (B).(TIFF) pone.0131071.s002.tiff (8.8M) GUID:?1E419894-1058-4F1D-B096-391920D3B2BF S3 Fig: Itpkb is required for the upregulation of activation markers on double-positive thymocytes and not required for T-independent antibody responses. (A) Circulation cytometry of thymocytes from WT, mice stained with antibodies to CD4, CD8, TCRb, and CD3. CD4+CD8+ cells were gated, and the percentage of cells expressing TCRb (top) or CD3 (bottom) is shown. The figures in the plots show the percentages of each gated populace. (B) Sera from WT and mice that were immunized with the T-independent antigen, TNP-Ficoll in Fig 2A, were tested for TNP-specific IgG2b antibody levels by ELISA on day 12 post-immunization. Data shown are one representative experiment (**, P 0.01).(TIFF) pone.0131071.s003.tiff (3.2M) GUID:?B38D5E06-0188-4906-916D-7361D1CF96D0 S4 Fig: Itpkb-deficient mature B lymphocytes proliferate normally, yet exhibit enhanced SOC entry. (A) B220+ cells were stimulated with numerous concentrations of F(ab)2 anti-IgM, anti-CD40, or LPS, and proliferation was measured by Cell Titer Glo. (B) Splenocytes gated on B220 were stimulated with F(ab)2 anti-IgM in the presence of exogenous calcium(C), or in the absence of exogenous calcium, followed by calcium re-addition (D). Data is usually shown as the mean fluorescent ratio of Fluo-3 and Fura-Red. The data are representative of five independent experiments.(TIFF) pone.0131071.s004.tiff SRT3190 (4.5M) GUID:?A94A73FA-F982-46CD-A3E8-1D0D45DC5978 S5 Fig: Itpkb negatively regulates activation-induced cell death of T lymphocytes via FasL. (A) Purified CD4+ cells were labeled with CFSE and stimulated with anti-CD3/28 beads in the presence of anti-FasL or an isotype control Ig. 72 hours following stimulation, CFSE dilution versus Annexin V staining was followed to determine whether Annexin V positivity required cell division. Numbers in the top right quadrant indicate the percentage of cells that died prior to cell division. Data shown are representative of four independent experiments.(TIFF) pone.0131071.s005.tiff (3.6M) GUID:?3BD2EC97-CA87-4EA4-B898-23D8ED39F214 S6 Fig: Itpkb does not control cytokine production. Itpkb-deficient T cells which survive primary stimulation do not possess any cytokine defects upon secondary stimulation. WT and Itpkb-deficient CD4+ T cells were stimulated with anti-CD3/28 beads in either Th1- or Th2-skewing conditions in the presence of exogenous IL-2. After 6 days in culture, live cells were re-stimulated and stained intracellularly for either IL-2 and IFN (Th1 cells) or IL-4 (Th2 cells). The bar graph represents the percentage of CD4+ cells which are positive for the respective cytokine. Data shown is representative of three independent experiments.(TIFF) pone.0131071.s006.tiff (6.5M) GUID:?539EA58B-31F4-46AD-8D15-B744D0D765DD S7 Fig: GNF362 does not exhibit activity on other protein or lipid kinases The activity of GNF362 was tested across a panel of 159 protein and lipid kinases. The percent of kinase inhibition at a concentration of 5M is shown.(TIFF) pone.0131071.s007.tiff (2.3M) GUID:?DE4C46AC-902D-410F-9766-8983696E57A0 S8 Fig: GNF362 specifically blocks IP4 production. Jurkat T cells were labeled with 3H-myo-inositol and activated through the T cell receptor for 5 minutes. The inositol phosphates IP3, IP4, and IP5 were resolved by HPLC using an in-line -ram detector. Raw HPLC traces from cells stimulated with anti-CD3 + anti-CD28 in the absence or presence of GNF362 are shown in (A). The area under the peaks corresponding to IP4 and IP5 were quantified, and data was normalized to IP5 levels, as this remained unchanged with stimulation. Normalized IP4 levels as a function of GNF362 concentration with an IC50 of 20nM is shown in (B). Data shown is one representative experiment.(TIFF) pone.0131071.s008.tiff (4.8M) GUID:?4F6E404A-C48B-42CA-B910-E26F10753D2E S9 Fig: GNF362 enhances SOC entry in thymocytes and mature T lymphocytes. The effect of GNF362 on Ca2+ responses was measured using the Ca2+ sensitive dyes Fluo-4 and Fura Red following.The complete floxed allele was obtained after excision of the FRT-flanked neo cassette through crossing to FLPeR transgenic mice. to FLPeR transgenic mice. The structure of the deleted allele obtained after Cre-mediated excision of the floxed region is also sketched. (B) DNA from the targeted CJ7 ES cells was digested with the indicated restriction enzymes and subjected to southern blot analysis with either a probe recognizing the short arm (SA) or long arm (LA). The short arm of the targeted allele yields a 5kb fragment upon EcoRI digestion. Similarly, the long arm of the targeted allele yields a 17kb fragment upon EcoRV and NotI digestion.