SO₄⁼ uptake in human hemoglobin-free resealed ghosts of erythrocytes under H₂O₂ treatment.

<p>Time course of SO₄⁼ uptake in hemoglobin-free resealed ghosts of erythrocytes measured in control conditions (untreated) or treated with either 300 μμM H₂O₂, or 10 μ M DIDS applied at the beginning of the incubation in SO₄⁼ medium. Points represent the mean ± SEM from separate experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146485#pone.0146485.t002" target="_blank">Table 2</a>), where ***p<0.001 significant <i>versus</i> control or ^§§§p<0.001 significant <i>versus</i> 10 μ M DIDS and n.s. not significant <i>versus</i> control, as determined by one way ANOVA followed by Bonferroni's <i>post hoc</i> test, by comparing all values of theoretical curves, at all time points.</p

H₂O₂-Induced Oxidative Stress Affects SO₄^{= - Fig 4} Transport in Human Erythrocytes

<p><b>Erythrocytes morphology under H</b>_<b>2</b><b>O</b>_<b>2</b><b>plus GSH treatment</b> Light microscope observations (400x magnification) of: <b>A)</b> 300 μM H₂O₂ treated erythrocytes, observed after 5 min incubation in SO₄⁼ medium and <b>B</b>) erythrocytes treated with 2 mM GSH and then with 300 μM H₂O₂, observed after 5 min incubation in SO₄⁼ medium; <b>C)</b> 300 μM H₂O₂ treated erythrocytes, observed after 90 min incubation in SO₄⁼ medium and <b>D)</b> erythrocytes treated with 2 mM GSH and then with 300 μM H₂O₂ observed after 90 min incubation in SO₄⁼ medium. Arrows indicate that morphological changes after H₂O₂ treatment are not inhibited by GSH.</p

Erythrocytes morphology under H₂O₂ treatment.

<p>Light microscope observations (400x magnification) of: <b>A</b>) Untreated erythrocytes; <b>B</b>) 300 μM H₂O₂ treated erythrocytes at 5 min of SO₄⁼ medium incubation; <b>C</b>) 300 μM H₂O₂ treated erythrocytes at 90 min of SO₄⁼ medium incubation. Arrows indicate significant morphological changes in both B) and C) compared to the control (untreated) erythrocytes A).</p

SO₄⁼ uptake in human erythrocytes under H₂O₂ treatment.

<p>Time course of SO₄⁼ uptake in human erythrocytes measured in control conditions (untreated erythrocytes) or treated with 10 μM DIDS, applied at the beginning of incubation in SO₄⁼ medium. Points represent the mean ± SEM from separate experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146485#pone.0146485.t001" target="_blank">Table 1</a>), where ***p<0.001 significant <i>versus</i> control and ^§§p<0.01 significant <i>versus</i> 100 μM H₂O₂, as determined by one way ANOVA followed by Bonferroni's <i>post hoc</i> test, by comparing all values of theoretical curves, at all time points.</p

SO₄⁼ uptake in human erythrocytes under H₂O₂ plus GSH treatment.

<p>Time course of SO₄⁼ uptake in human erythrocytes measured in control conditions (untreated erythrocytes) or treated with 300 μM H₂O₂, or treated with 2 mM GSH and then with 300 μM H₂O₂. Points represent the mean ± SEM from separated experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146485#pone.0146485.t001" target="_blank">Table 1</a>), where ***p<0.001 significant <i>versus</i> control and ^¥¥p<0.01 significant <i>versus</i> 300 μM H₂O₂, as determined by one way ANOVA followed by Bonferroni's <i>post hoc</i> test, by comparing all values of theoretical curves, at all time points.</p

MDA levels and –SH groups estimation under H₂O₂ treatment.

<p>A) MDA levels observed in control (untreated erythrocytes) and in erythrocytes treated with 300 μM H₂O₂. Data are presented as means ± SEM from at least 3 experiments, where n.s. is not significantly different <i>versus</i> control, as determined by <i>t-</i>Student test. B) Percentage of –SH groups measured in control (untreated erythrocytes), in erythrocytes treated with either 300 μM H₂O₂ or 2 mM NEM. Bars represent the mean ± SEM from at least 3 experiments, where n.s. is not significant <i>versus</i> control, ^§§§ p<0.001 significant <i>versus</i> control and 300 μM H₂O₂-treated erythrocytes, as determined by one way ANOVA followed by Bonferroni's <i>post hoc</i> test.</p

SO₄⁼ uptake in human erythrocytes under H₂O₂ plus curcumin treatment.

<p>Time course of SO₄⁼ uptake in human erythrocytes measured in control conditions (untreated erythrocytes) or treated with 300 μM H₂O₂ preceded or not by 10 μM curcumin application. Points represent the mean ± SEM from separate experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146485#pone.0146485.t001" target="_blank">Table 1</a>), where ***p<0.001 significant <i>versus</i> control or **p<0.01 significant <i>versus</i> control and ^#p<0.05 significant <i>versus</i> 300 μM H₂O₂, as determined by one way ANOVA followed by Bonferroni's <i>post hoc</i> test, by comparing all values of theoretical curves, at all time points.</p

Image_2_Novel POU3F4 variants identified in patients with inner ear malformations exhibit aberrant cellular distribution and lack of SLC6A20 transcriptional upregulation.tif

Hearing loss (HL) is the most common sensory defect and affects 450 million people worldwide in a disabling form. Pathogenic sequence alterations in the POU3F4 gene, which encodes a transcription factor, are causative of the most common type of X-linked deafness (X-linked deafness type 3, DFN3, DFNX2). POU3F4-related deafness is characterized by a typical inner ear malformation, namely an incomplete partition of the cochlea type 3 (IP3), with or without an enlargement of the vestibular aqueduct (EVA). The pathomechanism underlying POU3F4-related deafness and the corresponding transcriptional targets are largely uncharacterized. Two male patients belonging to a Caucasian cohort with HL and EVA who presented with an IP3 were submitted to genetic analysis. Two novel sequence variants in POU3F4 were identified by Sanger sequencing. In cell-based assays, the corresponding protein variants (p.S74Afs*8 and p.C327*) showed an aberrant expression and subcellular distribution and lack of transcriptional activity. These two protein variants failed to upregulate the transcript levels of the amino acid transporter gene SLC6A20, which was identified as a novel transcriptional target of POU3F4 by RNA sequencing and RT-qPCR. Accordingly, POU3F4 silencing by siRNA resulted in downregulation of SLC6A20 in mouse embryonic fibroblasts. Moreover, we showed for the first time that SLC6A20 is expressed in the mouse cochlea, and co-localized with POU3F4 in the spiral ligament. The findings presented here point to a novel role of amino acid transporters in the inner ear and pave the way for mechanistic studies of POU3F4-related HL.</p

Data_Sheet_2_Novel POU3F4 variants identified in patients with inner ear malformations exhibit aberrant cellular distribution and lack of SLC6A20 transcriptional upregulation.xlsx

Hearing loss (HL) is the most common sensory defect and affects 450 million people worldwide in a disabling form. Pathogenic sequence alterations in the POU3F4 gene, which encodes a transcription factor, are causative of the most common type of X-linked deafness (X-linked deafness type 3, DFN3, DFNX2). POU3F4-related deafness is characterized by a typical inner ear malformation, namely an incomplete partition of the cochlea type 3 (IP3), with or without an enlargement of the vestibular aqueduct (EVA). The pathomechanism underlying POU3F4-related deafness and the corresponding transcriptional targets are largely uncharacterized. Two male patients belonging to a Caucasian cohort with HL and EVA who presented with an IP3 were submitted to genetic analysis. Two novel sequence variants in POU3F4 were identified by Sanger sequencing. In cell-based assays, the corresponding protein variants (p.S74Afs*8 and p.C327*) showed an aberrant expression and subcellular distribution and lack of transcriptional activity. These two protein variants failed to upregulate the transcript levels of the amino acid transporter gene SLC6A20, which was identified as a novel transcriptional target of POU3F4 by RNA sequencing and RT-qPCR. Accordingly, POU3F4 silencing by siRNA resulted in downregulation of SLC6A20 in mouse embryonic fibroblasts. Moreover, we showed for the first time that SLC6A20 is expressed in the mouse cochlea, and co-localized with POU3F4 in the spiral ligament. The findings presented here point to a novel role of amino acid transporters in the inner ear and pave the way for mechanistic studies of POU3F4-related HL.</p

Image_1_Novel POU3F4 variants identified in patients with inner ear malformations exhibit aberrant cellular distribution and lack of SLC6A20 transcriptional upregulation.tif

Hearing loss (HL) is the most common sensory defect and affects 450 million people worldwide in a disabling form. Pathogenic sequence alterations in the POU3F4 gene, which encodes a transcription factor, are causative of the most common type of X-linked deafness (X-linked deafness type 3, DFN3, DFNX2). POU3F4-related deafness is characterized by a typical inner ear malformation, namely an incomplete partition of the cochlea type 3 (IP3), with or without an enlargement of the vestibular aqueduct (EVA). The pathomechanism underlying POU3F4-related deafness and the corresponding transcriptional targets are largely uncharacterized. Two male patients belonging to a Caucasian cohort with HL and EVA who presented with an IP3 were submitted to genetic analysis. Two novel sequence variants in POU3F4 were identified by Sanger sequencing. In cell-based assays, the corresponding protein variants (p.S74Afs*8 and p.C327*) showed an aberrant expression and subcellular distribution and lack of transcriptional activity. These two protein variants failed to upregulate the transcript levels of the amino acid transporter gene SLC6A20, which was identified as a novel transcriptional target of POU3F4 by RNA sequencing and RT-qPCR. Accordingly, POU3F4 silencing by siRNA resulted in downregulation of SLC6A20 in mouse embryonic fibroblasts. Moreover, we showed for the first time that SLC6A20 is expressed in the mouse cochlea, and co-localized with POU3F4 in the spiral ligament. The findings presented here point to a novel role of amino acid transporters in the inner ear and pave the way for mechanistic studies of POU3F4-related HL.</p