Journal: bioRxiv

Article Title: Remodeling of Il4-Il13-Il5 locus underlies selective gene expression

doi: 10.1101/2022.07.22.501024

Figure Lengend Snippet: a , REs identified in this study were selectively deleted (red bars) to generate 6 mouse strains, and subjected to papain induced lung inflammation ( b ) to compare their type 2 response. WT (naïve; n=24, papain; n=37), KHS-KO (naïve; n=8, papain; n=11), 5HS-I(a+b+c+d)-KO (naïve; n=4, papain; n=10), 5HS-Ie KO (naïve; n=12, papain; n=8), 5HS-II KO (naïve; n=14, papain; n=11), and 5HS-III(a+b+c) KO (naïve; n=11, papain; n=16) were analyzed in two to five independent experiments. c , PAS staining of the lung from mice treated with papain. A representative image from each genotype is shown. d , Histological scores were calculated for each genotype group and compared with statistical evaluation (asterisks). e , Cell numbers recovered from the lungs were compared among 6 genotypes (color code as in d) in naïve and papain challenged conditions. Cells evaluated were eosinophils (CD11b + Siglec-F + ), neutrophils (CD11b + Gr1 + ), Th2 (CD3ε + TCRβ + CD4 + CD44 + Foxp3 - GATA3 + ) and ILC2s (Lin - Thy1 + CD127 + GATA3 + ). f , Cytokine production from ILC2s (top panels) and Th2 (bottom panels) were evaluated by FACS for IL-4, IL-13 and IL-5. g , Graphical summary of impact on cytokine production due to RE deletion in mice. Cell type dependent differential impact was noted for some REs. Statistical significance is depicted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 (Student’s t test).

Article Snippet: The following antibodies were used for flow cytometry and cell purification in this study: Biotin-CD3ε (145-2C11, Biolegend), Biotin-TCRβ (H57-597, Biolegend), Biotin-CD4 (GK1.5, Biolegend), Biotin-CD8α (53-6.7, Biolegend), Biotin-CD19 (6D5, Biolegend), Biotin-CD11c (N418, Biolegend), Biotin-CD11b (M1/70, Biolegend), Biotin-TCRγδ (GL3, Biolegend), Biotin-FcεRI (MAR-1, Biolegend), Biotin-Ly6G/C (RB6-8C5, Biolegend), Biotin-NK1.1 (PK136, Biolegend), Biotin-Ter119 (TER-119, Biolegend), Biotin-EpCAM (G8.8, Biolegend), BV785-CD45 (30-F11, Biolegend), V500-CD90.2 (53-2.1, BD Bioscience), PE-CF594-CD127 (SB/199, BD Bioscience), PE-Cy7-KLRG1 (2F1, Biolegend), BUV395-GATA3 (L50-823, BD Bioscience), BUV496-CD44 (IM7, BD Bioscience), BUV615-TCRβ (H57-597, BD Bioscience), BUV737-CD4 (RM4-5, BD Bioscience), BV421-CD8α (53-6.7, Biolegend), eFlour450-Foxp3 (FJK-16s, Thermo Fisher Scientific), BV480-Siglec-F (E50-2440, BD Bioscience), BV510-IFNγ (XMG1.2, Biolegend), BV570-CD11b (M1/70, Biolegend), BV605-TCRγδ (GL3, Biolegend), BV711-CD11c (N418, Biolegend), BV750-CD90.2 (53-2.1, BD Bioscience), BV786-CD19 (1D3, BD Bioscience), AF488-IL-4 (11B11, Biolegend), PerCP-eFluor710-IL-13 (eBio13A, Thermo Fisher Scientific), PE-IL-5 (TRFK5, Biolegend), PE-CF594-KLRG1 (2F1, BD Bioscience), PE/Fire 640-MHCII (M5/114.15.2, Biolegend), PE-Cy5-CD127 (SB/199, Biolegend), PE/Fire 700-CD45 (30-F11, Biolegend), PE-Cy7-T-bet (4B10, Biolegend), AF700-NK1.1 (PK136, BD Bioscience), APC-Cy7-CD3ε (17A2, BD Bioscience), APC-Fire810-Ly6G/C (RB6-8C5, Biolegend).

