Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 1. Effector-Stage Tcf7hi CD8+ T Cells Resemble Central Memory Cells B6 (CD45.1/2) mice were adoptively transferred with Tcf7GFP P14 cells (CD45.2) and infected with LCMV WE. (A) Tcf7GFP expression by splenic P14 cells at the indicated time points post-infection (p.i.).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Infection, Expressing

Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 5. Vaccination of Mice and Humans Generates Effector-Phase Tcf1hi CD8+ T Cells (A–H) B6 Tcf7GFP mice were vaccinated as indicated with a modified Ovalbumin peptide and Pam3CSK4 in Montanide (Ova) or with Pam3CSK4 in Montanide (Ø). (B, C, and E) Abundance and (D and F) Tcf7GFP and CD62L expression by KbOva+ CD8+ T cells in the blood on d21 and d35 (B–D) and in the spleen on d35 post- vaccination (E–G). (H) IL-2 and TNF-a production by d35 splenic IFN-g+ cells. (I–K) Healthy volunteers received a yellow fever vaccine, and peripheral blood CD8+ T cells were analyzed 14 days later. (I) TCF1 expression by A2/LLW tetramer+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Expressing

Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 6. Tcf1 Is Essential for the Stemness of d8 Tcf7GFPhi CD8+ T Cells (A–D) B6 mice (CD45.1/2) were transplanted with WT or Tcf7/ (KO) Tcf7GFP P14 cells (CD45.2) and infected with LCMV WE. (A) Abundance of P14 cells in the spleen at d8 p.i. and (B) expression of Tcf7GFP. Phenotype (C) and cytokine production (D) by WT and KO Tcf7GFPhi P14 cells. (E–G) Recall response of sorted d8 WT and KO Tcf7GFPhi P14 cells. (E) Abundance of P14 cells in the spleen and (F) Tcf7GFP expression 8 days later (d8+8). (G) Abundance of secondary Tcf7GFPhi cells compared to input. Data are representative of 3 experiments with 4 mice per group (A–C), representative of 2 experiments with 5–6 mice per group (D), or compiled from 2 ex- periments with 5–7 mice per group (E–G). Mean ± SD are shown. Statistics: non-paired t test (A–D and F) or one-way ANOVA with Tukey’s test (E and G) with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; and (ns) p > 0.05. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Infection, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

doi: 10.1136/jitc-2024-010239

Figure Lengend Snippet: 15–4P Stimulation increases NKG2D expression and murine NK cell proliferation compared with IL-15/IL-15Rα stimulation alone. ( A ) Schematic showing AgN2a-4P and NK cell receptor-ligand interactions. B6 NK cells are isolated and cultured with IL-15/IL-15Rα conjugate and with or without irradiated AgN2a-4P cells at a 1:1 ratio for 12 days. All expression markers were examined on CD3 − NK1.1 + NK cells. ( B ) Dot plots of NKG2D (top) and NKp46 (bottom) on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with percentage of gated cells. ( C ) Per cent of NK cells positive for NKG2D (left) and NKp46 (right) and MFI below. ( D ) Fold change of IL-15 NK cells and 15–4P stimulated NK cell expansion at day 12 compared with day 0. ( E ) Dot plots of proliferation marker Ki-67 on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with ( F ) percentage and MFI. ( G ) Percentage and MFI of TRAIL (top) and Fas-L (bottom) were assessed for each group. Data are representative of an experiment that was replicated two times. Representative dot plots examples of one replicate out of a minimum of three replicates. All bar graphs show individual experimental replicates plotted with mean and SEM (n=3–5). Two-sided two-sample t-tests were performed where indicated (*p<0.05, **p<0.01, ***p<0.001, ns=not significant). AgN2a, aggressive variant of the Neuro-2a murine neuroblastoma cell line; MFI, median fluorescence intensity; NK, natural killer.

Article Snippet: T cells were depleted using a CD3 MicroBead Kit (Miltenyi Biotec) by negative selection on the AutoMACs Pro Separator (Miltenyi Biotec) at day+0.

Techniques: Expressing, Isolation, Cell Culture, Irradiation, Marker, Variant Assay, Fluorescence

Journal: bioRxiv

Article Title: Atf3 Integrates Lipid and Cytoskeletal Remodeling to Drive Macrophage Fusion

doi: 10.64898/2026.04.01.715652

Figure Lengend Snippet: (A-B) Representative confocal micrographs of IL-4 stimulated BMDMs stained against the membrane-localized integrin CD11b show numerous DAPI-stained nuclei within a single control BMDM (A-A’’). In contrast, Atf3 KO BMDMs form multilayered aggregates, with mononuclear cells clearly separated by cell membranes (B-B’’). White arrowheads indicate the positions of the cross-sections. (C-D) 3D confocal projection of z-stack images of control (C) BMDMs stained for F-actin (Phal) after three days of IL-4 stimulation shows prominent ruffling (R), while Atf3 KO BMDMs display scarce thin filopodia (Fl, arrow). White arrowheads indicate the positions of the cross-sections, which are presented below (C’, D’). (E-F) Electron microscopy images highlight long filopodia in control BMDMs treated with IL-4 for three days (e), compared to the shorter projections in Atf3 KO cells (F). (G-J) Compared to the rich cortical actomyosin network in control cells, visualized by staining for F-actin (Phal) (G, H, corresponding intensity heatmaps G’, H’) and phospho-Myosin (S19), actomyosin cables in Atf3 KO BMDMs (I, J) are concentrated beneath the plasma membrane and extend along the branches. (K-L) Confocal micrographs of BMDMs expressing Lifeact-EGFP reveal actin-rich podosome rings (K, asterisks), lamellipodia (K, arrowhead) and filopodia (K, arrow) in control cells while these structures are notably reduced in Atf3 KO BMDMs (l). (M-O) Representative confocal micrographs (M, N) and quantification (O) of IL-4 stimulated BMDMs showing increased WASP fluorescence intensity in Atf3 KO macrophages (N) compared with control cells (M). In contrast to the primarily nuclear signal in control BMDMs, Atf3 KO BMDMs display WASP signal in both nucleus and cytoplasm. (P) Representative western blot of IL-4 treated cells shows marked decrease of F-actin in Atf3 KO BMDMs compared to control . (Q-R) Representative western blots of WASP, phospho-Cofilin (p-Cofilin) and total Cofilin protein levels (Q) and quantification (R) in IL-4-stimulated BMDMs of the indicated genotypes. ATP5α was used as a loading control. (S-T) Representative confocal micrographs reveal differential localization and enrichment of Filamin A (FLNA) in the nucleus (arrow, N) of control (S, S’) cells and in perinuclear foci (PN) of Atf3 KO (T, T’) BMDMs stimulated by IL-4. Data information: Data represent means ± s.d.; **p<0.01, ****p<0.0001 (O, R). Statistical significance was determined using Mann-Whitney test. Each dot represents individual cell from three independent experiments (O, n = 3) or individual animal (R, n = 8 left, n = 15 right) Micrographs are z-projections of multiple confocal sections. DAPI labels nuclei. Scale bars: 10 µm (A-D, G-L, S, T), 50 µm (M, N), and 0.5 µm (E, F).

Article Snippet: Peritoneal Macrophages (PMs) were isolated from the peritoneal cavity of 8-10-week-old mice according to a previously described protocol ( Herb et al , 2019 ) using paramagnetic CD11b MicroBeads (MiltenyiBiotec #130-049-601).

Techniques: Staining, Membrane, Control, Electron Microscopy, Clinical Proteomics, Expressing, Fluorescence, Western Blot, MANN-WHITNEY

