Journal: bioRxiv

Article Title: STING activation promotes robust immune response and tumor regression in glioblastoma models

doi: 10.1101/2022.02.28.481908

Figure Lengend Snippet: a , Immunoblot of cGAS, STING, TBK1, IRF-3 and GAPDH levels in patient GBM tissue, patient-derived GBM cells and murine GL261 and CT-2A glioma cells. b , Quantification of tissue microarray (TMA) sections for STING and pTBK1, as percentage of positive pixels. AA, n = 77; GBM, n = 49; oligo, n = 10; brain, n = 16. P values calculated by oneway ANOVA. c , Representative TMA sections immunostained for STING and pTBK1 in normal brain and d , GBM. Scale bars = 400 and 100 μm. e , Immunofluorescence staining showing partial activation of the STING cascade in GL261 tumors and the colocalization of pTBK1 within the vasculature (CD31 and α-SMA). Left panel: white arrows point to blood vessels. Right panel: Split channels show colocalization of pTBK1 with the vascular markers CD31 and α-SMA. Scale bar = 100 μm. f , Multiplex immunofluorescence staining on a whole brain section from a GL261 tumor bearing mouse showing DAPI (blue), CD31 (green), STING (yellow) and F4/80 (red). Scale bar = 1 mm. g , h and i , Immunofluorescence staining of the tumor zone showing selected markers as indicated. Scale bars = 400 and 100 μm.

Article Snippet: Protein extracts were subjected to polyacrylamide gel electrophoresis using either Criterion or Mini-Protean TGX pre-cast gels, transferred to nitrocellulose (Millipore) membranes, and immunoblotted using antibodies (Cell Signaling Technologies, Danvers, MA) that specifically recognize STING (clone D2P2F, Cat# 13647S), cGAS (clone D1D3G, Cat# 15102), TBK1 (clone E8I3G, Cat#38066), human or mouse anti-GAPDH (ab9484-200, Abcam, Cambridge, MA, USA) and peroxidase-conjugated secondary antibodies (Jackson Laboratories, Bar Harbor, ME, USA).

Techniques: Western Blot, Derivative Assay, Microarray, Immunofluorescence, Staining, Activation Assay, Multiplex Assay