Journal: Nature Communications
Article Title: Treg-γδ T cell axis determines sexual dimorphism in hepatocarcinogenesis
doi: 10.1038/s41467-026-69603-w
Figure Lengend Snippet: a Schematic flow of mouse orthotopic HCC implantation after gonadectomy. Liver SVG Vector by SVG Repo, licensed under CC0 1.0 Universal. Scissors SVG Vector by SVG Repo, licensed under CC0 1.0 Universal. b Representative flow plots (left) and quantification (right) of intratumoral Tregs after gonadectomy ( n = 5). c The relative mRNA expression of Ccl2 in orthotopic HCC measured by qPCR ( n = 5). d Representative immunofluorescent staining for AFP, Ccl2 and DAPI. scale bars, 50 μm (left). Quantification of AFP + Ccl2 + cells in HCC (right) ( n = 7). e HepG2 and Hepa1-6 cells were treated with testosterone (TS) at different concentrations. Representative fluorescence intensity plots (left) and quantification (right) of Ccl2 after treatment ( n = 4, 6). f The correlation analysis between the relative mRNA expression of CCL2 and NFKB1 in human HCC samples from the GEPIA database ( n = 398). g The immunoblots of Integrin α6, PAK1, AR, p-NFκB and NFκB ( n = 3). h–j Genetically deletion of AR in Hepa1-6 using CRISPR/Cas9 followed by orthotopic Hepa1-6 implantation. Representative liver image (left) and tumour volume (right) ( n = 5) ( h ). Representative immunofluorescent staining for AFP, Ccl2 and DAPI. scale bars, 50 μm (left). Quantification of Ccl2 intensity in HCC (right) ( n = 4) ( i ). Representative flow cytometry plots (left) and the statistical quantification (right) of Tregs ( n = 4) ( j ). Cut&Tag analysis was used to assess the interaction between NFκB and Ccl2 in HepG2 cells. Genomic Regions Enrichment of Annotations Tool (GREAT) (upper). Annotation of all peaks (lower) ( k , n = 3). The chromatin accessibility heatmap showing NFκB binding sequence regions with or without testosterone (TS) treatment (upper). Cut&Tag tracks of Ccl2 locus in HepG2 cells (lower) ( l , n = 3). Top2 de novo motifs (#1 and #2) of NFκB enriched by HOMER using all called peaks ( m , n = 3). The relative expression of Ccl2 with specific primers targeting motif #2 by qPCR ( n , n = 3). Data represent three independent experiments. Data are shown as the mean ± SEM. P value of Pearson r correlation ( f ) or one-way ANOVA followed by Bonferroni’s multiple comparisons test ( e and n ) or two-way ANOVA followed by Bonferroni’s multiple comparisons test ( b – d and h – j ). For additional details, see also Supplementary Fig. . Source data are provided as a Source Data file.
Article Snippet: The antibodies used were as follows: Integrin α6 (Huabio, SR45-00l, 1:1000), PAK1 (Baijia, IPB5302, 1:1000), Androgen receptor (Huabio, PO00-29, 1:1000), Phospho-NFκB p65 (Ser468, Proteintech, 6N1, 1:1000), NFκB p65 (Proteintech, 4C7, 1:1000), Hif1α (Servicebio, GB111339 , 1:1000), S100a4 (Huabio, SD200-08, 1:1000), HSP90 (Servicebio, GB15284, 1:1000), ZAP70 (Zenbio, R26132 , 1:1000), phospho-ZAP70 (Tyr319, Zenbio, 310211, 1:1000), Akt (HUABIO, ET1609-51, 1:1000), phospho-Akt (Ser473, SinoBiological, 110852-R0069, 1:1000), and Tubulin (Huabio, 1-B11, 1:5000).
Techniques: Plasmid Preparation, Expressing, Staining, Fluorescence, Western Blot, CRISPR, Flow Cytometry, Binding Assay, Sequencing
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