Journal: Nature Communications

Article Title: Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates

doi: 10.1038/s41467-025-62776-w

Figure Lengend Snippet: a , b Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with BMS-CPP1 ( a ) or BMS-CPP2 ( b ) at indicated concentration, or indicated time. c , d Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with PD-LYSO ( c , d ) or BMS-CPP1 ( d ) for 8 h at indicated concentration. e Western blot analysis of HA-PD-L1 levels in HeLa cells (stably expressing HA-PD-L1) treated with BMS-CPP1 or BMS-CPP2 for 8 h at indicated concentration. f , g Immunofluorescence analysis of PD-L1 (red) degradation on cell membrane ( f ) or in whole cells ( g ). HeLa cells stably expressing PD-L1 were treated with BMS-CPP1, BMS-8, CPP1, or a combination of BMS-8 and CPP1. The nuclei were labeled by DAPI (blue). Scale bar, 10 μm. h Western blot analysis of PD-L1 levels in different cell lines treated with 25 nM of BMS-CPP1, BMS-8 or CPP1 for 8 h. i , j Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with 25 nM of BMS-CPP1 ( i ) or 5 nM of BMS-CPP2 ( j ) for 8 h along with bafilomycin A1 (100 μM) or MG132 (5 μM). k , l Western blot analysis of the inhibitory effect of Nystatin (50 μM), 7-KC (7-keto-cholesterol, 30 μM), CPZ (chlorpromazine, 10 μg/mL) or EIPA (ethylisopropylamiloride, 10 μg/mL) on the degradation of PD-L1 mediated by BMS-CPP1 ( k ) or BMS-CPP2 ( l ). Source data are provided as a Source Data file.

Article Snippet: The membranes were respectively incubated overnight with primary antibodies [PD-L1 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; PD-L1 antibody, Proteintech, mouse, 1:5000; CA9 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; CB 2 R antibody, Abcam, rabbit, 1:200; GAPDH antibody, Proteintech, mouse, 1:10000) at 4 o C with slightly shaking.

Techniques: Western Blot, Concentration Assay, Stable Transfection, Expressing, Immunofluorescence, Membrane, Labeling

Journal: Nature Communications

Article Title: Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates

doi: 10.1038/s41467-025-62776-w

Figure Lengend Snippet: a Schematic illustration of the tumor-inhibition study and the general treatment procedure. Created in BioRender. OU, ZI. (2025) https://BioRender.com/8m9xnsp . b Body weight change curves of mice from day 6 to day 18. Data represent the mean ± SEM ( n = 5 mice per group). c Tumor growth curves for each group. Comparison of the tumor size after 18 days of different treatments. Data represent the mean ± SEM ( n = 5 mice per group). The statistical significance was assessed using two-way ANOVA. d Photograph of the peeled-off tumors of all four groups. e Comparison of the tumor weight after tumor dissection. Data represent the mean ± SEM ( n = 5 mice per group). The statistical significance was assessed using two-tailed Student’s t -tests. f Metastasis of tumor in the spleens of each group. g Western blot analysis of PD-L1 in B16F10 tumor tissues. Densitometry was used to calculate protein levels, and data were normalized to control. Data represent the mean ± SEM ( n = 3 mice per group). Statistical significance was assessed using two-tailed Student’s t -tests. h Representative immunohistochemical staining of PD-L1 in tumor tissues. Scale bar, 50 μm. Source data are provided as a Source Data file.

Article Snippet: The membranes were respectively incubated overnight with primary antibodies [PD-L1 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; PD-L1 antibody, Proteintech, mouse, 1:5000; CA9 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; CB 2 R antibody, Abcam, rabbit, 1:200; GAPDH antibody, Proteintech, mouse, 1:10000) at 4 o C with slightly shaking.

Techniques: Inhibition, Comparison, Dissection, Two Tailed Test, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates

doi: 10.1038/s41467-025-62776-w

Figure Lengend Snippet: a , b Western blot analysis of PD-L1 levels in MDA-MB-231 cells ( a ) or 4T1 cells with low expression level of αvβ3 integrin ( b ) treated with BMS-CPP1 or BMS-RGD for 8 h at indicated concentration. c , d Western blot analysis of PD-L1 levels in MDA-MB-231 cells treated with BMS-RGD ( c ) or BMS-CPP1 ( d ) for 8 h at indicated concentration. Source data are provided as a Source Data file.

Article Snippet: The membranes were respectively incubated overnight with primary antibodies [PD-L1 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; PD-L1 antibody, Proteintech, mouse, 1:5000; CA9 antibody, Cell Signaling Technology (CST), rabbit, 1:1000; CB 2 R antibody, Abcam, rabbit, 1:200; GAPDH antibody, Proteintech, mouse, 1:10000) at 4 o C with slightly shaking.

