Journal: bioRxiv

Article Title: Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

doi: 10.1101/2022.11.29.518315

Figure Lengend Snippet: (a) Immunoblotting of cleaved caspase 7 following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNα and 1000 U/mL TNFα) (Mix) for 96 h. Tubulin was used as loading control (n=3). (b) Immunoblotting of IκBα, phosphorylated STAT1, and phosphorylated JNK following exposure to cytokines (50 U/mL IL-1β, 1000 U/mL IFNγ and 1000 U/mL TNFα) alone or in combination (Mix) for 30 min. GAPDH was used as loading control. Representative image (n=3). Lysates from INS-1E cells with/without cytokine exposure were used as positive control. Gene expression of (c) DDIT3/CHOP, (d) HRK/DP5 and (e) CXCL10 in cells exposed to individual cytokines for 48 h. GAPDH was used as housekeeping gene. Data are means ± SEM (n=4), *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-MHC-I (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-GAPDH (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076) or anti-rabbit (#7074) (Cell Signaling) IgG antibodies.

Techniques: Western Blot, Control, Positive Control, Gene Expression

Journal: Nature immunology

Article Title: PTPN2 regulates the generation of exhausted CD8 + T cell subpopulations and restrains tumor immunity

doi: 10.1038/s41590-019-0480-4

Figure Lengend Snippet: (a-c) Quantification of (a) Slamf6 + Tim-3 – , (b) Slamf6 + Tim-3 + , and (c) Slamf6 – Tim-3 + subsets following in vitro stimulation (αCD3/CD28) of control or Ptpn2 -deleted naive CD8 + T cells in the presence of indicated cytokines or blocking antibodies. Representative of two pooled experiments, n ≥ 4 technical replicates. (d) Quantification of pSTAT1 expression of splenic CD8 + T cells day 6 post LCMV Clone 13 infection for co-transferred control and Ptpn2 -deleted cells following ex vivo restimulation with IFN-α. Representative of two independent experiments, n = 5 biological replicates. (e-f) Quantification of pSTAT1 in (e) Slamf6 + or (f) Tim-3 + cells following ex vivo IFN-α restimulation of co-transferred control and Ptpn2 -deleted cells as in (d). Representative of two independent experiments, n = 5 biological replicates. (g) Quantification of frequencies of co-transferred control and Ptpn2 -deleted CD8 + T cells day 4 post LCMV Clone 13 infection following treatment with isotype (left graph) or IFNAR blocking antibody (right graph). Frequencies at day 4 were normalized to input frequencies at day 0. Representative of two independent experiments, n = 3 biological replicates. (h) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFNAR blocking antibody (right). Representative of two independent experiments, n = 5 biological replicates. (i) Quantification of Slamf6 + Tim-3 – , Slamf6 + Tim-3 + , and Slamf6 – Tim-3 + subsets day 4 post LCMV Clone 13 infection in mice that received co-transferred control and Ptpn2- deleted P14 CD8 + T cells and were treated with isotype (left) or IFN-γ neutralizing antibody (right). Representative of two independent experiments, n = 5 biological replicates. Bar graphs represent mean and error bars represent standard deviation. Statistical significance was assessed by two-way ANOVA (a-c) or two-sided Student’s paired t-test (d-i) (ns p>.05, * p≤.05, ** p≤.01, *** p≤.001, **** p≤.0001). See also .

