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GCDCA induces M2-type polarization in macrophages through activation of <t>S1PR2</t> receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.
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GCDCA induces M2-type polarization in macrophages through activation of <t>S1PR2</t> receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.
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GCDCA induces M2-type polarization in macrophages through activation of <t>S1PR2</t> receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.
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GCDCA induces M2-type polarization in macrophages through activation of <t>S1PR2</t> receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.
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GCDCA induces M2-type polarization in macrophages through activation of S1PR2 receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Journal: Frontiers in Immunology

Article Title: GCDCA promotes hepatocellular carcinoma progression through S1PR2/PI3K/AKT-mediated polarization of M2-type macrophages

doi: 10.3389/fimmu.2026.1640450

Figure Lengend Snippet: GCDCA induces M2-type polarization in macrophages through activation of S1PR2 receptors. (A) Single-cell sequencing to analyze bile acid receptor expression in M2 macrophages. (B) Activation of the S1PR2 receptor by GCDCA was detected using the Tango GPCR assay (n = 3 in each group). (C, D) TCGA database analysis of S1PR2 correlation with KRT19 , EPCAM , SOX9 , CD68 , MRC1 , CD163 . (E) Representative immunohistochemical staining of CD68, CD163, and S1PR2-positive cells in the livers of different groups of rats, scale bar = 100 μm (n = 3 in each group). (F) Representative immunofluorescence staining of S1PR2 (red) co-localized with CD163- (green) and CD206-(green) positive cells in the livers of different groups of rats, scale bar = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Article Snippet: The following antibodies were used for immunohistochemistry: EPCAM (1:100, ab71916; Abcam), KRT19 (1:4000, 10712-1-AP; Proteintech), SOX9 (1:2000, ab185230; Abcam), CD163 (1:4000, ab213612; Abcam), S1PR2 (1:100, sc-365963; Santa Cruz Biotechnology), and CD68 (1:3000, ab31630; Abcam).

Techniques: Activation Assay, Single Cell, Sequencing, Expressing, Immunohistochemical staining, Staining, Immunofluorescence

Pharmacological inhibition of S1PR2 by JTE-013 reverses GCDCA-induced stemness. (A) Flow chart of animal experiments. (B) Kaplan–Meier plots of DEN-induced primary hepatocellular carcinoma in rats after administration of GCDCA and the receptor inhibitor JTE-013 ( n = 5 in each group). (C) Representative images of liver tumors in different groups of rats (scale bar = 1 cm) and HE-stained images (scale bar = 200 μm). (D, E) Analysis of maximum diameter and number of tumors (n = 4 in each group). (F–H) Serum AST and ALT activities and TBA levels in different groups of rats (n = 4 in each group). (I) Representative immunohistochemical staining of KRT19, EPCAM, and SOX9-positive cells in the livers of different groups of rats, scale bar = 200 μm (n = 3 in each group). (J) Representative immunofluorescence staining of KRT19, EPCAM, and SOX9 co-localized positive cells in the livers of different groups of rats, scale bar = 50 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Journal: Frontiers in Immunology

Article Title: GCDCA promotes hepatocellular carcinoma progression through S1PR2/PI3K/AKT-mediated polarization of M2-type macrophages

doi: 10.3389/fimmu.2026.1640450

Figure Lengend Snippet: Pharmacological inhibition of S1PR2 by JTE-013 reverses GCDCA-induced stemness. (A) Flow chart of animal experiments. (B) Kaplan–Meier plots of DEN-induced primary hepatocellular carcinoma in rats after administration of GCDCA and the receptor inhibitor JTE-013 ( n = 5 in each group). (C) Representative images of liver tumors in different groups of rats (scale bar = 1 cm) and HE-stained images (scale bar = 200 μm). (D, E) Analysis of maximum diameter and number of tumors (n = 4 in each group). (F–H) Serum AST and ALT activities and TBA levels in different groups of rats (n = 4 in each group). (I) Representative immunohistochemical staining of KRT19, EPCAM, and SOX9-positive cells in the livers of different groups of rats, scale bar = 200 μm (n = 3 in each group). (J) Representative immunofluorescence staining of KRT19, EPCAM, and SOX9 co-localized positive cells in the livers of different groups of rats, scale bar = 50 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Article Snippet: The following antibodies were used for immunohistochemistry: EPCAM (1:100, ab71916; Abcam), KRT19 (1:4000, 10712-1-AP; Proteintech), SOX9 (1:2000, ab185230; Abcam), CD163 (1:4000, ab213612; Abcam), S1PR2 (1:100, sc-365963; Santa Cruz Biotechnology), and CD68 (1:3000, ab31630; Abcam).

Techniques: Inhibition, Staining, Immunohistochemical staining, Immunofluorescence

