Journal: Scientific Reports

Article Title: NF-κB/ICAM-1 signaling regulates vascular dysfunction in depressive hypertension rats

doi: 10.1038/s41598-025-27188-2

Figure Lengend Snippet: The inhibition of NF-κB decreased the differentiation of endothelial progenitor cells into smooth muscle cells in chronic depressive SHRs. ( A and B ) CD33 + CD133 + endothelial progenitor cells in the carotid artery with immunofluorescence double staining. Magnification, 200x. ( C and D ) Western blot with statistics for expression of MMP3, MMP9, CXCL16 and MCP-1 in carotid arteries. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. ** P < 0.01 vs. Control group. # P < 0.05, ## P < 0.01 vs. SHR group. && P < 0.01 vs. SHR + CUMS group. n = 6 in each group.

Article Snippet: CXC motif chemokine ligand 16 (CXCL16) , BS-1441R , bioss , 1:2000.

Techniques: Inhibition, Immunofluorescence, Double Staining, Western Blot, Expressing, Control

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a KEGG pathway enrichment of DEGs between FACS-sorted TNFR2 + and TNFR2 − CD4 + T cells from MPE ( n = 3). b , c Volcano plot ( b ) and heatmap ( c ) of DEGs related to the chemokine signaling pathway. d , e Flow cytometry histograms ( d ) and comparisons of chemokine receptor expression (CXCR6, CCR4 and CCR6) ( e ) on TNFR2 + T reg cells and TNFR2 − T reg cells ( n = 13). f Schematic of Transwell chemotaxis assay testing TNFR2 − T reg chemotaxis toward MPE supernatant (by Figdraw). g , h Transwell chemotaxis assay comparing TNFR2 + T reg frequencies between freshly isolated cells and cells that migrated toward MPE supernatant after 4 h incubation. i Concentrations of CXCL16, CCL17, CCL22 and CCL20 in MPE and PB were quantified using ELISA ( n = 16). j Schematic of chemotaxis assay to investigate the chemotatic axis to attract TNFR2 + T reg cells in MPE. k , l Flow cytometry histograms and comparisons of chemotaxis of TNFR2 + T reg cells in response to MPE in the presence of anti-CXCL16, anti-CCL17, anti-CCL22 or anti-CCL20 mAbs. Data shown in d , e , g – i , k and l are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using paired two-tailed Student’s t -test ( e and h ), Wilcoxon test ( i ) or one-way ANOVA ( l ). * P < 0.05, ** P < 0.01, **** P < 0.0001. ns not significant, mAbs monoclonal antibodies.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Flow Cytometry, Expressing, Chemotaxis Assay, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Bioprocessing

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a Flow cytometry analysis comparing of CXCL16 expression on CD45 + and CD45 − cells from MPE ( n = 9). b CXCL16 expression on major cell populations of MPE: CAFs (CD45 − FAP + ), neutrophils (CD45 + CD11b + Ly6G + ), macrophages (CD45 + CD68 + ), T lymphocytes (CD45 + CD3 + ) and B lymphocytes (CD45 + CD19 + ) from MPE ( n = 8). c Schematic of CAF purification from MPE using MACS (by Figdraw). d Schematic of Transwell assay testing TNFR2 + T reg chemotaxis toward CXCL16-supplemented medium or CAF culture supernatant, with or without anti-CXCL16 mAbs (by Figdraw). e , f Representative flow cytometry plots ( e ) and comparisons ( f ) of TNFR2 + T reg frequencies recruited under different conditions ( n = 3). g Schematic diagram of the MPE mouse model. h Representative images showing mouse MPE and pleural cavity tumors. i – k Bioluminescence images depicting the growth ( n = 5) ( i ), MPE volume ( n = 5) ( j ) and Kaplan–Meier survival plot ( k ) of MPE mice ( n = 9–10 per group). l Concentrations of CXCL16 in MPE from mouse models ( n = 6). m , n Frequencies of TNFR2 + cells among T reg cells in murine MPE of each group. o , p Frequencies of CD8 + T cells from mouse MPE in each group. Data shown in a , b , e , f and h – p are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using paired two-tailed Student’s t -test ( a ), unpaired two-tailed Student’s t -test ( l ), one-way ANOVA ( b , f , j , n and p ) or log-rank test ( k ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant, CS culture supernatant, MACS magnetic-activated cell sorting, α-CXCL16 CXCL16 neutralizing antibody.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Flow Cytometry, Expressing, Purification, Transwell Assay, Chemotaxis Assay, Two Tailed Test, FACS