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Disruptors of sestrin-MAPK interactions rejuvenate T cells and expand TCR specificity

doi: 10.1101/2024.05.17.594698

Figure Lengend Snippet: (A) Senescence-associated β-galactosidase expression in DOS-treated (DOS-juvenated) or untreated T sen . Cells were purified and cultured for one week in the presence of anti-CD3 (0.5 μg/mL) and rh-IL-2 (5 ng/mL), then stained to detect β-galactosidase activity. Representative image on the inverted phase-contrast microscope (left) and relative quantification (right, n = 8 donors). (B) Population doublings of human T sen (transduced with irrelevant scramble) and sestrin null CD4 + T sen (transduced by triple lentiviral depletion of sestrins) and cultured as in (A) (left). Donor-matched CD27 + CD28 + CD4 + T cells (herafter, T erl ) were cultured in parallel but activated with anti-CD3 and anti-CD28 ( n = 5 donors). Cells were cultured over two weeks with restimulation every 7 days. DOS-driven population doublings (right) were calculated as delta between DOS treated and DOS untreated T cells, with or without depletion of sestrins. (C) DOS-juvenation of human CD4 + T cells. Terminally differentiated effector memory CD45RA - CD28 - CD27 - CD4 + T cells (hereafter, T EM ) were purified and cultured over 20 days, as in (A). At day 1 (18 hours), 7, and 21 cell phenotypes were assessed by flow cytometry. Quantifications of rejuvenated stem like (CD28 + CD45RA + CCR7 + CD95 + CD62L + TCF1 + ) among human CD4 + T cells are shown (left; n = 5 donors). Decay of T EM and CD28 - CD27 - CD45RA + CD4 + T cells (hereafter, T EMRA ) undergoing rejuvenation is shown (right). (D) Adoptive transfer of DOS-juvenated T cells, experimental design. Donor T cells were derived from twenty-month-old mice 15 days after Fluad vaccination with or without DOS treatment (0.1 mg/Kg throughout), labeled with Cell Trace Violet (CTV) dye or congenic CD45.1 tracking, then transferred into young naïve CD45.2 recipients (3 months). In parallel, young mice (3 months) were used as young donor control. Recipient animals were rested for 28 days, then analysed for donor T cell persistence and maintenance of stem phenotype after transfer. (E) Maintenance of donor DOS-juvenated CD45.1 CD4 + T cells, their aged-matched controls, and that of young donor T cells, 28 days after transfer (day 43) in recipient mouse lymph nodes. Representative flow cytometry plots and poled data ( n = 5 mice per group) are shown. (F) Assessment of mouse T cell memory programs in stem like transferred T cells (among CD45.1 CD44 - CD62L + CD95 + CD4 + T cells) and terminally differentiated cells (TE, among CD45.1 CD44 - CD62L - CD4 + T cells) following adoptive transfer as in (D) ( n = 5 mice). (G) IL7R gene expression and (H) CD95 and Sca-1 mean fluorescent intensity (MFI; throughout) in stem cells derived from CD4 + CD45.1 + transferred stem T cells among lymph nodes of recipient CD45.2 mice, 28 days after transfer ( n = 5 mice). (I) Representative plots of CD45.1 transferred cells in quiescent state before and after transfer assessed by cycle related intra-nuclear Ki67 staining. Representative of n = 5 mice per group. (J) G1 (Ki67 + ) to G0 (Ki67 - ) transition in stem like CD45.1 CD4 + T cells before and after adoptive transfer as indicated ( n = 5 mice per group). (K) Assessment of T cell longevity following DOS-juvenation. Cells from lymph nodes were stained using the Annexin-PI Apoptosis detection Kit 28 days after transfer. Representative FACS plot (left) and quantification of dead CD45.1 + CD4 + transferred T cells (right). (L) DOS-juvenated T cell maintenance, in vivo model. In (A and B, right) two tailed paired T test was used. In (B, left-C, E-H and J-K) one-way Anova with Bonferroni post-correction for multiple comparisons was used, *p<0,05, **P<0,01; ***P<0,001; ****P<0,0001. Error bars indicate SEM.

Article Snippet: Primary human CD4 + T cells were isolated using CD4 MicroBeads (130-045-101; Miltenyi); primary human senescent CD4 + CD27 - CD28 - T cells (thereafter T sen ) were isolated using CD4 + T Cell Isolation Kit (130-096-533; Miltenyi) followed by depletion of CD27 + and CD28 + T cells using CD27 MicroBeads (130-051-601; Miltenyi) and CD28 MicroBead Kit (130-093-247; Miltenyi).

