Fig 1: miR-210 mitigates the hypoxia-induced oligomerization and insertion of BAX and BAK into the outer mitochondrial membrane (OMM). Isolated mitochondria were subjected to a 0.1 M sodium carbonate (Na2CO3) treatment to produce the alkali-resistant OMM-inserted (OMM-embedded) protein fraction and the alkali-soluble OMM-tethered (OMM-anchored) protein fraction. (A–D) ELISA immunoassays determining the abundance of BAX (A,C) and BAK (B,D) performed on the denatured lysates from the respective OMM-inserted protein fraction (A,B) and OMM-tethered protein fraction (C,D). Experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm) were first expressed as fold-change and subsequently normalized to either TOM40 expression levels (for the OMM-inserted protein fraction) or HK2 expression levels (OMM-tethered protein fraction). The expression levels of TOM40 and HK2 in the respective fractions are reported in Supplementary Figure S6A,B. miR-210 expression levels in the respective whole-cell lysates were determined by the miR-210 hybridization immunoassay (as described in Section 4.3) and are reported in Supplementary Figure S7A. The validation of the ectopic expression of the HA-tagged ca-GSK3ß-S9A mutant in the pertinent experimental groups was performed by ELISA immunoassay and is reported in Supplementary Figure S7B. GSK3ß kinase activity (as described in Section 4.4) was measured in all experimental groups to corroborate and validate the translative effects of the ectopic expression of the ca-GSK3ß-S9A mutant (reported in Supplementary Figure S7C). Data are represented as double-normalized ratiometric values (BAX/TOM40 and BAX/HK2 as well as BAK/TOM40 and BAK/HK2), expressed as mean ± S.D fold-change, 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; O.D: optical density; S.D: standard deviation; OMM: outer mitochondrial membrane.