Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Related to . (A) PBMCs from chilblain patients (PC, n = 13) or HD (controls, n = 16) were stimulated with the TLR7-selective agonist IMQ for 24 h. Secreted cytokines were evaluated in a LEGENDplex assay. Freshly isolated PBMCs were used in these experiments. (B) THP1-Dual reporter cells were treated with various doses of recombinant IFN-α (rIFN-α2b), ranging from 10 to 1,000 IU/ml. Left panel: luciferase activity was quantified with a luminometer, and RLA is represented as fold induction over untreated reporter cells. Right panels: the expression of ISG15 and SIGLEC1 was analyzed by flow cytometry with intracellular staining. Graphs show MFI as fold induction over basal levels in THP1 cells. The dashed lines represent the mean type I IFN activity levels for supernatants from the TLR7-stimulated leukocytes of HD (black) (controls, n = 12) and chilblain patients (red) (PC, n = 12). (C) PBMCs from chilblain patients (PC, n = 15) or HD (controls, n = 16) were stimulated with viral nucleic acid surrogates for 24 h. Secreted IFN-β levels were evaluated in a LEGENDplex assay after stimulation with liposome-encapsulated poly(I:C), cGAMP (STING agonist) or poly(dA:dT) (cytosolic DNA sensors), or naked CpG-A (TLR9 agonist). Freshly isolated PBMCs were used in these experiments. (D) PBMCs from PC patients or controls were stimulated for 24 h with SARS-CoV-2 ( n = 8) or IAV ( n = 12). Secreted IFN-β levels were evaluated in a LEGENDplex assay. Cryopreserved PBMCs were used in these experiments. (E) PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12) were stimulated for 24 h with HSV-1. Secreted IFN-α and IFN-β levels were evaluated in a LEGENDplex assay. (F) Distribution of lymphoid and myeloid subsets among PBMCs from PC patients (PC, n = 12) and HD (controls, n = 14) was determined by flow cytometry. Data are represented as percentages of total CD45 + cells. CM, central memory T cells; EM, effector memory T cells; EMRA, CD45RA + effector memory T cells; NK, natural killer cells; cDC CD1c + , CD1c + conventional dendritic cells; cDC CD1c − , CD1c − conventional dendritic cells. (G) Frequency of CD123 + BDCA2 + pDCs in PBMCs from PC patients ( n = 12) and controls ( n = 14), as analyzed in F, is shown on a separate graph. Bars represent the mean ± SD. The adjusted P values in A, C–E, and G were determined in Mann–Whitney tests with the Bonferroni correction for multiple testing. *P < 0.05; ns = nonsignificant. IMQ, imiquimod; RLA, relative luciferase activity; MFI, mean fluorescence intensity; IAV, influenza A virus.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: CD45 Alexa Fluor 350 (clone 2D1, FAB1430U, AB_3646482, 1:200 dilution; R&D Systems); HLA-DR BUV496 (clone L243, 753685, 1:5,000 dilution; BD Biosciences); CD56 BUV737 (clone TULY56, RRID:AB_2895975, 1:400 dilution; Thermo Fisher Scientific); CD11c eFluor 450 (clone 3.9, AB_11218498; Thermo Fisher Scientific, 1:400 dilution); CD123 BV510 (clone 6H6, AB_2562068, 1:800 dilution; BioLegend); CD16 BV570 (clone 3G8, AB_10915988, 1:200 dilution; BioLegend); BDCA-2 BV785 (clone 201A, AB_2572146, 1:800 dilution; BioLegend); CD14 Spark Blue 550 (clone 63D3, AB_2832724, 1:60,000 dilution; BioLegend); CD3 NovaFluor B610-70S (clone SK7, AB_3098363, 1:2,000 dilution; Thermo Fisher Scientific); CD1c PerCP-eFluor 710 (clone L161, AB_10545854, 1:2,000 dilution; Thermo Fisher Scientific); TLR7 PE (clone S18024F, AB_2910431, 1:400 dilution; BioLegend); TLR8 APC (clone S16018A, AB_2801050, 1:400 dilution; BioLegend); TLR9 BV421 (clone S16013D, AB_2801039, 1:800 dilution; BioLegend); and CD19 APC-Fire810 (clone HIB19, AB_2860770, 1:200 dilution; BioLegend), in the presence of Human TruStain FcX (AB_2818986; BioLegend), True-Stain Monocyte Blocker (Cat. 426102; BioLegend), and CellBlox Plus (C001T06F01; Thermo Fisher Scientific) to minimize nonspecific binding.