Article Snippet: Antibodies and dyes were purchased from BD Biosciences (7-AAD Cat# 559925 (1:100 dilution), Slamf6 Clone 13G3 Cat# 561540 (1:100 dilution), BrdU Clone 3D4 Cat# 552598 (1:50 dilution), Ki67-PerCP-Cy5.5 Clone B56 Cat# 561284 (1:100 dilution)); Biolegend (B220 Clone RA3–6B2 Cat# 103208, 103226 (1:100 dilution), CD11b Clone M1/70 Cat# 101208, 101216 (1:100 dilution), CD127 Clone A7R34 Cat# 135014, 135024 (1:100 dilution), CD25 Clone PC61 Cat# 101904 (1:100 dilution), CD3ε Clone 17A2 Cat# 100220, 100308, 100336, 100328 (1:100 dilution), CD4 Clone RM4–5 Cat# 100516, 100531, 100543 (1:100 dilution), CD44 Clone IM7 Cat# 103008, 103028, 103030 (1:100 dilution), CD45.1 Clone A20 Cat# 110708, 110716, 110741 (1:100 dilution), CD45.2 Clone 104 Cat# 109824, 109832, 109830 (1:100 dilution), CD5 Clone 53–7.3 Cat# 100608 (1:100 dilution), CD62L Clone MEL-14 Cat# 104417 (1:100 dilution), CD8α Clone 53–6.7 Cat# 100737 (1:100 dilution), CD8β Clone YTS156.7.7 Cat# 126606, 126608, 126610, 126620, 126614 (1:100 dilution), c-Kit Clone ACK2 Cat# 135108 (1:100 dilution), CXCR5 Clone L138D7 Cat# 145509 (1:50 dilution), Gr-1 Clone RB6–8C5 Cat# 108408 (1:100 dilution), Granzyme B Clone GB11 Cat# 515403, 515406 (1:100 dilution), I-A/I-E Clone M5/114.15.2 Cat# 107614 (1:100 dilution), IFN-γ Clone XMG1.2 Cat# 505810 (1:100 dilution), IFNAR1 Clone MAR1–5A3 Cat# 127314 (1:100 dilution), Ly-6c Clone HK1.4 Cat# 128007 (1:100 dilution), PD-1 Clone 29F.1A12 Cat# 135206, 135209 (1:100 dilution), Sca-1 Clone D7 Cat# 108108, 108128 (1:100 dilution), TCR Vα2 Clone B20.1 Cat# 127814, 127806 (1:100 dilution), TCR Vβ5 Clone MR9–4 Cat# 139506 (1:100 dilution), TCR Vβ8 Clone KJ16–133.18 Cat# 118406 (1:100 dilution), TER-119 Clone TER-119 Cat# 116208 (1:100 dilution), TCF1 Clone 7F11A10 Cat# 655208 (1:100 dilution), Tim-3 Clone RMT3–23 Cat# 119703, 119723 (1:100 dilution), TNF Clone MP6-XT22 Cat# 506322 (1:100 dilution), TruStain fcX Clone 93 Cat# 101320 (1:50 dilution), Rat IgG2a κ Isotype Clone RTK2758 Cat# 400508 (1:100 dilution), Rat IgG2b κ Isotype Clone RTK4530 Cat# 400612 (1:100 dilution), Streptavidin Cat# 405225 (1:400)); Invitrogen anti-GFP (Polyclonal) Cat#A21311 (1:350 dilution); Thermo Fisher Scientific (Foxp3 Clone FJK-16s Cat# 48-5773-82 (1:50 dilution), Near-IR Fixable Live/Dead Cat# L34976 (1:500 dilution)); and Cell Signaling Technology pSTAT1 Clone 58D6 Cat# 9174 (1:100 dilution).

Techniques: In Vitro, Blocking Assay, Expressing, Infection, Ex Vivo, Standard Deviation

Journal: eLife

Article Title: Mitochondrial respiration contributes to the interferon gamma response in antigen-presenting cells

doi: 10.7554/eLife.65109

Figure Lengend Snippet:

Article Snippet: Antibody , Phospho-Stat1 Tyr701, clone 58D6 (rabbit monoclonal) , Cell Signaling Technology , Cat# 5375; RRID: AB_10860071 , WB (1:1000).

Techniques: Virus, Transduction, Clone Assay, Recombinant, Selection, Modification, CRISPR, Purification, Neutralization, One Step RT-PCR, Viability Assay, Software, Cell Isolation, Enzyme-linked Immunosorbent Assay, Sequencing