Journal: bioRxiv

Article Title: LSD1 inhibitors induce neuronal differentiation of Merkel cell carcinoma by disrupting the LSD1-CoREST complex and activating TGFβ signaling

doi: 10.1101/2020.04.14.041657

Figure Lengend Snippet: a , Bulk mouse skin labeled with IgM-PE mouse isotype control. b , Bulk mouse skin labeled with NCAM-PE. DAPI - cells were sorted. c , Merkel cells labeled with IgM-PE mouse isotype control. d , Merkel cells labeled with NCAM-PE. DAPI - /NCAM + cells were sorted. Each plot shows the subpopulation gated for in the preceding plot to the left.

Article Snippet: Merkel cells were stained with anti-PSA-NCAM-PE antibody (Miltenyi Biotec, Cat. 130-117-394) or with mouse IgM-PE isotype control (Miltenyi Biotec, Cat. 130-120-070) according to manufacturer’s instructions, for 10 min at 4°C.

Techniques: Labeling, Control

Journal: Immunity

Article Title: Transcription Factor IRF4 Promotes CD8 + T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

doi: 10.1016/j.immuni.2017.11.021

Figure Lengend Snippet: Figure 4. IRF4 and NFAT Form a Positive Feedback Circuit during T Cell Exhaustion (A) Expression of Nfatc1 isoforms as identified by RNA sequencing. Graph shows expression relative to naive T cells of the short isoform (NFATc1aA, represented by exon 1–3 junctions, skipping exon 2) and the long isoform (represented by exon 2–3 junctions) in P14 T cells isolated from acutely (WE) and chronically (Docile) LCMV-infected mice at day 30 post infection (p.i.). (B) Western blot showing NFATc1, NFATc2, IRF4, BATF, and Actin (loading control) expression in CD8+ T cells flow cytometry sorted as CD62L from acutely and as CD62LPD-1+ from chronically LCMV-infected mice at day 30 p.i. The arrow marks the short isoform of NFATc1. (C) Binding of NFATc1 and NFATc2 to the Irf4 and Pdcd1 (encoding PD-1) promoters demonstrated by chromatin immunoprecipitation using specific antibodies or control IgG and RT-qPCR in total CD44+CD8+ T cells isolated from spleens of acutely and chronically LCMV-infected mice at day 8 p.i. Enrichment is expressed as percentage of total chromatin input and compares isotype control and NFATc1- and NFATc2-specific antibodies. (D) Western blot showing NFATc1, NFATc2, IRF4, BATF, and p50 NF-kB (loading control) expression in polyclonal CD8+ T cells deficient (KO) in NFATc1 (Nfatc1fl/flCd4Cre), NFATc2 (Nfatc2/), or both (DKO) after in vitro activation with anti-CD3 and anti-CD28 for 48 hr. (E) Western blot showing NFATc1, IRF4, BATF, and Actin (loading control) in polyclonal CD8+ T cells activated with anti-CD3, anti-CD28, and IL-2 with or without cyclosporine A (CsA) for 24 hr. Data in (A)–(C) are representative of 2 independent experiments and data in (D) and (E) are representative of 3 independent experiments. Error bars denote mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Fluorochrome-conjugated antibodies directed against the following antigens were used for analysis by flow cytometry: CD8a (53-6.7), CD62L (MEL-14), CD71 (R17217), CD98 (RL388), Ly5.1 (A20), 2B4 (eBio244F4), PD-1 (J43), Lag3 (T47-530), TIGIT (GIGD7), CTLA4 (14D3), TIM-3 (RMT3-23), CD127 (A7R34), CXCR5 (SPRCL5), IL-2 (JES6-5H4), IFNg (XMG1.2) (from Thermo Fisher Scientific), CD44 (IM7), Ly5.2 (104), TNF (MP5-XT22) (from BD Biosciences), IRF4 (REA201) (from Miltenyi), TCF1 (C63D9), BATF (D7C5) (from Cell Signaling Technology), IRF4 (M17) (from Santa Cruz Biotechnology), and anti-goat IgG coupled to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) and anti-rabbit IgG coupled to Alexa Fluor 647 (Thermo Fisher Scientific). e3 Immunity 47, 1–13.e1–e5, December 19, 2017 Propidium iodide, SytoxBlue or fixable viability dye eFluor506 (all Thermo Fisher Scientific) were used to exclude dead cells.

