Journal: Nature Communications

Article Title: A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs

doi: 10.1038/s41467-024-45612-5

Figure Lengend Snippet: a – c Representative confocal IF images of the liver of XCR1 Venus/+ rAAV-treated recipient mice at 12-days post rAAV, showing large clusters of XCR1 + cDC1s, gBT-1 and P25 T cells in PT regions Scale bars 50 μm ( a ), 30 μm ( b ), 4 μm/2 μm ( c ). d , e Quantification of the average distance (μm) separating XCR1 + cells with gBT-1 T cells and ( d ), or with P25 T cells ( e ) in portal tracts (PT), peri-central vein (PCV) and sinusoidal (S) liver regions. Data points represent a single region of interest from n = 7 mice from 2 independent experiments. f , g Hepatic XCR1 + cDC1 isolated from recipient mice treated, 12-days prior, with rAAV gB-Ag85b or PBS, were assessed for their ability to activate and induce proliferation (assessed by CD44 upregulation and CTV dilution), of naive gBT-1 ( f ) or P25 ( g ) T cells co-cultured for 72 h. h , i Same as ( f , g ) but recipient mice were also treated with isotype control (black) or anti-CD40L blocking mAb (red) to assess the role of CD40/CD40L interactions. Representative flow plots ( h ), and proportion of proliferating CD44 high CTV low gBT-1 T cells ( i ). j Co-stimulatory molecule expression levels in hepatic cDC1s isolated from rAAV gB-Ag85b –treated mice that also received isotype control (black) or anti-CD4 depleting mAb (red) at 12-days post rAAV. k , l Quantification of Ki67 + gB T-1 T cell numbers forming clusters in PT ( k ) and PCV regions ( l ) at 12-days post-rAAV treatment in XCR1 +/+ (black) and XCR1 DTR/+ (red) recipient mice treated with DTx, 9-days prior. m , n Quantification of total numbers of intrahepatic ( m ) and splenic ( n ) effector gBT-1 cell in recipient mice treated with rAAV, 15-days prior. Comparison between diphtheria toxin treated XCR1 +/+ (black) and XCR1 DTR/+ (red) recipient mice at 9-days post-rAAV.Data representative of two independent experiments. n = 5 ( a – c ), n = 7 ( d , e ), n = 3 or 4 ( f , g ), n = 7 ( i ), n = 4 ( j ) n = 5 ( k , l ) and n = 6 ( m , n ) mice per group. Error bars indicate ± SEM. Analysed with a Kruskal-Wallis test with multiple comparisons ( d , e ), a two-tailed unpaired Student’s t test ( i , j ) or a two tailed unpaired Mann–Whitney U test ( k – n ); ns not statistically significant. Source data are provided as a Source Data file.

Article Snippet: For CD4 + - CD8 + T cell and T cell-XCR1 + cDC1 proximity analysis, CD45.1 + gBT-1 T cells, GFP + P25 T cells and Venus + XCR1 cells were segmented in Imaris 9.6 using the surface function and the average distance between cells was calculated in portal tracts, PCV regions and the sinusoidal compartment using the XTension plugin “Spots and Surfaces Distance” MATLAB version: 9.13.0 (The MathWorks Inc. 2022.

Techniques: Isolation, Cell Culture, Control, Blocking Assay, Expressing, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Distinct interactions stabilize EGFR dimers and higher-order oligomers in cell membranes.

doi: 10.1016/j.celrep.2023.113603

Figure Lengend Snippet: Figure 2. TM domain dimerization motifs do not influence EGFR diffusion (A) Mutations introduced to disrupt Sm-x-x-x-Sm dimerization motifs40 in the EGFR TM domain. The three Sm-x-x-x-Sm motifs are marked, where Sm is any small amino acid (red): T-g-m-v-G; G-m-v-g-A; and A-l-g-i-G. All Sm residues were replaced with valine in TM3X. (B) SPT data for wild-type EGFR (gray) and a variant with the TM3X TM domain shown in (A) (blue). Unliganded receptors were tracked using QD-HA (open diamonds), and ligand-bound receptors were tracked using 200 pM QD-EGF (filled diamonds). EGF induces a similar slowdown for wild-type EGFR (n = 40 cells without ligand, 42 with; p = 6.7 3 106) and the TM3X variant (n = 45 without ligand, 46 with; p = 1.9 3 107). Plots show variability and mean (±SD) across n cells. Unpaired two-sided Welch’s t tests were used to calculate p values (**p < 1 3 103; ***p < 1 3 106). See also Table S2. (C) pEGFR immunoblots of wild-type and TM3X EGFR activated with 16 nM EGF for 5 min and probed with anti-pY1173 (upper), anti-EGFR (middle), and anti-GRB2 (lower) as loading control. Representative of three biological re- peats.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER N-terminally HaloTagged EGFR(DC-Tail) in pcDNA3.1 This study N/A pHTC HaloTag CMV-neo Vector Promega Cat#: G7711 pFastbac Vector Kit ThermoFisher Scientific Cat#: 10360014 pFastbac sEGFR Ferguson et al.3 N/A pFastbac EGFR TKD [672–998] Park et al.59 N/A pFastbac EGFR JM-TKD [645–998] Red Brewer et al.9 N/A pFastbac EGFR JM-TKD [645–998], V924R This study N/A pFastbac EGFR TKD [672–998], I682Q This study N/A pSpCas9(BB)-2A-GFP (PX458) Ran et al.74 Addgene Cat#: 48138 Software and algorithms DBSCAN Ester et al.75 N/A SMITE (Single Molecule Imaging Toolbox Extraordinaire) K. Lidke lab76 https://github.com/LidkeLab/smite MATLAB version: 9.13.0 (R2022b) The MathWorks Inc., Natick, MA, USA https://www.mathworks.com Origin 2021b (9.8.5.201) OriginLab Corporation, Northampton, MA, USA https://www.originlab.com/ PyMol Molecular Graphics System (Version 2.5.0) Schrödinger, LLC http://www.pymol.org GraphPad Prism 9.5.1 GraphPad Software, La Jolla California USA https://www.graphpad.com/ ExPASy ProtParam Tool Gasteiger et al.77 https://web.expasy.org/protparam/ Broad Institute gRNA designer Broad Institute https://portals.broadinstitute.org/gppx/ crispick/public Adobe Photoshop 24.2.0 Adobe Inc. https://www.adobe.com/products/ photoshop.html

Techniques: Diffusion-based Assay, Variant Assay, Western Blot, Control