Techniques: Staining

Journal: Journal of cell science

Article Title: Microtubule-associated protein-4 controls nanovesicle dynamics and T cell activation

doi: 10.1242/jcs.199042

Figure Lengend Snippet: (A) Immunoblots showing phosphorylation of the indicated molecules (CD3ζ Y83, P-ERK1/2 and LAT Y191) in control or MAP4KD Jurkat T cells activated with SEE-pulsed Raji B cells (SEE-APCs) for the indicated times. α-tubulin, GAPDH and β-actin are loading controls. (B) Quantification (a.u., arbitrary units) of CD3ζ, ERK2 and LAT phosphorylation from experiments as in A (n=6, 7 and 6 for CD3ζ Y83, P-ERK1/2 and LAT Y191, respectively). *P<0.05; ns, not significant (paired t-test). (C) Immunoblot of CD3ζ phosphorylation (CD3ζ Y83) in control or MAP4KD cells activated with crosslinked anti-CD3 and -CD28 antibodies for the indicated times. β-actin, loading control. (D) Quantification of CD3ζ phosphorylation from experiments as in C (n=6). *P<0.05; ns, not significant (paired t-test). (E) Left panel, map of trajectories of CD3ζ–mCherry-bearing vesicles in control and MAP4KD cells spreading over anti-CD3 and -CD28-coated glass-bottom chambers. A maximal projection of a time-lapse (Δt=65 s) and initial bright field image (t=0) are shown. Right panel, quantification of the displacement length (μm) from experiments as in E (n=16 and 17 for control and MAPKD, respectively). **P<0.01 (Student’s t-test). (F) Quantification by FACS of the surface basal levels of TCR (Vβ8) and CD3ε in control and MAP4KD Jurkat T cells (n=3). ns, not significant (paired t-test). (G) Quantification of CD3ε internalization (left panel) and recycling (right panel) by FACS of control or MAP4KD cells activated with anti-CD3/CD28-coated plates (n=5). ns, not significant [ paired t-test (G); Wilcoxon test (F)]. All graphs represent mean±s.d.

Article Snippet: Antibodies and reagents Antibodies used in this study were: anti-CD3ζ-pY83 (ab68236; 1:1000), anti-CD3ζ (ab190728 1:1000), anti-MAP4 [ab89650;1:800 western blotting (WB), 1:200 immunofluorescence (IF)]; anti-LAT-pY132 (ab4476 1:1000); anti-LAT-pY191 (ab59197 1:1000) (all Abcam); FITC-conjugated anti-α-tubulin (F2168; 1:100), anti-α-tubulin (T6199; 1:2000 WB), anti-β-actin (A2228; 1:1000) (all Sigma); anti-PKCθ (610090; 1:1000), anti-CD4 V450 (560346; 1:100 FACS), anti-CD3ε V500 (561416; 1:100), FITC-conjugated anti-Vβ8 (catalog number: 555606), 1:100), anti-human CD28 (555725, 3 μg/ml) (all BD-Pharmingen); anti-ERK1/2-pT202/Y204 (44285; Calbiochem; 1:1000); anti-p65 (sc372; 1:1000) (all Santa Cruz Biotechnology); anti-PKCθ-pT538 (9377 S; 1:1000), anti-PLCγ1 (2822S; 1:1000), anti-PLCγ1-pY783 (#2821L; 1:1000), anti-ERK1/2 (91075; 1:1000) (all Cell Signaling); anti-SMC1 (A300-055, 1:100) (Bethyl); anti-human CD3ε (300314, 5 μg/ml) and anti-GAPDH (649202,1:1000) (both Biolegend); Percp-Cy5.5-conjugated anti-CD19 (65-0199; 1:100); GHOST Dye Violet 510 (13-0870; 1:500 FACS) (TONBO Biosciences); FITC-conjugated anti-CD69 (21620693; 1:100) (ImmunoTools), and anti-detyrosinated tubulin (Tub Glu; Andrés-Delgado et al., 2012 ).

Techniques: Western Blot