Techniques: Expressing, Purification, Cell Culture, Staining, Activity Assay, Microscopy, Quantitative Proteomics, Transduction, Flow Cytometry, Adoptive Transfer Assay, Derivative Assay, Labeling, Control, Gene Expression, In Vivo, Two Tailed Test

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 1. The expression of CD133 in lung cancer and normal lung tissues. Original values are presented as log2 ratios. Spots showing the level of CD133 mRNA in NSCLC and normal tissues. (A) GSE10072. (B) GSE40275. (C) GSE63459; *p<0.05, **p<0.01 and ***p<0.001. (D) Correlation between CD133 and CXCR4 in GSE30219.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 2. CD133 and CXCR4 are highly expressed in NSCLC patients with metastasis. (A) Representative images of immunohistochemistry staining (mag- nification, x400) of NSCLC patients. (a) CD133 low expression; (b) CD133 high expression; (c) CXCR4 low expression; (d) CXCR4 high expression. Positive staining ratio of CD133 (B)/CXCR4 (C) in metastatic and non-metastatic NSCLC patients. Positive staining score of CD133 (D)/CXCR4 (E) in metastatic and non-metastatic NSCLC patients. (F) Correlation results of CD133 and CXCR4. (G) Correlation analysis between CD133/CXCR4 co-expression and disease- free survival (red and black curves indicate high and low CD133/CXCR4 co-expression groups of patient death, respectively; *p<0.05).

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 3. CD133 enhances A549 cells proliferation. (A) Effectiveness of magnetic cell sorting was verified by immunofluorescence assay (magnification, x400). Red and blue indicate CD133 expression and cell nucleus, respectively. (B) Formation of colonies by A549 cell lines after 2-week incubation. (C and D) Cell proliferation capacity was determined in A549 cells, CD133+ A549 cells and CD133-A549 cells by CCK8 assays.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: FACS, Immunofluorescence, Expressing, Incubation

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 4. CXCR4 is upregulated by CD133. Silencing effectiveness of CD133 siRNA was verified by qPCR (A) and western blot analysis (B). (C) Expression of CXCR4 after treatment with CD133 siRNA (50 nM, 48 h) by qPCR analysis. (D) Western blot analysis for the expression of CXCR4 after treatment with CD133 siRNA (50 nM, 72 h). β-actin as a loading control. (E) The statistics of (D). All experiments were performed in triplicate. ***p<0.001.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Western Blot, Expressing, Control

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 5. CD133+CXCR4+ promotes EMT process in NSCLC cells. (A) Transwell assay for the invasion of CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (B) Western blot assay for the expression of E-cadherin and Vimentin in CD133-CXCR4+, CD133+CXCR4- and CD133+CXCR4+ cells. (C) qPCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC, A549/CD133+ shRNA-CXCR4 and A549/CD133+ with amd3100. (D and E) RT-PCR for the expression of E-cadherin, Vimentin, Snail, Slug and Twist in A549/CD133+, A549/CD133+ shRNA-NC and A549/CD133+

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Transwell Assay, Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: CXCR4 is involved in CD133-induced EMT in non-small cell lung cancer.

doi: 10.3892/ijo.2016.3812

Figure Lengend Snippet: Figure 6. Vimentin is positively associated with CD133/CXCR4 co-expression. (A) Representative images of immunohistochemistry staining (magnification, x400) of NSCLC patients. (a) E-cadherin low expression; (b) E-cadherin high expression; (c) Vimentin low expression; (d) Vimentin high expression. Positive staining ratio of E-cadherin (B)/Vimentin (C) in metastatic and non-metastatic NSCLC patients. Correlation analysis for E-cadherin (D) or Vimentin (E) with CD133/CXCR4 co-expression in NSCLC patients.

Article Snippet: Cells were incubated at 4 ̊C with 100 μl FcR blocking reagent and 100 μl CD133 Micro Beads (130-097-049, Miltenyi Biotec, Germany) for 30 min.

Techniques: Expressing, Immunohistochemistry, Staining