Techniques: Western Blot, Expressing, Concentration Assay

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Primers used for plasmid construction.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Plasmid Preparation

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Primers used for qRT-PCR.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques:

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Expression profile and regulatory role of PDL1 in plasma exosomes of HCC. (A) IHC staining showing the protein levels of PDL1 and CD8 α in representative cancer tissues of HCC. Scale bar 40 μm. (B) Statistical analysis of IHC staining with PDL1 or CD8 α antibody in tissue sections of HCC, which was evaluated by Spearman's correlation ( ρ ). (C and D) TEM showing the morphology (C) and NTA showing the size (D) of isolated exosomes derived from plasma of HCC patients. Scale bar in (C) is 200 nm. (E) Western blot for protein levels of PDL1 and exosomal markers including CD63, CD9, and TSG101. (F) Scatter plots showing the expression profiles of exosomal PDL1 protein in plasma of HCC patients and healthy individuals, as examined by ELISA. Data were analyzed by Student's two-sided t -test and are represented as mean ± SD. ∗∗∗ p <0.001. (G) Statistical analysis of PDL1 level examined by IHC staining or CD8 α level examined by ELISA of HCC, which was evaluated by Pearson's correlation ( r ). (H) Kaplan–Meier analysis showing the influence of exosomal PDL1 on 5-year overall survival of HCC, as determined by log-rank test. The high and low level of exosomal PDL1 level was determined according to the median value.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Expressing, Clinical Proteomics, Immunohistochemistry, Isolation, Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Clinicopathologic features of different levels of PDL1 protein in plasma exosomes of HCC patients.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Clinical Proteomics

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Regulatory effect of exosomal PDL1 on proliferation of CD8 + T and hepatic cancer cells. (A–D): qRT-PCR (A and C) and western blot (B and D) for PDL1 level in Huh7 cells with the overexpression or knockdown of PDL1. (E) Western blot for exosomal PDL1 level in supernatants of PDL1-silenced Huh7 cells. (F and G): TEM (F) and NTA (G) for isolated exosomes derived from the supernatants of PDL1-silenced Huh7 cells. (H) Proliferation of Huh7 cells with the overexpression or silencing of PDL1, as assayed by CCK8. (I) Proliferation of CD8 + T cells with the treatments of exosomes derived from the supernatants of Huh7 cells, as assayed by CCK8. PDL1 was overexpressed or silenced in Huh7 cells. (J) ELISA for TNF α and IFN- γ levels in supernatants of human CD8 + T cells after treatments with exosomes. Data were analyzed by Student's two-sided t -test and are represented as mean ± SD. ∗∗ p <0.01, ∗∗∗ p <0.001.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Isolation of exosomes derived from the supernatants of cultured Hepa1–6 cells with different levels of PDL1. (A–D): qRT-PCR (A and C) and western blot (B and D) for PDL1 level in Hepa1–6 cells with the overexpression or knockdown of PDL1. (E) Western blot for protein level of PDL1, TSG101, and GAPDH in exosomes derived from the supernatants of PDL1-silenced Hepa1–6 cells. (F and G) TEM and NTA analysis for the isolated exosomes derived from the supernatants of Pdl1-silenced Hepa1–6 cells. (H) Proliferation of Hepa1–6 cells with the overexpression or silencing of Pdl1, as assayed by CCK8. (I) Proliferation of CD8 + T cells after treatments of exosomes derived from the supernatants of Hepa1–6 cells, as assayed by CCK8. Pdl1 was overexpressed or silenced in Hepa1–6 cells. (J) ELISA for TNF α and IFN- γ levels in supernatants of mice CD8 + T cells after treatments with exosomes. Data were analyzed by Student's two-sided t -test and are represented as mean ± SD. ∗∗∗ p <0.001.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Isolation, Derivative Assay, Cell Culture, Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Influence of exosomal Pdl1 on growth of tumors formed by Hepa1–6 cells. (A) The formed tumors by Hepa1–6 cells in C57L mice. After subcutaneous injection of Hepa1–6 cells in mice for 10 days, exosomes derived from the supernatants of Hepa1–6 cells that stably transfected with Flag-Pdl1 plasmid or Flag vector or stably infected with shControl or shPdl1 lentivirus were administrated into mice by tail vein injection every 3 days for four consecutive injections. The smallest scale of the ruler, 1 mm. (B and C) Statistical analysis for tumor volume and tumor weight in (A). (D) Representative images of IHC staining for protein levels of Pdl1 and CD8 in mouse tumor tissues. Scale bar 50 μm. Data were analyzed by Student's two-sided t -test and are represented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Injection, Derivative Assay, Stable Transfection, Transfection, Plasmid Preparation, Infection, Immunohistochemistry

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Exosomal PDL1 Suppresses the Anticancer Activity of CD8 + T Cells in Hepatocellular Carcinoma

doi: 10.1155/2024/1608582

Figure Lengend Snippet: Statistical analyses for protein levels of Pdl1 and CD8a in mice tumors examined by IHC staining.

Article Snippet: ELISA plates (96-well) (Biolegend) that coated with Human PDL1 antibody (DY156, R&D SYSTEMS, Minneapolis, MN, USA), Human TNF α ELISA Kit (KIT10602, SinoBiology, Beijing, China), Mouse TNF α Matched ELISA Antibody Pair Set (SEKA50349, SinoBiology), Human IFN gamma ELISA Kit (KIT11725A, SinoBiology), and Mouse IFN gamma ELISA Kit (KIT50709, SinoBiology) were used for detecting protein levels of PDL1, TFN α , and IFN- γ in samples according to manufactures' instructions.

Techniques: Staining, Plasmid Preparation