cgamp  (MedChemExpress)

Journal: bioRxiv

Article Title: PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages

doi: 10.1101/2020.07.17.198598

Figure Lengend Snippet: (A) Syngeneic mice were engrafted with Myc-CAP (STING lo ) cells, and tumors were harvested when the tumor volume reached 400 mm 3 . Single cell suspensions of the tumors were treated with the indicated drug(s), in the presence or absence of GW4869 (MV biogenesis inhibitor). (B) Bone marrow derived macrophage (BMDMs) were co-cultured for 30 hours with supernatants (-/+ exogenous 50 units of DNase I pre-treatment for 30 minutes) from Myc-CAP cells that were treated with the indicated drug(s) for 36 hours. (C) cGAMP (10 μg/ml) was pre-incubated with 10 units of DNAse I or mock control for 30 minutes and then treated with BMDMs for 30 hours. (D-G) STING +/+ /STING -/- BMDMs were co-cultured for 36 hours with supernatants from Myc-CAP and B6-Myc cancer cells, that were treated with the indicated drug(s) for 36 hours. Supernatants were collected from A-G at the end of treatment and analyzed for IFNβ1 by ELISA. (H) Myc-CAP (STING lo ) tumor-bearing mice were treated with the indicated drug(s) until tumors first reached approx. 2500 mm 3 . For the degerelix/rucaparib/buparlisib combination, additional cohorts of mice underwent concomitant treatment with STING antagonist H-151 or GW4869. Tumor volume was recorded daily for duration of experiment. For in vitro experiments, n=2 independent experiments and for in vivo experiments n= 3-4 animals /group. Significance/p-values were calculated by one-way ANOVA and are indicated as follows, *p < 0.05; **p < 0.01, ***p < 0.001; **** p< 0.0001; ns = not statistically significant;

Article Snippet: To rule out cGAMP as the mediator of STING pathway activation within BMDMs, 10 μg of cGAMP disodium salt (MedChem Express) was reconstituted in RNA/DNAse free water and pre-treated with 10 units of DNase I.

Techniques: Derivative Assay, Cell Culture, Incubation, Control, Enzyme-linked Immunosorbent Assay, In Vitro, In Vivo

Journal: bioRxiv

Article Title: PARP and PI3K inhibitor combination therapy eradicates c-MYC-driven murine prostate cancers via cGAS/STING pathway activation within tumor-associated macrophages

doi: 10.1101/2020.07.17.198598

Figure Lengend Snippet: (A) Experimental schema: Myc-CAP (STING lo ) cells were treated with rucaparib (0.5 µM) for 36 hours, followed by isolation of MVs from supernatant (R-MVs), which were then co-cultured with BMDMs in the presence/absence of buparlisib (1 µM) for 36 hours. BMDMs were directly treated with DMXAA (50 µg/ml). (B-C) Cellular metabolites and proteins extracted from co-cultured BMDMs (as per schema in A ) were used for cGAMP ELISA (B) and for assessment of activation of STING pathway by western blotting using indicated antibodies (C ), respectively. (D-E) Supernatants collected from experiment in (A) were used for IFN-α (D) and IFN-β ELISA (E) . ( F) Model for cGAS/STING pathway activation within suppressive macrophages (M2) following treatment with buparlisib and MVs (isolated from rucaparib-treated Myc-CAP cells); n=2 independent experiments. Significance/ p-values were calculated by one-way ANOVA and are indicated as follows, *p < 0.05; **p < 0.01, ***p < 0.001, **** p< 0.0001, ns = not statistically significant.

Article Snippet: To rule out cGAMP as the mediator of STING pathway activation within BMDMs, 10 μg of cGAMP disodium salt (MedChem Express) was reconstituted in RNA/DNAse free water and pre-treated with 10 units of DNase I.

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot