Fig 1: miR-210 increased the hypoxia-reoxygenation (H-R)-induced DISC-IIa complex formation, thereby activating the extrinsic apoptosis pathway. (A–F) Co-IP coupled with tandem ELISA determining the quantitative abundance of procaspase-8 and c-FLIP in the FADD immunoprecipitates from RIPK1-precleared lysates were performed as a surrogate measure of the abundance of the DISC-IIa complex formation and the subsequent activation of the extrinsic apoptosis pathway. (A,C) ELISA immunoassays unequivocally show that overexpression of miR-210 significantly increased the H-R-induced procaspase-8 abundance ((A) inset c), while having no effect on c-FLIP abundance ((C) inset c), in the FADD immunoprecipitates from RIPK1-precleared lysates. (B,D) ELISA immunoassays unequivocally demonstrated that the ectopic expression of the miR-210-3p decoy/inhibitor significantly reduced the H-R-induced procaspase-8 abundance (C inset c), while eliciting no effect on c-FLIP abundance ((D) inset c), in the in the FADD immunoprecipitates from RIPK1-precleared lysates. (E,F) ELISA determining the quantitative abundance of FADD ((E) inset c, (F) inset c) in the FADD immunoprecipitates from RIPK1-precleared lysates. ((A) inset b—(F) inset b) ELISA showing the absence of procaspase-8 (inset b in (A,B), c-FLIP (inset b in (C,D), and FADD (inset b in (E,F)) in the mouse IgG immunoprecipitates from RIPK1-precleared lysates, thereby demonstrating the specificity of the FADD Co-IP assays. ((A) inset a—(F) inset a) ELISA determining the quantitative abundance of procaspase-8 (inset a in (A,B), c-FLIP (inset a in (B,D)), and FADD (inset a in (E,F) in the RIPK1 precleared lysates serving as input (10%). The validity and integrity of the RIPK1-precleared lysates in all experimental groups were determined by ELISA (as described in Section 2.11) and are reported in Figure S3A,B. miR-210 expression levels in the respective cell lysate inputs were determined by the miR-210 hybridization immunoassay (as described in the Section 2.3) and are reported in Figure S3C,D. Data are expressed as experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm). Data are expressed as mean ± S.D from three technical replicates for each of the four biological replicates belonging to each experimental group (n = 4).* p = 0.05; ** p = 0.01; *** p = 0.001; **** p = 0.0001; ns: not significant (p > 0.05); OE: miR-210 overexpression; KD: miR-210-3p decoy/inhibitor; H-R: hypoxia-reoxygenation O.D: optical density; S.D: standard deviation.