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a GSVA of signaling pathways enriched in CXCL16 + CAFs compared with CXCL16 − CAFs. b , c Gene set enrichment analysis (GSEA) was performed on gene sets related to the hypoxia signaling pathway ( b ) and the glycolysis signaling pathway ( c ). A positive NES indicates higher expression in CXCL16 + CAFs. d , e Lactate levels in CAFs cultured under normoxia (21% oxygen) or hypoxia (1% oxygen) for 48 h or at indicated time points ( n = 3). f , g CXCL16 concentrations in CAFs supernatant under normoxia or hypoxia for 48 h or at indicated time points ( n = 3). h Western blot of CXCL16 expression in CAFs under hypoxia at designated times. i Schematic of metabolic modulators targeting glycolysis or lactate production. j , k Lactate levels ( j ) and CXCL16 concentrations ( k ) in CAFs treated with indicated glycolysis modulators for 48 h ( n = 3). l – n Dose-dependent effects of DCA ( l ), oxamate ( m ) or rotenone ( n ) on lactate and CXCL16 levels in CAFs supernatant. o Western blot analysis of CXCL16 expression in CAFs treated with the indicated concentrations of glycolysis modulators for 48 h. Data shown in d – h and j – o are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t -test ( d and f ) or one-way ANOVA ( e , g and j – n ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant, NES normalized enrichment score, DMSO dimethylsulfoxide, DCA dichloroacetate.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Protein-Protein interactions, Expressing, Cell Culture, Western Blot, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a Western blot analysis of the indicated proteins in CAFs treated with increasing concentrations of exogenous lactate or glucose for 48 h. b CXCL16 concentrations in CAFs supernatant after exogenous lactate treatment. c , d Lactate ( c ) and CXCL16 ( d ) levels in CAFs supernatant after treatment with glucose for 48 h. e Western blot analysis of the indicated proteins in CAFs treated with DCA, oxamate or rotenone for 48 h. f Schematic of LDHA knockdown in CAFs. g Lactate in the culture supernatant of LDHA-knockdown CAFs. h Western blot analysis of the indicated proteins in LDHA-knockdown CAFs. i CXCL16 levels in the supernatant of LDHA-knockdown CAFs. j Western blot analysis of the indicated proteins in CAFs treated with increasing concentrations of β-alanine, lactylation modulator, for 48 h. k CXCL16 concentrations in CAFs supernatant after β-alanine treatment. Data shown in a – e and g – k are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using one-way ANOVA ( b – d , g , i and k ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant, KD knockdown, sh-LDHA shRNA against LDHA, sh-NC negative control.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Western Blot, Knockdown, shRNA, Negative Control

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a IGV tracks presenting H3K18la enrichment at the CXCL16 gene locus in CAFs from MPE by CUT&Tag analysis. b ChIP–qPCR validation of H3K18la binding at the CXCL16 promoter. c – e Predicted FOXO3 binding sites at the CXCL16 promoter using JASPAR ( http://jaspar.genereg.net ). f ChIP–qPCR validation of FOXO3 binding at the CXCL16 gene promoter in CAFs. g Western blot analysis of FOXO3 and CXCL16 expression following FOXO3 knockdown or overexpression in CAFs. h , i CXCL16 levels in CAFs supernatants after FOXO3 knockdown ( h ) or overexpression ( i ). j Schematic of dual-luciferase reporter assay, drawn by Figdraw. k Luciferase assays assessing FOXO3-mediated regulation of CXCL16 transcription in CAFs. l , m CUT&Tag ( l ) and ChIP–qPCR ( m ) analyses showing H3K18la enrichment at the FOXO3 promoter. n Western blot analysis of the indicated proteins in CAFs treated with glucose (0–20 mm/l), DCA (0–20 mm/l), oxamate (0–20 mm/l), retenone (0–50 nm/l) or β-alanine (0–20 mm/l) for 48 h. o Western blot analysis of the indicated proteins in LDHA-knockdown CAFs. Data shown in b , f – i , k and m – o are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t -test ( h and i ) or one-way ANOVA ( b , f , k and m ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant, IGV Integrative Genomics Viewer, OE overexpression.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: ChIP-qPCR, Biomarker Discovery, Binding Assay, Western Blot, Expressing, Knockdown, Over Expression, Luciferase, Reporter Assay, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a Schematic illustrating the the generation of LDHA-knockout NIH/3T3 fibroblasts (LDHA −/− 3T3) using CRISPR–Cas9, drawn by Figdraw. b Western blot validation of LDHA knockout in LDHA −/− 3T3 fibroblasts. c , d Lactate ( c ) and CXCL16 ( d ) levels in the culture supernatant of LDHA −/− 3T3 fibroblasts. e Western blot analysis of the indicated proteins in LDHA −/− 3T3 fibroblasts cultured with or without glucose. f Schematic of MPE mouse model. g Representative images showing mouse MPE and thoracic tumors in each group. h – j, Bioluminescence images depicting the growth ( n = 5) ( h ), MPE volume ( n = 6) ( i ) and Kaplan–Meier survival curves (n = 10 per group) ( j ) of MPE-bearing mice. Data shown in c – e and g – j are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t -test ( c and d ), one-way ANOVA ( i ) and log-rank test ( j ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant, LDHA−/−3T3 LDHA-knockout NIH/3T3 fibroblasts.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Knock-Out, CRISPR, Western Blot, Biomarker Discovery, Cell Culture, Two Tailed Test

