Fig 2: An illustrated schematic depicting the inhibition of GSK3ß kinase activity as the molecular pivot that underlies the miR-210-elicited mitigation of hypoxia-induced intrinsic apoptosis cascade. miR-210 attenuates the hypoxia-induced GSK3ß kinase activity (1) that decreases the hypoxia-induced increase in the abundance of the OMM (outer mitochondrial membrane)-tethered pool of BAX (2). Subsequently, the miR-210-elicited inhibition of the hypoxia-induced increase in GSK3ß kinase activity results in a significant decrease in the hypoxia-induced abundance of the OMM-inserted pool of BAX (3) and BAK (4) that constitute the MAC (mitochondrial apoptosis-induced channel). The miR-210-elicited inhibition of the hypoxia-induced increase in GSK3ß kinase activity translates into a decrease in the hypoxia-induced abundance of MAC formation that culminates in a commensurate mitigation of the hypoxia-induced increase in Cytochrome C release from the mitochondria into the cytosol (5). The miR-210-evoked GSK3ß inhibition-mediated reduction in the hypoxia-induced increase in Cytochrome C release into the cytosol translates into a decrease in the hypoxia-induced caspase-3 activity (6) and the ensuing DFF40 endonuclease activity (7) that culminates in the attenuation of hypoxia-induced apoptotic cell death (8). This illustration was created using BioRender.com (https://app.biorender.com/illustrations/627d566667f7b4db938c909f, accessed on 8 August 2022).

Fig 3: miR-210 attenuates the hypoxia-driven intrinsic apoptosis pathway through the inhibition of GSK3ß kinase activity. (A,B) Quantitative ELISA immunoassays determining the Cytochrome C abundance in the mitochondrial fractions (A) and cytosolic fractions (B), expressed as experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm). (C–F) Quantitative ELISA immunoassays determining the abundance of BAX (C,D) and BAK (E,F) in the mitochondrial fractions (C,E) and cytosolic fractions (D,F), expressed as experimental blank-corrected absorbances (O.D) measured at ?450 (450 nm) normalized to fold-change values. Quantitative ELISA immunoassays determining the abundance of BAX and BAK in whole-cell fractions are reported in Supplementary Figure S4A,B. The validity of the integrity of the respective subcellular compartments is reported in Supplementary Figure S3A–C. miR-210 expression levels in the respective cell lysates were determined by the miR-210 hybridization immunoassay (as described in Section 4.3) and are reported in Supplementary Figure S1A. 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 S1C. 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 S1E). All 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; O.D: optical density; S.D: standard deviation.

Fig 4: Effect of cadmium and/or linagliptin administration on testicular apoptosis in rats. The co-treatment with linagliptin curbed testicular pro-apoptotic events that were turned on by cadmium metal. This was demonstrated by downregulated protein expression of Bcl-2 associated x protein (Bax) as detected by immunohistochemistry that shows up as a brown color at 400× magnification. (A,B) The immunostaining of testicular Bax protein demonstrates minimal expression in the control and control + LIN groups. (C) The immunostaining of the pro-apoptotic Bax was upregulated in the cadmium-intoxicated group. (D) The immunostaining of Bax was downregulated in the Cd + LIN group. (E) Quantitative analysis of Bax immunostaining in the testicular tissue presented as the area % of the immunoreactivity/full microscopic field area. Six non-overlapping fields were inspected for each specimen. In addition, co-treatment with linagliptin diminished the activity of testicular executioner caspase 3 (F). * p < 0.05, ** p < 0.01, and *** p < 0.001 show statistical significance compared to the group of control rats; # p < 0.05, and ## p < 0.01 show statistical significance compared to the group of cadmium-intoxicated rats. Values are stated as the mean ± standard error of the mean for n = 6 per group. LIN, linagliptin; Cd, cadmium chloride.

Fig 5: 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.