Techniques: Isolation, Recombinant, Luciferase, Activity Assay, Expressing, Flow Cytometry, Staining, MANN-WHITNEY, Fluorescence, Virus

Journal: The Journal of Experimental Medicine

Article Title: Enhanced TLR7-dependent production of type I interferon by pDCs underlies pandemic chilblains

doi: 10.1084/jem.20231467

Figure Lengend Snippet: Enhanced TLR7-mediated I-IFN production by pDCs in chilblain patients. (A) Intracellular levels of IFN-α (upper panel), TNF (middle panel), and IL-6 (lower panel) in leukocyte subsets were evaluated by flow cytometry after the stimulation of PBMCs for 5 h with the TLR7-selective agonist CL087 and the dual TLR7/8 agonist R848 in the presence of brefeldin A. The subsets studied were as follows: CD14 + monocytes (orange), CD123 + BDCA2 + pDCs (red), CD1c + conventional dendritic cells (DC CD1c + ) (purple), CD1c − conventional dendritic cells (DC CD1c − ) (light purple), CD19 + B cells (blue), CD3 + T cells (green). Representative FACS plots from a single patient are shown. The gating strategy is shown in . (B) Intracellular levels of IFN-α (upper panel), TNF (middle panel), and IL-6 (lower panel) in leukocyte subsets were evaluated by flow cytometry following the stimulation of PBMCs from PC patients (PC, n = 12) or HD (controls, n = 12), as described in panel A. Graphs represent the percentage of cytokine-positive cells. (C) pDCs are the primary source of IFN-α produced in response to stimulation with TLR7 agonists. The upper panel shows the expression of IFN-α by total CD45 + PBMCs stimulated with CL087 and R848. The middle panel shows the expression of pDC markers, CD123 and BDCA2, by IFN-α–producing cells. The lower graph shows the percentage of CD123 + BDCA2 + pDCs among IFN-α–producing cells from the PBMCs of PC patients ( n = 12) stimulated with CL087 and R848. (D and E) Intracellular levels of TLR7 (left panel), TLR8 (middle panel), and TLR9 (right panel) in leukocyte subsets were evaluated by flow cytometry on PBMCs. The histograms in D show representative data from a single patient. The graphs in E represent the MFI in PBMCs from female PC patients (PC, n = 8) or healthy female donors (controls, n = 11). Cryopreserved PBMCs were used in A–E. The adjusted P values in B and E were obtained in Mann–Whitney tests with the Bonferroni correction for multiple testing for non-normally distributed datasets, or in Student’s t tests with the Bonferroni correction for multiple testing for normally distributed datasets. Normality was assessed with Shapiro–Wilk and Kolmogorov–Smirnov tests. **P < 0.01; *P < 0.05; ns = nonsignificant; MFI, mean fluorescence intensity.

Article Snippet: Permeabilized cells were incubated overnight at 4°C with the following antibodies: CD45 Alexa Fluor 350 (clone 2D1, FAB1430U, AB_3646482, 1:200 dilution; R&D Systems); HLA-DR BUV496 (clone L243, 753685, 1:5,000 dilution; BD Biosciences); CD56 BUV737 (clone TULY56, RRID:AB_2895975, 1:400 dilution; Thermo Fisher Scientific); CD11c eFluor 450 (clone 3.9, AB_11218498; Thermo Fisher Scientific, 1:400 dilution); CD123 BV510 (clone 6H6, AB_2562068, 1:800 dilution; BioLegend); CD16 BV570 (clone 3G8, AB_10915988, 1:200 dilution; BioLegend); BDCA-2 BV785 (clone 201A, AB_2572146, 1:800 dilution; BioLegend); CD14 Spark Blue 550 (clone 63D3, AB_2832724, 1:60,000 dilution; BioLegend); CD3 NovaFluor B610-70S (clone SK7, AB_3098363, 1:2,000 dilution; Thermo Fisher Scientific); CD1c PerCP-eFluor 710 (clone L161, AB_10545854, 1:2,000 dilution; Thermo Fisher Scientific); TLR7 PE (clone S18024F, AB_2910431, 1:400 dilution; BioLegend); TLR8 APC (clone S16018A, AB_2801050, 1:400 dilution; BioLegend); TLR9 BV421 (clone S16013D, AB_2801039, 1:800 dilution; BioLegend); and CD19 APC-Fire810 (clone HIB19, AB_2860770, 1:200 dilution; BioLegend), in the presence of Human TruStain FcX (AB_2818986; BioLegend), True-Stain Monocyte Blocker (Cat. 426102; BioLegend), and CellBlox Plus (C001T06F01; Thermo Fisher Scientific) to minimize nonspecific binding.