Techniques: Expressing, RNA Sequencing, Isolation, Infection, Western Blot, Control, Cytometry, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, In Vitro, Activation Assay, Two Tailed Test

Journal: Immunity

Article Title: Transcription Factor IRF4 Promotes CD8 + T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

doi: 10.1016/j.immuni.2017.11.021

Figure Lengend Snippet: Figure 5. IRF4 and BATF Cooperate with NFAT to Establish T Cell Exhaustion (A) Venn diagram showing overlap between genes bound by IRF4, BATF, and NFAT as identified by chromatin immunoprecipitation (ChIP) sequencing of effector CD8+ T cells (Kurachi et al., 2014; Man et al., 2013; Martinez et al., 2015). (legend continued on next page) Immunity 47, 1–13, December 19, 2017 7

Article Snippet: Fluorochrome-conjugated antibodies directed against the following antigens were used for analysis by flow cytometry: CD8a (53-6.7), CD62L (MEL-14), CD71 (R17217), CD98 (RL388), Ly5.1 (A20), 2B4 (eBio244F4), PD-1 (J43), Lag3 (T47-530), TIGIT (GIGD7), CTLA4 (14D3), TIM-3 (RMT3-23), CD127 (A7R34), CXCR5 (SPRCL5), IL-2 (JES6-5H4), IFNg (XMG1.2) (from Thermo Fisher Scientific), CD44 (IM7), Ly5.2 (104), TNF (MP5-XT22) (from BD Biosciences), IRF4 (REA201) (from Miltenyi), TCF1 (C63D9), BATF (D7C5) (from Cell Signaling Technology), IRF4 (M17) (from Santa Cruz Biotechnology), and anti-goat IgG coupled to fluorescein isothiocyanate (Jackson ImmunoResearch Laboratories) and anti-rabbit IgG coupled to Alexa Fluor 647 (Thermo Fisher Scientific). e3 Immunity 47, 1–13.e1–e5, December 19, 2017 Propidium iodide, SytoxBlue or fixable viability dye eFluor506 (all Thermo Fisher Scientific) were used to exclude dead cells.

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing

Journal:

Article Title: DNA-PKcs function regulated specifically by protein phosphatase 5

doi: 10.1073/pnas.0307765100

Figure Lengend Snippet: Phosphorylation status of DNA–PKcs after irradiation in PP5 overexpressing clones. PP5.C4 and PP5.C13 subclones were irradiated with IR (11.5 Gy) and recovered for 30–410 min. The undamaged control (UD) is shown on the left. Nuclear extracts were prepared, and 20–60 μg of protein was applied to a SDS gel. After Western blotting, the membrane was stained with rabbit antibody to DNA–PKcs pT2609 (A), rabbit antibody to DNA–PKcs pS2056 (B), or rabbit antibody to ATM pS1981 (C). For loading controls the same membranes were stripped and incubated with antibody to total DNA–PKcs or to DNA polymerase δ. PP5 overexpresssion was visualized by anti-PP5 antibody. * and ** denote phosphorylated DNA–PKcs degradation products.

Article Snippet: The proteins were transferred by Western blotting onto a nitrocellulose membrane (Bio-Rad) and incubated with antibody 18-2 to DNA–PKcs (Neomarkers, Fremont, CA), antibody to pT2609, antibody to pS2056 site, antibody to DNA polymerase δ (Transduction Laboratories, Lexington, KY), antibody to PP5 (Transduction Laboratories), or antibody to ATM pS1981 (Rockland, Gilbertsville, PA).

Techniques: Phospho-proteomics, Irradiation, Clone Assay, Control, SDS-Gel, Western Blot, Membrane, Staining, Incubation