Journal: eLife

Article Title: A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

doi: 10.7554/eLife.80277

Figure Lengend Snippet: Phenotypic analyses of thymocytes from control and CD2-Lis1 cKO mice. ( A ) Dot plots show CD4 versus CD8 surface staining on thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of four independent experiments each including n = 3–4 mice per group. ( B ) Dot plots show CD44 versus CD25 surface staining on CD4-CD8- [DN] thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the numbers of thymocytes in each indicated subset from mice of the indicated genotype. Data are mean ± SD and represent a pool of five independent experiments each including n = 4–5 mice per group. ( C ) Dot plots show CD27 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD27 hi thymocytes in DN3 thymocytes. Data are mean ± SD and represent a pool of two independent experiments each including n = 2–3 mice per group. ( D ) Dot plots show CD5 versus TCRβ intracellular staining on DN3 thymocytes from control and CD2-Lis1 cKO mice. Histogram bars represent the percentages of TCRβ hi CD5 hi thymocytes in DN3 thymocytes and the MFI of TCRβ and CD5 in DN3 TCRβ hi CD5 hi thymocytes from mice of the indicated genotype. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( E ) Histogram graphs show IL-7R, CD71 surface staining and forward-scatter (FSC) on DN3 thymocytes expressing the TCRβ chain. Histogram bars represent the MFI of IL-7R, CD71, and FSC in the indicated DN3 thymocytes subsets. Data are mean ± SD and represent a pool of two independent experiments each including n = 3 mice per group. ( F ) Histogram graphs show DNA intracellular staining on DN3 thymocytes from the indicated subsets. The percentages represent cells in the G2/M phase of cell cycle. Histogram bars represent the percentages of DN3 TCRβ hi CD5 hi thymocytes in the G2/M phase of cell cycle. Data are mean ± SD and represent a pool of three independent experiments each including n = 1 mouse per group. Unpaired two-tailed Mann–Whitney t tests were performed for all analyses. **p<0.01; ***p<0.001; ****p<0.0001. Figure 1—source data 1. LIS1 is required for T-cell development following the β-selection checkpoint.

Article Snippet: Antibody , Anti-CD5 (rat monoclonal) , Thermo Fisher Scientific , Clone 53-7.3 , Conjugated to FITC (1/1000).

Techniques: Staining, Expressing, Two Tailed Test, MANN-WHITNEY, Selection

Journal: eLife

Article Title: A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage

doi: 10.7554/eLife.80277

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-CD5 (rat monoclonal) , Thermo Fisher Scientific , Clone 53-7.3 , Conjugated to FITC (1/1000).

Techniques: Staining, Purification, Binding Assay, Magnetic Beads, Microscopy, Software

Journal: bioRxiv

Article Title: The Cul5 E3 Ligase Complex Is a Key Negative Feedback Regulator of TCR/IL2 Signaling and Anti-Tumor Activity in CD8 + T Cells

doi: 10.1101/2022.11.16.516824

Figure Lengend Snippet: a, DIA-MS signals of the CUL5 protein in NC (Black) or Cul5 (Red) KO primary CD8 + T cells at the T0, T8 and T16 conditions determined by mass spectrometry (MS). Data are shown as mean ± SEM (**p < 0.01 and ****p < 0.0001 by multiple t-test, n=3). b, Flow cytometry analysis of Cul5 expression in primary CD8 + T cells before (Black) or after (Blue) 2-day activation by anti-CD3/CD28 beads. Data are shown as mean ± SEM (****p < 0.0001, by unpaired t-test, n=3). c, Western blot analysis of CUL5 expression in primary CD8 + T cells treated as in b . The ratio of neddylated to non-neddylated Cul5 (Bottom) was quantified. d, Volcano plot of differentially expressed proteins between Cul5 KO and non-targeting (NC) primary CD8 + T cells at T0 (Left) and T16 (Right) conditions as quantified by DIA-MS (n=3). e,f, Flow cytometry validation of immunological markers (CD25, CD5, CD137, ICOS, PD1, CTLA4 and CD62L) altered in Cul5 KO primary CD8 + T cells at T0 and/or T16 conditions. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). h, Live cell numbers of NC (Black) and Cul5 (Red) KO primary CD8 + T cells before and after 16-hour anti-CD3 plus anti-CD28 stimulation. Data are shown as mean ± SEM (****p < 0.0001 by unpaired t-test, n=3). i, Growth curve of tumors from E.G7-OVA cells inoculated s.c. into C57BL/6 mice. Data are shown as mean ± SEM. The back arrow indicates the time of sub-lethal irradiation followed by immediate adoptive transfer of Cas9/OT-I cells with NC (Black), Ctla4 (Blue), Cul5 (Pink) and Ctla4/Cul5 double (Red) KO. (*p<0.05, ***p<0.001 and ****p<0.0001 by two-way ANOVA; n=4-5).

Article Snippet: Antibodies used for flow cytometry are as follows: anti-mouse: Biolegend: CD8a-PE-Cy7 (Cat.100721), CD122-APC (Cat.105911), CD127-APC-Cy7 (Cat.135039), GZMB-APC (Cat.372203), IFNg-APC-Cy7 (Cat.505849), TNF-PE-Cy7 (Cat.506323), IL2-PB (Cat.503820), CD25-APC-Cy7 (Cat.101917), CD5-PB (Cat.100641), ICOS-APC (Cat.107711), PD1-PE-Cy7 (Cat.135215), CTLA4-APC (Cat.106309), CD62L-PE-Cy7 (Cat.104417), Vb5-PB (Cat.139515), Va2-PE (Cat.127807); Thermo Scientific: CD137-PB (Cat.48-1371-82); BD: CD107a-APC (Cat.560646), pSTAT5-Alexa 647 (Cat.562076), anti-human (Biolegend): GZMB-APC (Cat.372203), IFNg-PE (Cat.502508), CTLA4-APC (Cat.369611), anti-rabbit: IgG-PE (Thermo Scientific, Cat. P-2771MP), IgG-Alexa Fluor 350 (Thermo Scientific, Cat.A-11069), anti-pERK1/2 (Cell Signaling Technology, Cat. 9101), Rabbit polyclonal IgG anti-Cul5 (Thermo Scientific, Cat.A302-173A); SIINFEKL-H-2K(b) tetramer-BV421 (NIH Tetramer Core Facility, Cat.53995)

Techniques: Mass Spectrometry, Flow Cytometry, Expressing, Activation Assay, Western Blot, Irradiation, Adoptive Transfer Assay