Techniques: Flow Cytometry, Produced, Expressing, MANN-WHITNEY, Fluorescence

Journal: Toxins

Article Title: Lebetin 2, a Snake Venom-Derived B-Type Natriuretic Peptide, Provides Immediate and Prolonged Protection against Myocardial Ischemia-Reperfusion Injury via Modulation of Post-Ischemic Inflammatory Response

doi: 10.3390/toxins11090524

Figure Lengend Snippet: Effects of Lebetin 2 (L2) and B-type natriuretic peptide (BNP) on post-ischemic leukocyte infiltration. Myocardial infarction was induced by 35 min of ischemia followed by a 2-day reperfusion period. ( a ) Infiltrated leukocytes were assessed by CD45 immunolabeling in the infarcted area and its border zone (viable area), as well as in the septum (non-ischemic area) of rats treated with saline (1 µL/g), BNP (50 ng/g) or L2 (100 ng/g) intraperitoneally, 5 min before reperfusion. On each section, several fields were photographed (12–30 images/rat, n = 5–6) and the mean number of leukocytes per field was calculated; ( b ) representative microphotographs showing variable density of the leukocytes (CD45 in red and nuclei in blue) in the infarcted area from control and treated animals. Data are mean ± SEM. ***, p < 0.001 vs. saline (control) corresponding group, †††, p < 0.001 vs. corresponding other LV areas. Scale bars = 50 µm. ×20. DAPI, 4′, 6′-diamidino-2-phenylindole.

Article Snippet: Leukocyte infiltration was detected using rat anti-mouse CD45 antibody (1:200; Santa Cruz, Dallas, TX, USA) reacting against most leukocytes.

Techniques: Immunolabeling, Saline, Control

Journal: International Journal of Molecular Sciences

Article Title: ERK1/2 Inhibition via the Oral Administration of Tizaterkib Alleviates Noise-Induced Hearing Loss While Tempering down the Immune Response

doi: 10.3390/ijms25126305

Figure Lengend Snippet: Tizaterkib treatment lowers the number of CD45-positive cells in the cochlea 4 days post noise exposure. ( A ) Representative low-magnification images of cochlear cryosections stained with CD45 (red) and DAPI (blue). The treatment protocol shown in A was utilized and mice were sacrificed 4 days after noise exposure, 1 h after the final tizaterkib treatment. ( B ) Quantification of the CD45-positive cells in the cochlear sections in ( A ). ( C ) Higher magnification of the images shown in ( A ) of the scala tympani. ( D ) Quantification of CD45-positive cells in the walls of the scala tympani as presented in ( C ). ( E ) Representative images of cochlear cryosections of the stria vascularis following noise and tizaterkib treatment. ( F ) Quantification of CD45-positive cells per experimental group from the images in ( E ). Carrier (black), tizaterkib alone (blue), noise alone (red), noise + tizaterkib (green). Data shown as means ± SEM, * p < 0.05 and *** p < 0.001 compared to noise alone by one-way ANOVA with a Bonferroni post hoc test. n = 3–6 mice, with 3 sections each per mouse.