Inhibition of S1PR2 receptor reverses GCDCA-induced polarization of M2 macrophages. (A) Expression of M2 macrophage marker genes Mrc1 , Cd163 , and Arg1 in NR8383 cells after inhibition of the S1PR2 receptor (n = 3 in each group). (B) Expression of CD206, CD163, and ARG1 proteins in NR8383 cells after inhibition of the S1PR2 receptor. (C) Expression of M2 macrophage marker genes MRC1 , ARG1 , and TGF-β in THP-1 cells after inhibition of the S1PR2 receptor (n = 3 in each group). (D) Expression of CD206, CD163, and ARG1 proteins in THP-1 cells after inhibition of the S1PR2 receptor. (E) Expression of stemness genes Krt19 , Epcam , and Sox9 in RH35 cells after co-culture with GCDCA-induced NR8383 cells pretreated with JTE-013 (n = 3 in each group). (F) Expression of KRT19, EPCAM, and SOX9 proteins in RH35 cells after co-culture with GCDCA-induced NR8383 cells pretreated with JTE-013. (G) Expression of stemness genes KRT19 , EPCAM , and SOX9 in Huh7 cells after co-culture with GCDCA-induced THP-1 cells pretreated with JTE-013 (n = 3 in each group). (H) Expression of KRT19, EPCAM, and SOX9 proteins in Huh7 cells after co-culture with GCDCA-induced THP-1 cells pretreated with JTE-013. (I-J) Representative immunofluorescence staining of M2 macrophage markers CD206 (white) and CD163 (white) co-localized with KRT19- (red) and EPCAM- (green) positive cells in the livers of different groups of rats, scale bar = 100 μm. GAPDH is used as an internal reference. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Journal: Frontiers in Immunology

Article Title: GCDCA promotes hepatocellular carcinoma progression through S1PR2/PI3K/AKT-mediated polarization of M2-type macrophages

doi: 10.3389/fimmu.2026.1640450

Figure Lengend Snippet: Inhibition of S1PR2 receptor reverses GCDCA-induced polarization of M2 macrophages. (A) Expression of M2 macrophage marker genes Mrc1 , Cd163 , and Arg1 in NR8383 cells after inhibition of the S1PR2 receptor (n = 3 in each group). (B) Expression of CD206, CD163, and ARG1 proteins in NR8383 cells after inhibition of the S1PR2 receptor. (C) Expression of M2 macrophage marker genes MRC1 , ARG1 , and TGF-β in THP-1 cells after inhibition of the S1PR2 receptor (n = 3 in each group). (D) Expression of CD206, CD163, and ARG1 proteins in THP-1 cells after inhibition of the S1PR2 receptor. (E) Expression of stemness genes Krt19 , Epcam , and Sox9 in RH35 cells after co-culture with GCDCA-induced NR8383 cells pretreated with JTE-013 (n = 3 in each group). (F) Expression of KRT19, EPCAM, and SOX9 proteins in RH35 cells after co-culture with GCDCA-induced NR8383 cells pretreated with JTE-013. (G) Expression of stemness genes KRT19 , EPCAM , and SOX9 in Huh7 cells after co-culture with GCDCA-induced THP-1 cells pretreated with JTE-013 (n = 3 in each group). (H) Expression of KRT19, EPCAM, and SOX9 proteins in Huh7 cells after co-culture with GCDCA-induced THP-1 cells pretreated with JTE-013. (I-J) Representative immunofluorescence staining of M2 macrophage markers CD206 (white) and CD163 (white) co-localized with KRT19- (red) and EPCAM- (green) positive cells in the livers of different groups of rats, scale bar = 100 μm. GAPDH is used as an internal reference. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.

Article Snippet: The following antibodies were used for immunohistochemistry: EPCAM (1:100, ab71916; Abcam), KRT19 (1:4000, 10712-1-AP; Proteintech), SOX9 (1:2000, ab185230; Abcam), CD163 (1:4000, ab213612; Abcam), S1PR2 (1:100, sc-365963; Santa Cruz Biotechnology), and CD68 (1:3000, ab31630; Abcam).

Techniques: Inhibition, Expressing, Marker, Co-Culture Assay, Immunofluorescence, Staining

GCDCA induces macrophage polarization toward M2 via the S1PR2/PI3K/AKT signaling pathway. (A) Volcano plot of differentially expressed genes (DEGs) between GCDCA-treated and control groups (n = 3 in each group). (B) Histogram of KEGG enrichment results of DEGs between GCDCA-treated and control groups. (C) Bubble plots of GO enrichment results of DEGs between GCDCA-treated and control groups. (D) Western blotting for p-AKT, AKT, p-PI3K, and PI3K protein expression in GCDCA-treated NR8383 cells. (E) Western blotting was used to detect the expression of p-AKT, AKT, p-PI3K, PI3K, CD206, and ARG1 proteins after pretreatment with LY294002 inhibitor.

Journal: Frontiers in Immunology

Article Title: GCDCA promotes hepatocellular carcinoma progression through S1PR2/PI3K/AKT-mediated polarization of M2-type macrophages

doi: 10.3389/fimmu.2026.1640450

Figure Lengend Snippet: GCDCA induces macrophage polarization toward M2 via the S1PR2/PI3K/AKT signaling pathway. (A) Volcano plot of differentially expressed genes (DEGs) between GCDCA-treated and control groups (n = 3 in each group). (B) Histogram of KEGG enrichment results of DEGs between GCDCA-treated and control groups. (C) Bubble plots of GO enrichment results of DEGs between GCDCA-treated and control groups. (D) Western blotting for p-AKT, AKT, p-PI3K, and PI3K protein expression in GCDCA-treated NR8383 cells. (E) Western blotting was used to detect the expression of p-AKT, AKT, p-PI3K, PI3K, CD206, and ARG1 proteins after pretreatment with LY294002 inhibitor.

Article Snippet: The following antibodies were used for immunohistochemistry: EPCAM (1:100, ab71916; Abcam), KRT19 (1:4000, 10712-1-AP; Proteintech), SOX9 (1:2000, ab185230; Abcam), CD163 (1:4000, ab213612; Abcam), S1PR2 (1:100, sc-365963; Santa Cruz Biotechnology), and CD68 (1:3000, ab31630; Abcam).

Techniques: Control, Western Blot, Expressing