Impact of hypoxia on proteins of the mitochondrial ISC assembly machinery.

<p>(A) Total protein extracts from HeLa cells grown in normoxia (Nx, 21% O₂) or hypoxia (Hx, 1% O₂) conditions for the indicated times were analyzed by immunoblotting using VDACs poly antibody and anti-CAIX, -ISCU, -FXN, -NFS1, -HSC20 antibodies. β-Actin was used as loading control. (<i>right panel)</i> Bar graph represents the amount of the indicated proteins relative to ß-actin level determined by quantification of n = 3 immunoblot analysis using the Odyssey System Imager. Mean and standard deviation of 3 independent experiments are shown (* p < 0.05, n = 3). (B) HeLa cells were either untreated or treated with CoCl₂ for 2 days. Total proteins were analyzed by western blotting using anti-HIF-1α, -ISCU, -FXN antibodies. Vinculin was used as loading control. (C) HeLa cells grown in normoxia (Nx, 21% O₂) or hypoxia (Hx, 1% O₂) conditions for 3 days and mRNA levels of <i>iscu</i>, <i>fxn</i> and <i>vegf A</i> were determined by RT-qPCR and normalized to 18S ribosomal rRNA levels. The bar graph presents the ratio between the relative level of each mRNA under hypoxic and normoxic conditions with a logarithmic scale. Mean and standard deviation of 6 independent experiments are shown (* p < 0.05, n = 6). (D) Total protein extracts from HeLa cells grown under normoxic (Nx, 21% O₂) or hypoxic (Hx, 1% O₂) conditions for the indicated times were analyzed by immunoblotting using anti-CIAPIN1, -NUBP1, -NARFL antibodies. β-Actin was used as loading control.</p

Iron depletion and nitric oxide stress induce the accumulation of the truncated VDAC1 form.

<p>(A) Total protein extracts of HeLa cells treated for 16 h with FAC or DFO were analyzed by immunoblotting using VDACs poly antibody. Prohibitin was used as loading control. (B) Total protein extracts of HeLa cells treated with DMSO (control), DFO or SIH for the indicated times were analyzed by immunoblotting using VDACs poly antibody. β-Actin antibody was used as loading control. (C) HepG2 cells were treated for 24 h with DMSO (control) or SIH. Immunoblotting was carried out using VDACs poly antibody on total protein extracts (TE), and on mitochondrial (Mito) and cytosolic (Cyto) fractions. mitoNEET and NUBP1 were used as mitochondrial and cytosolic markers, respectively. (D). HeLa cells were treated for 16 h with DMSO (control), SIH or DETA-NO (NO). Total protein extracts were analyzed by immunoblotting using VDACs poly antibody and anti-HIF-1α and -CAIX antibodies. β-Actin was used as loading control. (E) HeLa cells were grown in normoxia (Nx) or 1% O₂ hypoxia (Hx) conditions for 3 days, or grown in normoxia conditions and treated for 24 h with DMSO (Control, Co), SIH or DETA-NO (NO), or grown in normoxia (21% O₂) conditions and transfected with either negative control (NC) or <i>iscu</i>-siRNA for 3 days. Total protein extracts were analyzed by immunoblotting using antibodies against the N-terminus of VDAC1 isoform (VDAC1-Nter), the three VDAC isoforms (VDACs poly), ISCU and CAIX. (F) HeLa cells were grown in hypoxia (Hx, 1% O₂) conditions and transfected with <i>iscu-</i> or NC-siRNA for 3 days, or grown in normoxia (Nx, 21% O₂) and treated or not with DFO for 16 h. Total proteins were analyzed by western blot using VDACs poly antibody and anti-HIF-1α, -CAIX, -ISCU antibodies. β-Actin was used as loading control.</p

Protein depletion in the mitochondrial ISC machinery confers cell protection against STS-induced apoptosis.