(TIFF) pone.0131071.s001.tiff (3.3M) GUID:?6188C477-C152-4B85-A9DE-2DDEA63DCF17 S2 Fig: PCR Genotyping strategy for Rabbit Polyclonal to MRPS18C mice. A schematic of lengths of the expected PCR products (A) are shown. Tail DNA from mice with the indicated genotypes that were either untreated or tamoxifen-treated, and representative PCR results are shown (B).(TIFF) pone.0131071.s002.tiff (8.8M) GUID:?1E419894-1058-4F1D-B096-391920D3B2BF S3 Fig: Itpkb is required for the upregulation of activation markers on double-positive thymocytes and not required for T-independent antibody responses. (A) Flow cytometry of thymocytes from WT, mice stained with antibodies to CD4, CD8, TCRb, and CD3. CD4+CD8+ cells were gated, and the percentage of cells expressing TCRb (top) or CD3 (bottom) is shown. The numbers in the plots indicate the percentages of each gated population. (B) Sera from WT and mice that were immunized with the T-independent antigen, TNP-Ficoll in Fig 2A, were tested for TNP-specific IgG2b antibody levels by ELISA on day 12 post-immunization. Data shown are one representative experiment (**, P 0.01).(TIFF) pone.0131071.s003.tiff (3.2M) GUID:?B38D5E06-0188-4906-916D-7361D1CF96D0 S4 Fig: Itpkb-deficient mature B lymphocytes proliferate normally, yet exhibit enhanced SOC entry. (A) B220+ cells were stimulated with various concentrations of F(ab)2 anti-IgM, anti-CD40, or LPS, and proliferation was measured by Cell Titer Glo. (B) Splenocytes gated on B220 were stimulated with F(ab)2 anti-IgM in the presence of exogenous calcium(C), or in the absence of exogenous calcium, followed by calcium re-addition (D). Data is shown as the mean fluorescent ratio of Fluo-3 and Fura-Red. The data are representative of five independent experiments.(TIFF) pone.0131071.s004.tiff (4.5M) GUID:?A94A73FA-F982-46CD-A3E8-1D0D45DC5978 S5 Fig: Itpkb negatively regulates activation-induced cell death of T lymphocytes via FasL. (A) Purified CD4+ cells were labeled with CFSE and stimulated with anti-CD3/28 beads in the presence of anti-FasL or an isotype control Ig. 72 hours following stimulation, CFSE dilution versus Annexin V staining was SRT3190 followed to determine whether Annexin V positivity required cell division. Numbers in the top right quadrant indicate the percentage of cells that died prior to cell division. Data shown are representative of four independent experiments.(TIFF) pone.0131071.s005.tiff (3.6M) GUID:?3BD2EC97-CA87-4EA4-B898-23D8ED39F214 S6 Fig: Itpkb does not control cytokine production. Itpkb-deficient T cells which survive primary stimulation do not possess any cytokine defects upon secondary stimulation. WT and Itpkb-deficient CD4+ T cells were stimulated with anti-CD3/28 beads in either Th1- or Th2-skewing conditions in the presence of exogenous IL-2. After 6 days in culture, live cells were re-stimulated and stained intracellularly for either IL-2 and IFN (Th1 cells) or IL-4 (Th2 cells). The bar graph represents the percentage of CD4+ cells which are positive for the respective cytokine. Data shown is representative of three independent experiments.(TIFF) pone.0131071.s006.tiff (6.5M) GUID:?539EA58B-31F4-46AD-8D15-B744D0D765DD S7 Fig: GNF362 does not exhibit activity on other protein or lipid kinases The activity of GNF362 was tested across a panel of 159 protein and lipid kinases. The percent of kinase inhibition at a concentration of 5M is shown.(TIFF) pone.0131071.s007.tiff (2.3M) GUID:?DE4C46AC-902D-410F-9766-8983696E57A0 S8 Fig: GNF362 specifically blocks IP4 production. Jurkat T cells were labeled with 3H-myo-inositol and activated through the T cell receptor for 5 minutes. The inositol phosphates IP3, IP4, SRT3190 and IP5 were resolved by HPLC using an in-line -ram detector. Raw HPLC traces from cells stimulated with anti-CD3 + anti-CD28 in the absence or presence of GNF362 are shown in (A). The area under the peaks corresponding to IP4 and IP5 were quantified, and data was normalized to IP5 levels, as this remained unchanged with stimulation. Normalized IP4 levels as a function of GNF362 concentration with an IC50 of 20nM SRT3190 is shown in (B). Data shown is one representative experiment.(TIFF) pone.0131071.s008.tiff (4.8M) GUID:?4F6E404A-C48B-42CA-B910-E26F10753D2E S9 Fig: GNF362 enhances SOC entry in thymocytes and adult T lymphocytes. The effect of GNF362 on Ca2+ reactions was measured using the Ca2+ sensitive dyes Fluo-4 and Fura Red following TCR-mediated cross-linking either in the presence or absence of exogenous Ca2+. (A) CD4+8+, CD4+, or CD8+ thymocytes pre-incubated with DMSO or 1M of GNF362, were treated with anti-CD3-biotin, followed by cross-linking with streptavidin in the presence of exogenous calcium (remaining column), or in the absence of exogenous calcium, followed by calcium re-addition to examine SOC channel function (ideal column). (B) Similarly, CD4+ or CD8+.