Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) dsDNA expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of dsDNA were generated by Alexa Fluor 488–conjugated secondary antibodies. Quantification of dsDNA in the cytoplasm of the B16F10 cells ( B ) and 4T1, H22, KPC, and CT26 cells ( C ). n = 3 biologically independent samples. ( D ) Schematic diagram of STING pathway activation upon recognition of cytosolic dsDNA. ELISA revealed cGAMP production in B16F10 cells ( E ) and 4T1, H22, KPC, and CT26 cells ( G ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( F ) ELISA revealed cGAMP production in B16F10 cells after incubation of PRO P+B with or without DNase I addition. ( H ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in B16F10 cells treated with PBS, PRO P , PRO B , and PRO P+B for 24 hours. ELISA revealed IFN-β production in B16F10 cells ( I ) and 4T1, H22, KPC, and CT26 cells ( J ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( K ) PD-L1 expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of PD-L1 were generated by Alexa Fluor 488–conjugated secondary antibodies. ( L ) Flow cytometry analysis of PD-L1 expression in 4T1, H22, KPC, and CT26 cells treated with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. (K) is the quantification of ( M ). Results are presented as the means ± SD.

Article Snippet: WB was performed as above using antibodies against cGAS (1:1000; CST, no. 15102), p-STING (Ser 366 : 1:500; CST, no. 72971), and p-IRF3 (Ser 396 : 1:500; CST, no. 4947).

Techniques: Expressing, Immunofluorescence, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) dsDNA expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of dsDNA were generated by Alexa Fluor 488–conjugated secondary antibodies. Quantification of dsDNA in the cytoplasm of the B16F10 cells ( B ) and 4T1, H22, KPC, and CT26 cells ( C ). n = 3 biologically independent samples. ( D ) Schematic diagram of STING pathway activation upon recognition of cytosolic dsDNA. ELISA revealed cGAMP production in B16F10 cells ( E ) and 4T1, H22, KPC, and CT26 cells ( G ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( F ) ELISA revealed cGAMP production in B16F10 cells after incubation of PRO P+B with or without DNase I addition. ( H ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in B16F10 cells treated with PBS, PRO P , PRO B , and PRO P+B for 24 hours. ELISA revealed IFN-β production in B16F10 cells ( I ) and 4T1, H22, KPC, and CT26 cells ( J ) 24 hours after indicated treatments (PBS, PRO P , PRO B , and PRO P+B ). ( K ) PD-L1 expression levels detected by immunofluorescence in B16F10 cells after incubating with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. The signals of PD-L1 were generated by Alexa Fluor 488–conjugated secondary antibodies. ( L ) Flow cytometry analysis of PD-L1 expression in 4T1, H22, KPC, and CT26 cells treated with indicated treatments (PBS, PRO P , PRO B , and PRO P+B ) for 24 hours. (K) is the quantification of ( M ). Results are presented as the means ± SD.

Article Snippet: WB was performed as above using antibodies against cGAS (1:1000; CST, no. 15102), p-STING (Ser 366 : 1:500; CST, no. 72971), and p-IRF3 (Ser 396 : 1:500; CST, no. 4947).

Techniques: Expressing, Immunofluorescence, Generated, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) Schematic diagram of STING pathway activation and maturation of DCs. ( B ) Representative flow cytometric analysis image and relative quantifications of mature DC markers CD80/CD86 in BMDCs co-incubated with B16F10 cells treated with PBS, PRO P , PRO B , and PRO P+B for 24 hours. ( C ) Schematic diagram of STING pathway activation and maturation of DCs. Production of cGAMP in DC2.4 cells ( D ) and THP1 cells ( E ), which were co-incubated with B16F10, 4T1, H22, KPC, and CT26 cells after the tumor cells were treated with PBS, PRO P , PRO B , and PRO P+B , respectively, measured by ELISA assay. ( F ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in DC2.4 cells that were co-incubated with B16F10, 4T1, H22, KPC, and CT26 cells after the tumor cells were treated with PBS, PRO P , PRO B , and PRO P+B , respectively. Results are presented as the means ± SD.

Article Snippet: WB was performed as above using antibodies against cGAS (1:1000; CST, no. 15102), p-STING (Ser 366 : 1:500; CST, no. 72971), and p-IRF3 (Ser 396 : 1:500; CST, no. 4947).

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: PROTAC-based synthetic lethality strategy endogenously activates systemic STING to boost antitumor immunity

doi: 10.1126/sciadv.ado7448

Figure Lengend Snippet: ( A ) IC 50 (median inhibitory concentration) and CI values of different cell lines after incubation with LNP@PRO P , LNP@PRO B , and LNP@PRO P+B . ( B and C ) Representative pictures of clonogenic assay in B16F10, 4T1, H22, KPC, and CT26 cells treated with PBS (G1), LNP@PRO P (G2), LNP@PRO B (G3), or LNP@PRO P+B (G4) for 24 hours. ( D ) Colony-forming efficiency of B16F10, 4T1, H22, KPC, and CT26 cells. ( E ) Schematic illustration of the timeline for B16F10 tumor inoculation and treatments in a C57BL/6 mouse model. Mice with ~100-mm 3 subcutaneous tumors were intravenously (iv) administered with LNP@PRO P , LNP@PR B , and LNP@PRO P+B five times at a PRO B equivalent dose of 10 mg kg −1 . sc, subcutaneously. ( F ) Tumor growth of B16F10 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . n = 7 biologically independent samples. ( G ) Survival of B16F10 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . ( H ) Immunofluorescence staining of BRD4, PARP1, PD-L1, and γ-H2AX expression levels in B16F10 tumor after indicated treatment. ( I ) WB analysis of cGAS, p-STING, and p-IRF3 protein levels in B16F10 tumor after indicated treatment. ( J ) Schematic illustration of the timeline for 4T1/CT26 tumor inoculation and treatments in a BALB/C mouse model. Tumor growth ( K ) and survival ( L ) of 4T1 tumor–bearing mice treated with LNP@PRO P , LNP@PRO B , or LNP@PRO P+B . n = 7 biologically independent samples. Tumor growth ( M ) and survival ( N ) of CT26 tumor–bearing mice treated with PBS (G1), LNP@PRO P (G2), LNP@PRO B (G3), or LNP@PRO P+B (G4). n = 7 biologically independent samples. Results are presented as the means ± SEM. ( O ) Schematic illustration of the timeline for B16F10 tumor inoculation and treatments in a NCG mouse model. ( P ) Tumor growth of B16F10 tumor–bearing mice treated with PBS or LNP@PRO P+B . n = 6 biologically independent samples. ( Q ) Photographs of the excised tumors. ( R ) Tumor weights of different groups after treatments. ( S ) Tumor inhibition of different groups after treatments. Results are presented as the means ± SD.

Article Snippet: WB was performed as above using antibodies against cGAS (1:1000; CST, no. 15102), p-STING (Ser 366 : 1:500; CST, no. 72971), and p-IRF3 (Ser 396 : 1:500; CST, no. 4947).

Techniques: Concentration Assay, Incubation, Clonogenic Assay, Immunofluorescence, Staining, Expressing, Inhibition