Article Snippet: Tissues were stained overnight at 4 °C with mouse CD45 antibody (1:200; Af114, R&D Systems; Minneapolis, MN, USA).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: ERK1/2 Inhibition via the Oral Administration of Tizaterkib Alleviates Noise-Induced Hearing Loss While Tempering down the Immune Response

doi: 10.3390/ijms25126305

Figure Lengend Snippet: Tizaterkib treatment lowers the amount of CD45 and CD68 in the cochlea 6 days post noise exposure. ( A ) Western blots showing the amount of CD45 and CD68 in the cochlea following noise exposure and tizaterkib treatment. The same treatment protocol shown in A was utilized and mice were sacrificed 6 days after noise exposure. ( B ) CD45/GAPDH ratio, normalized to the carrier-alone lane. Band intensities were measured using ImageJ software (version 1.54g). ( C ) CD68/GAPDH ratio, normalized to the carrier-alone lane. Data shown as means ± SEM, * p < 0.05 and ** p < 0.01 compared to noise alone by one-way ANOVA with a Bonferroni post hoc test. The experimental groups, from left to right, are as follows: carrier alone, tizaterkib alone, noise alone, and noise + tizaterkib. Each group had the cochleae from 5 mice (10 cochleae) pooled together to make the tissue lysate. n = 5.

Article Snippet: Tissues were stained overnight at 4 °C with mouse CD45 antibody (1:200; Af114, R&D Systems; Minneapolis, MN, USA).

Techniques: Western Blot, Software

Journal: The American Journal of Pathology

Article Title: Kidney Tertiary Lymphoid Structures in Lupus Nephritis Develop into Large Interconnected Networks and Resemble Lymph Nodes in Gene Signature

doi: 10.1016/j.ajpath.2020.07.015

Figure Lengend Snippet: T cells and B cells are prominent cells of tertiary lymphoid structures (TLS) and are organized into distinct T- and B-cell areas with a large network of blood and lymph vessels. A: Immunohistochemistry performed with anti-CD3 detecting T cells, anti-B220 detecting B cells, anti-CD21 detecting follicular dendritic cells (FDCs), double staining B220 (purple) and CD21 (brown), anti-F4/80 detecting macrophages, anti–peripheral lymph node addressin (PNAD) (MECA-79) detecting high endothelial venules (HEVs), anti–monoclonal anti–dendritic cell (DC) antibody (MIDC)-8 detecting activated DCs, and anti–B-cell lymphoma 6 (BCL6) detecting germinal center B and T cells. B: Immunofluorescence performed with anti–forkhead box P3 (FoxP3) detecting regulatory T cells and anti-B220 detecting B cells. C: Immunofluorescence performed with anti-B220 detecting B cells and anti– muscle, intestine and stomach expression (Mist)-1 detecting plasma cells. D: CD31 staining detecting endothelial cells reveals a network of different vessels with positive wispy cells in larger vessels ( white arrowheads ) and microcapillaries or thicker cells as in HEVs ( black asterisk ). The HEVs stained positively for PNAD ( white asterisks ), and some of the thin vessels are lymphatic vessel endothelial hyaluronan receptor 1 positive. E: The larger thin vessels ( white arrowheads ) are filled with leukocytes that were mostly CD3-positive T cells and B220-positive B cells and a few MIDC-8–positive DCs. None of the immune cells within the vessels were F4/80 or CD21-positive macrophages and FDCs, respectively. F: In young anti–double-stranded DNA–negative mice, CD3-positive T cells and F480-positive macrophages could be detected within the pelvic wall. Scale bars: 200 μm ( A , left column ); 100 μm ( B ; D , left column , E , and F ); 20 μm ( A and D , right columns ); 50 μm ( C ). Original magnification: ×10 ( A , left column ); ×20 ( B , and D , left column ); ×60 ( A , right column , and C ). G, glomerulus; v, vein.

