Fig 1: Effects of CP-25 on GRK2 and CXCR4 expression in HUVECs treated with CXCL12. Representative images of western blotting of (A) total, (D) cytoplasmic and (G) membrane expression of GRK2 and CXCR4. (B, C, E, F, H and I) Western blotting semi-quantification of GRK2 and CXCR4 expression. Data are expressed as the means ± standard deviation of three independent experiments. #P<0.05, ##P<0.01 vs. control; *P<0.05, **P<0.01 vs. CXCL12. CP-25, paeoniflorin-6′-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2.
Fig 2: Effects of CP-25 on ERK1/2 expression in HUVECs treated with CXCL12. (A) Representative images of western blotting of the expression of ERK1/2 and p-ERK1/2. (B and C) Western blotting semi-quantification of ERK1/2 and p-ERK1/2. (D) Ratio of p-ERK/total-ERK. (E) Representative images of western blotting for the co-expression of GRK2 and CXCR4, and GRK2 and p-ERK1/2. (F and G) Western blotting semi-quantification of the binding between GRK2 and CXCR4, and GRK2 and p-ERK1/2. Data are expressed as the means ± standard deviation of three independent experiments. ##P<0.01 vs. control; *P<0.05, **P<0.01 vs. CXCL12. CP-25, paeoniflorin-6‰-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2; p, phosphorylated.
Fig 3: Regulation of CXCR4 activity and signaling. (A) Upon ligand binding, CXCR4 could activate numerous signaling cascades, which may result in increased GRK2 membrane localization, weakening the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm and enhancing ERK1/2 phosphorylation. (B) CP-25 could inhibit ERK1/2 phosphorylation by reducing the membrane localization of GRK2 and enhancing the inhibitory effect of GRK2 on ERK1/2 in the cytoplasm. CP-25, paeoniflorin-6'-O-benzene sulfonate; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C chemokine receptor type 4; GRK2; G protein-coupled receptor kinase 2; p, phosphorylated.
Fig 4: a) Photoacoustic (PA) imaging of tumors from orthotopic GL261 tumor‐bearing mice collected before and after tail vein injection of CS‐J NPs or CS‐J@CM/6 NPs (dose: 5 mg kg−1) at different time points (tumors are highlighted by the red dashed circles). b) Imaging of tumor slices stained with rubeanic acid (RA) from GL261 tumor‐bearing mice administered with CS‐J NPs or CS‐J@CM/6 NPs (scale bar: 50 µm). c,d) Detection of GRK2 expression and S1PR1 expression on the CD3+ T cells in the BM of mice from different groups (n = 3). e,f) Flow cytometry analysis of TIM‐3+CD8+ T cells and their percentage in tumor from different group of mice (n = 5). g) Immunofluorescence images of PD‐L1 expression in tumor slices (scale bar: 25 µm). h) Schematic illustration of the specific interaction between ALCAM expressed on the endothelial cells of BBB and CD6 on the CS‐J@CM/6 NPs to make nanoparticles cross the BBB and target tumor.
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