Journal: Experimental & Molecular Medicine

Article Title: H3K18 lactylation in cancer-associated fibroblasts drives malignant pleural effusion progression via TNFR2 + T reg recruitment

doi: 10.1038/s12276-025-01557-3

Figure Lengend Snippet: a , b Lactate ( a ) and CXCL16 ( b ) levels measured in MPE from mice treated with LDHA −/− 3T3 fibroblasts or control fibroblasts. c – j Flow cytometry plots ( c , e , g and i ) and comparisons ( d , f , h and j ) of the frequencies of TNFR2 + T reg cells ( c and d ), IFN-γ + CD8 + T cells ( e and f ), granzyme B + CD8 + T cells ( g and h ) and perforin + CD8 + T cells ( i and j ) in MPE. k Schematic diagram illustrating the proposed mechanism: CAFs in MPE undergo glycolysis, leading to elevated endogenous lactate levels. This increase in lactate induces H3K18 lactylation modification at the promoter regions of both the CXCL16 gene and its transcription factor FOXO3, thereby promoting CXCL16 expression. TNFR2 + T reg cells, which express high levels of CXCR6, the only known receptor for CXCL16, are efficiently recruited into MPE. This recruitment dampens the antitumor response generated by CD8 + T cells, leading to the immunosuppression and progression of MPE (by Figdraw). Data shown in a – j are representative of at least three independent experiments (mean ± s.d.). Statistical analysis was performed using unpaired two-tailed Student’s t -test ( a and b ) and one-way ANOVA ( d , f , h and j ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant.

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature for 2 h, followed by overnight incubation with primary antibodies at 4 °C, including anti-Pan-Kla antibody (#PTM-1401, PTM BIO), anti-H3K18la antibody (#PTM-1406RM, PTM BIO), anti-H3K9la antibody (#PTM-1419RM, PTM BIO), anti-H3K27la antibody (#PTM-1428, PTM BIO), anti-H4K12la antibody (#PTM-1411RM, PTM BIO), anti-H4K8la antibody (#PTM-1415RM, PTM BIO), anti-CXCL16 antibody (#60123-1-Ig, Proteintech), anti-FOXO3 antibody(#10849-1-AP, Proteintech), anti-GAPDH antibody (#60004-1-Ig, Proteintech) and anti-histone H3 antibody (#17168-1-AP, Proteintech).

Techniques: Control, Flow Cytometry, Modification, Expressing, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: Remodeling of the immune microenvironment is linked to adverse outcome in pediatric T cell acute lymphoblastic leukemia

doi: 10.1038/s41467-025-65134-y

Figure Lengend Snippet: a Interaction specificity weights of ligand-receptor interactions between NC monocytes and DNαβ T cells in T-ALL group 2 patients (NATMI ). b Bubble heatmap showing expression of genes involved in the CXCL16-CXCR6 axis in group 2 T-ALL patients at diagnosis. c mRNA expression of CXCL16 , ADAM10 and ADAM17 in peripheral blood NC monocytes for group 2 T-ALL patients at diagnosis ( n = 1603 cells), prophase ( n = 740 cells) and remission ( n = 103 cells) and healthy donors ( n = 168 cells). A Kruskal-Wallis test followed by Dunn’s post-hoc test with Holm correction was used for statistical testing and only significant comparisons are shown. d Barplot showing concentration of soluble CXCL16 in serum of primary peripheral blood samples grouped by percentage of NC monocytes of monocytes. Data are presented as mean values +/- SEM (0-30%: n = 3, 30-60%: n = 2, 60–100%: n = 2) including technical duplicates. A Kruskal-Wallis test followed by Dunn’s post-hoc test with Holm correction was used for statistical testing and only significant comparisons are shown. e Experimental approach for in vitro induction of CD8 low cells. CD8 + T cells were activated with anti-CD3/CD28 beads and cultured with 150 U/ml IL-2 alone or IL-2 with 100 ng/ml CXCL16 for 7 days. Upon harvest, CD8 high , CD8 mid and CD8 low CD3 + T cells were sorted and processed for low-input RNA-seq using the Smart-seq2 protocol . f , Heatmap of scaled normalized expression of low-input RNA-seq using the Smart-seq2 protocol of genes in the DNαβ T cell transcriptome score for in vitro induced CD8 high ( n = 3), CD8 mid ( n = 4) and CD8 low T cells at day7 in the absence ( n = 2) or presence ( n = 4) of CXCL16. g Heatmap of regulon activity (RScenic ) of top DNαβ T cell regulons shown in Fig. for sorted CD8 high , CD8 mid and CD8 low T cells from cultured CD8 + T cells harvested at day 7. h Boxplots showing regulon activity for in vitro induced CD8 low T cells at day 7 in the presence or absence of CXCL16 shown in ( g ) for each hierarchical cluster ( n = 3, n = 3 and n = 5 regulons in each cluster, respectively). A paired t-test was used for statistical analysis; n.s.: not significant.

Article Snippet: Human CXCL16 was measured in duplicates using a multiplex assay (BosterBio, #SEST004-P0004) by BosterBio ( https://www.bosterbio.com/ Pleasenton, CA).

Techniques: Expressing, Biomarker Discovery, Concentration Assay, In Vitro, Cell Culture, RNA Sequencing, Activity Assay