Article Snippet: Anti-mouse CD45R (B220) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Immunohistochemistry, Double Staining, Immunofluorescence, Expressing, Clinical Proteomics, Staining

Journal: Journal of Biological Chemistry

Article Title: Early Murine T-lymphocyte Activation Is Accompanied by a Switch from N-Glycolyl- to N-Acetyl-neuraminic Acid and Generation of Ligands for Siglec-E

doi: 10.1074/jbc.m111.243410

Figure Lengend Snippet: FIGURE 6. Siglec-E-Fc binds subsets of sialylated proteins from activated T-lymphocytes. A, purified T cells were activated for 24 h and either untreated or sialidase-treated and then surface-labeled with biotin. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc com- plexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with streptavidin-HRP to detect surface biotinylated proteins. Arrows indicated proteins that bound to Siglec-E-Fc in a sialidase-sensitive manner (lane 4). B, purified T cells were activated for 24 h and treated or not with sialidase and lysates passed over MAL or SNA affinity columns. Bound sialylated proteins were eluted with SDS-PAGE sample buffer, separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with siglec-E-Fc precomplexed to anti-Fc-HRP. Arrows indicate proteins that are recognized by siglec-E-Fc in a sialidase-sensitive manner. C, purified T cells were activated for 24 h, cultured for 4 days and treated or not with sialidase. Cell lysates were passed over protein G-Sepharose coupled noncovalently to siglec-E-Fc/anti-Fc complexes and bound proteins eluted with SDS-PAGE sample buffer. Western blots were probed with goat anti-CD45 followed by HRP-conjugated anti-goat secondary antibody. The arrow indicates CD45 that is recognized by siglec-E-Fc in a sialidase-sensitive manner. The other bands represent nonspecifically bound proteins.

Article Snippet: For CD45 blots, membranes were blocked as above and then probed with 1 g/ml goat anti-mouse CD45 primary antibody and 1:10000 HRP-conjugated anti-goat IgG secondary antibody (R&D Systems) and detected as described previously.

Techniques: Purification, Labeling, SDS Page, Western Blot, Cell Culture

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 2.5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) five times at three-day intervals. Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05 and ** p < 0.01. Two-Way ANOVA test ( n = 4). B The resected tumors were weighed on Day 30. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. D The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). E The mRNA levels of Cd86 and Cd163 in resected tumors were analyzed by qRT‒PCR ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). F The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry ( n = 3-4). * p < 0.05 and *** p < 0.001. One-Way ANOVA test. G The quantification of the M1/M2 ratio is shown ( n = 3). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). H The frequencies of CD4 (CD4 + CD3 + CD45 + 7AAD - ) and CD8 (CD8 + CD3 + CD45 + 7AAD - ) tumor-infiltrating T cells were analyzed by flow cytometry ( n = 3-4). * p < 0.05. One-Way ANOVA test. I The quantification of CD8 + TILs is shown. * p < 0.05. One-Way ANOVA test ( n = 3). J The frequency of regulatory CD4 T cells (CD25 + CD127 - CD4 + CD3 + CD45 + 7AAD - ) was analyzed by flow cytometry ( n = 3-4). K The quantification of the CD8/Treg ratio is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). L The densities of dendritic cells (CD11c + ) and cytotoxic T cells (CD8 + ) in resected tumors were analyzed by immunofluorescence staining. The density of CD11c + DCs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). M The density of CD8 + TILs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3).

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Immunofluorescence, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 5 days and then intraperitoneally administered with anti-ENO1 antibodies (HuL001, 40 mg/kg) or clodronate liposomes (50 μL/mouse) on the indicated days ( n = 5). Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 4). B The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C 4T1 cells (5 ×10 4 ) were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) six times on the indicated days ( n = 5). Local radiotherapy was given on Days 10 and 12. The tumor volume was recorded every three days. * p < 0.05 and *** p < 0.001. Two-Way ANOVA test ( n = 4). D The resected tumors were weighed on Day 40. * p < 0.05. One-Way ANOVA test ( n = 4). E The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. F The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05. One-Way ANOVA test ( n = 3). G The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD1206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). H The quantification of the M1/M2 ratio is shown. * p < 0.05. One-Way ANOVA test ( n = 3). I A representative image of flow cytometric analysis of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells. J The frequency of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells was analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). K The density of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown. * p < 0.05. One-Way ANOVA test ( n = 3-4). L The density of IFNγ + (IFNγ hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown ( n = 3-4). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3-4). M The proposed mechanism of TGFβ1/TGFβR/Smad3/PRMT5-mediated ENO1 translocation for lactate release via MCT4.

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Liposomes, Immunofluorescence, Staining, Flow Cytometry, Translocation Assay