Journal: Nature medicine
Article Title: Tumor innate immunity primed by specific interferon-stimulated endogenous retroviruses
Figure Lengend Snippet: Discovery of an IFN-inducible subclass of ERVs. ( a ) Immunoblot of pTBK1, TBK1, IKKε, pIRF3, pERK, ERK, pAKT, AKT and tubulin levels in H69 and H69M cells after 24 or 72 h culture. ( b ) Log-2 fold change cytokine/chemokine differences between H69M/H69 CM. ( c ) H E and pTBK1 IHC of a patient-derived SCLC brain metastasis. Scale bar indicates 20 μm. ( d ) Isotype control versus PD-L1 or CD44 surface expression on H69 and H69M cells ± 200 ng/mL 24 h IFNγ stimulation (representative of n=3 biological replicates). ( e ) Immunoblot of pTBK1, TBK1, pERK, ERK, pAKT, AKT and β-actin levels in H69, H69M, H69M-PD-L1 low , and H69M-PD-L1 high cells. ( f ) Log-2 fold change cytokine/chemokines differences between H69M-PD-L1 high or H69M-PD-L1 low /H69 CM. ( g ) qRT-PCR of ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. Numeric values on each bar represent the fold change in expression of a DNMT regulated ERV enriched panel 11 , 12 of previously published ERVs. Error bars are mean ± s.e.m of n=3 biological replicates. TRIM22 promoter and antisense orientation of MLT1C49 in the 3′UTR are represented below the qRT-PCR graph. ( h ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 low cells transfected with pLX-307-GFP control or pLX_307-MLT1C49 construct for 72h. Mean ± s.e.m of n=3 biological replicates shown. ( i ) qRT-PCR of CXCL10 and CCL2 in H69M PD-L1 high cells transfected with scrambled negative control siRNA or siRNAs specific for MLT1C49. Mean ± s.e.m of n=3 biological replicates shown. ( j ) Overlap of 3′UTR antisense ERVs with H69M upregulated genes (log-2 fold change relative to H69 > 2) and table showing the fold change in expression of these genes/ERVs in H69M-PD-L1 high normalized to H69M-PD-L1 low cells. ( k ) Immunoblot of STING, MAVS, pTBK1, TBK1, pIRF3, IRF3, E-Cadherin, Vimentin and β-actin levels in H69M cells after CRISPR mediated deletion of MAVS and/or STING. ( l ) Log-2 fold change cytokine/chemokine differences in CM from H69M cells after CRISPR mediated deletion of MAVS and/or STING compared to sgCTRL (Scramble). ( m ) CXCL10 Luminex absolute levels (pg/mL) in Scramble, STING KO, MAVS KO and double KO (dKO) H69M cells. Mean ± s.e.m of n=2 biological replicates shown. *p
Article Snippet: After extraction, 1 μg total RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit, which includes both oligo-dT and random primers (Thermo Fisher Scientific, Waltham, MA).
Techniques: Immunohistochemistry, Derivative Assay, Expressing, Quantitative RT-PCR, Transfection, Construct, Negative Control, CRISPR, Luminex