<p>(A) HeLa cells were transfected with negative control (NC) or <i>iscu-</i>, <i>nfs1-</i>, <i>mfrn2-</i> or <i>hsc20</i>-siRNA for 6 days. Cells treated with staurosporine (STS) for 4 h were used as positive control for apoptosis induction. After staining with Hoechst 33342, cells were analyzed by epifluorescence microscopy. Scale bar: 100 μm. (B) HeLa cells were either transfected with NC or <i>iscu</i>-siRNA for 3 days or treated with STS for 4 h. Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase 3 (C3), PARP1 and ISCU. Vinculin was used as loading control. (C) Cells were transfected with negative control (NC) or, <i>nfs1-</i>, <i>mfrn2-</i> or <i>hsc20</i>-siRNA for 6 days or STS-treated for 4 h. Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase-3, PARP1, NFS1 and HSC20. Vinculin was used as loading control. (D) Schematic representation of the protocol used. <i>Phase 1</i>—HeLa cells were seeded the day before the transfection with <i>mfrn2-</i>, <i>iscu-</i> or NC siRNA and incubated for 6 days (144 hours) under normoxic conditions (21% O₂) with a transfection every 3 days. <i>Phase 2</i> –Cells were either untreated or treated with STS for 4 h. (E and F) Total protein extracts were analyzed by immunoblotting using antibodies against cleaved caspase-3 (C3) and ISCU. β-Actin was used as loading control. (<i>Lower panel)</i> The bar graph presents the ratio between the amounts of cleaved C3 over ß-actin determined by quantification of the immunoblot. Mean and standard deviation of n = 3 (<i>iscu</i>-siRNA) and n = 4 (<i>mfrn2</i>-siRNA) independent experiments (* p < 0.05).</p

Downregulation of mitochondrial Fe-S cluster assembly leads to C-terminal VDAC1 truncation and subsequent resistance to pro-apoptotic treatments.

<p>Red and black arrows represent links unveiled in the present study and in previous studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194782#pone.0194782.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194782#pone.0194782.ref018" target="_blank">18</a>], respectively. Hypoxia stabilizes and activates HIF-1α (1) and leads to drastic changes in mitochondrial morphology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194782#pone.0194782.ref017" target="_blank">17</a>] and VDAC1-ΔC accumulation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194782#pone.0194782.ref018" target="_blank">18</a>](2). In the present study, we showed that hypoxia leads to the downregulation of NFS1, FXN and ISCU, three components of the mitochondrial ISC core machinery (3). Whether this regulation directly involves HIF-1α remains to be explored (4). Subsequently, deficit in Fe-S cluster biogenesis leads to the loss of the mitochondrial network, appearance of enlarged mitochondria and accumulation of VDAC1-ΔC. We identified the MAM-anchored Fe-S protein CISD2 as a key protein for the cellular fate of VDAC1 (5). We still cannot exclude that CISD2 is the only Fe-S protein involved in this process (6). Interestingly, when components of the ISC core machinery were silenced by siRNA in hypoxia, VDAC1-ΔC levels were increased compared to hypoxia alone.</p

Knock-down of CISD2 induces the accumulation of the truncated VDAC1 form.

<p>(A) Total protein extracts of either non-transfected (-) or transfected HeLa cells for 3 days with scramble (NC)-, <i>cisd2</i>-, or <i>nfs1</i>-siRNA were analyzed by immunoblotting using antibodies against VDACs poly, CISD2, and α-tubulin, which was used as loading control. Eight independent experiments were performed and a representative western blot is shown. (B) Total mRNA was extracted from HeLa cells transfected with scramble (NC)-, <i>cisd2</i>-, or <i>nfs1</i>-siRNA. Twenty-four hours after transfection, the mRNA levels of <i>cisd2</i> and <i>nfs1</i> were determined by quantitative RT-qPCR. Data were normalized to <i>gapdh</i> mRNA levels. Means ± standard deviation of n = 4 independent experiments are presented (*** p < 0.001).</p

