Fig 1: The effect of hypoxia on the subcellular localization of HIG2A in the mitochondria and the nucleus of C2C12 cells. The representative immunofluorescence images in C2C12 cells; anti-HIG2A primary antibody (Abcam ab135399), DyLight® 488 secondary antibody (HIG2A green). MitoTrackerTM Red CM-H2XRos mitochondrial fluorescent marker (M red). Hoescht 33342 (blue signal—DNA). Z-axis series were obtained using confocal microscopy (Leica TCS SP8). The separation between each of the slices (Z step size) was 0.130 microns. The C2C12 cells were subjected to hypoxic stress (2% O2) for 24-h (A,C,E) and 48-h (B,D,F). The quantification of colocalization was performed using Manders’ coefficient. The fluorescence signal corresponding to HIG2A was quantified over the fluorescence signal of the nucleus (HIG2A/nucleus [M2]) (C,D) and over the signal of the mitochondria (HIG2A/mitochondria [M1]) (C,D). Each bar graph represents the mean ± SE, n = 4 biological replicates, 20 cells were analyzed per condition; they were analyzed by a one-tailed t-test (p < 0.05), followed by a Mann-Whitney test. Statistical differences were found with a significance of p-value (*** p = 0.0001 (C), ** p = 0.0073 (C), *** p = 0.0001 (D), *** p = 0.0002 (D), *** p = 0.0002 (E), *** p = 0.0004 (F), *** p = 0.0002 (F)). ** Very significant (0.001 to 0.01). *** Extremely significant (0.0001 to 0.001). White bars indicate a 5 µm scale.
Fig 2: Model of HIG2A behavior in response to stress. (A) The transcription factor E2F-1 controls the HIGD2A gene, which encodes the HIG2A protein (in orange) [12]. The model describes two “pools” of HIG2A protein. Once synthesized in the cytoplasm, the HIG2A protein is distributed at the mitochondria and the nucleus [12,17]. Currently, the function of HIG2A at the nucleus has not been described, whereas, at mitochondria, HIG2A is localized in the inner mitochondrial membrane where it participates in the formation of respiratory supercomplexes (SC), interacting with complex III (CIII) and complex IV (CIV), and promoting their stability [8,11,12]. The increase in the formation of respiratory supercomplexes has been associated with a decrease in mitochondrial reactive oxygen species (ROSmt) and an increase in mitochondrial energy generation (ATPmt) [1,2,4,6]. The distribution of HIG2A protein at the subcellular level will depend on the type of stress to which cells are exposed. (B) Mitochondrial stress by Rotenone (Complex I (CI) inhibitor) causes an increase of HIG2A at the nuclear level. Inhibition at the CI level causes an increase in superoxide anion (O2-) generation and a decrease in electron (e-) flow through the electron transport chain (ETC) and mtATP generation [67,68]. In contrast, FCCP causes a reduction at the nucleus, which could suggest (dotted arrow) a shift of HIG2A into the mitochondria to make up for the deficiency in mitochondrial membrane potential (??m) that is caused by the mitochondrial uncoupler FCCP [69], participating in SC formation, stabilizing electron flow, and maintaining mitochondrial energy generation (mtATP). In comparison, H2O2 (generalized stress) caused an increase in HIG2A at the mitochondria and cytoplasm. H2O2 causes an increase in ROS at the cellular level which may participate in signaling pathways. (C) It has been described that under hypoxic conditions, the transcription factor HIF-1a is stabilized and forms a heterodimer with the transcription factor HIF-1ß recognizing consensus sequences of the target genes that are involved in lactic acid formation [70]. At mitochondria, mtROS that are generated by complex III (CIII) can inhibit the enzyme prolyl hydroxylase (PHD), which hydroxylases (OH) the transcription factor HIF-1a, for subsequent degradation via the proteasome [71]. In addition, the decrease in oxygen (O2) levels causes an increase in the formation of SCs, which helps prevent electrons from escaping, thus decreasing mtROS generation [71,72,73]. The subcellular distribution of HIG2A by hypoxic stress that is caused by physical hypoxia of 2% O2 and by chemical hypoxia by CoCl2 (hypoxia equal to or less than 1% O2) causes the HIG2A protein to present a differential distribution. Hypoxia of 2% O2 causes an increase of HIG2A at the nucleus, which could indicate new functions that are not yet described at the nuclear level and which could be regulated by stress factors. Conversely, CoCl2 stress causes a decrease in HIG2A at the nucleus. This decrease could indicate (dotted arrow) a shift of HIG2A to the mitochondrial level to promote SC formation and stability, decrease mtROS generation, and provide more efficient electron transport through the mitochondrial transport chain.
Fig 3: The effect of hypoxia on the subcellular localization of HIG2A in the mitochondria and the nucleus of HEK293 cells. The representative immunofluorescence images in HEK293 cells; anti-HIG2A primary antibody (Abcam ab135399), DyLight® 488 secondary antibody (HIG2A green). MitoTrackerTM Red CM-H2XRos mitochondrial fluorescent marker (M red). Hoescht 33342 (blue signal—DNA). Z-axis series were obtained using confocal microscopy (Leica TCS SP8). The separation between each of the slices (Z step size) was 0.130 microns. The HEK293 cells were subjected to hypoxic stress (2% O2) for 24-h (A,C,E) and 48-h (B,D,F). The quantification of colocalization was performed by Manders’ coefficient. The fluorescence signal corresponding to HIG2A was quantified over the fluorescence signal of the nucleus (HIG2A/nucleus [M2]) (C,D) and over the signal of the mitochondria (HIG2A/mitochondria [M1]) (C,D). Each bar graph represents the mean ± SE, n = 4 biological replicates, 20 cells were analyzed per condition; they were analyzed by a one-tailed t-test (p < 0.05), followed by a Mann-Whitney test. Statistical differences were found with a significance of p-value (** p = 0.0011 (C), *** p = 0.0001 (D); ** p = 0.0029 (D)). ** Very significant (0.001 to 0.01). *** Extremely significant (0.0001 to 0.001). The white bars indicate a 5 µm scale.
Supplier Page from Abcam for Anti-HIGD2A antibody