<i>Mycobacterium avium</i> Infection Induces H-Ferritin Expression in Mouse Primary Macrophages by Activating Toll-Like Receptor 2

<div><p>Important for both host and pathogen survivals, iron is a key factor in determining the outcome of an infectious process. Iron with-holding, including sequestration inside tissue macrophages, is considered an important strategy to fight infection. However, for intra-macrophagic pathogens, such as <i>Mycobacterium avium</i>, host defence may depend on intracellular iron sequestration mechanisms. Ferritin, the major intracellular iron storage protein, plays a critical role in this process. In the current study, we studied ferritin expression in mouse bone marrow-derived macrophages upon infection with <i>M. avium</i>. We found that H-ferritin is selectively increased in infected macrophages, through an up-regulation of gene transcription. This increase was mediated by the engagement of Toll like receptor-2, and was independent of TNF-alpha or nitric oxide production. The formation of H-rich ferritin proteins and the consequent iron sequestration may be an important part of the panoply of antimicrobial mechanisms of macrophages.</p> </div

TLR-2 activation leads to increased expression of H-ferritin.

<p>A, B – BMM from C57Bl/6 (WT) and TLR-2^-/- mice were left uninfected or infected for 24h with <i>M. avium</i>. The H-ferritin fold increase in infected BMM in comparison with uninfected ones is shown at the protein level (A) and mRNA (B). C – BMM were treated with the TLR-2 agonist FSL-1 for 24h, and the levels of H- and L-ferritin was quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. Statistical differences as described in Figure 1.</p

Effect of <i>Mycobacterium avium</i> infection on intramacrophagic ferritin.

<div><p>Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with <i>M. avium</i>, as described in Material and Methods, or left uninfected. A - At different time points, macrophages were lysed and the amount of ferritin was quantified by ELISA. Data are presented as ng of ferritin per mg of total protein. The results are shown as average ± SD from one experiment performed in triplicate out of four independent experiments. Superscripts indicate statistical significance between M. avium-infected and uninfected, within the correspondent time-point, as follows: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. B – BMM uninfected or infected with <i>M. avium</i> for 24h were incubated for 6h with (⁵⁵Fe) ferric ammonium citrate. Total protein (18 µg) was loaded (in duplicates) in native PAGE and exposed to autoradiography to analyze protein-bound iron. A single band was detected corresponding to cytosolic H/L ferritin. The values indicate the average relative band intensity for each condition. C – BMM infected with <i>M. avium</i> for 4h, 1 and 3 days and respective uninfected controls were tested for IRP-IRE binding activity, by gel retardation assay. 2% of 2-mercaptoethanol (2-ME) fully activates IRP binding activity and shows equal loading. BMM treated with iron or deferoxamine (DFO) were tested in a separated gel to confirm the reliability of the assay. D – BMM were treated with the transcriptional inhibitor actinomycin D or with vehicle. After an 8h-infection with <i>M. avium</i>, the BMM were lysed and H- and L-ferritin were quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. ***<i>p</i><0.001, NS not significant. </p> <p>E – At different time points, total RNA was collected from macrophages and the expression levels of ferritin genes was quantified by qRT-PCR, and normalized to <i>Hprt1</i>. Results are shown as fold increase in <i>M. avium</i>-infected macrophages in comparison with uninfected ones. Data are presented as average ± SE from one experiment performed in triplicate from a total of two independent experiments. </p></div

Effect of <i>M. avium</i> infection on ferritin content in the absence of TNF-alpha, iNOS and TLR-2.

<p>Bone marrow-derived macrophages were obtained from C57Bl/6 (WT), TLR-2^-/-, TNF-alpha^-/- and NOS2^-/- mice. BMM were infected and the ferritin content was quantified as described in Figure 1. The results are shown as average ± SD from one experiment performed in triplicate out of two independent experiments.</p