Evaluation of SPIOn labeled cell biological properties and migration.

<p><b>a</b> Proliferative and <b>b</b> metabolic rates of hCVCs appeared not affected by SPIOn presence, as previously demonstrated by our group for hAFCs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078435#pone.0078435-Bigini1" target="_blank">[20]</a>; <b>c</b> hAFCs and hCVCs were incubated for 72 hours with SPIOn (35 µg/ml) and then migration assay was performed. Data are expressed as a percentage of migrated cells. ***p<0.001 versus respective (unlabeled, control cells, CTR); °°p<0.01 versus SPIOn labeled hCVCs. One way analysis of variance (ANOVA) as specified in the statistical section.</p

Migration and survival of hAFCs and hCVCs labeled with different SPIOn concentrations.

<p><b>a</b> Cells were labeled with different concentration of SPIOn for 72 hours before migration assay. Data are expressed as a percentage of migrated cells. *p<0.05, **p<0.01 and ***p<0.001 versus respective CTR (control, unlabeled cells). Two way analysis of variance (ANOVA); as specified in the statistical section; <b>b</b> hAFCs and hCVCs labeled with different SPIOn concentrations were used to test SPIOn toxicity by MTS assay. Data are expressed as a percentage of viable cells versus unlabeled cells used as control (CTR). Two way analysis of variance (ANOVA); as specified in the statistical section.</p

Flow cytometric analysis.

<p>Fluorescent cell linker labeled hCVCs (here PKH26) and hAFCs (here PKH67) were analyzed by flow cytometry in order to investigate differences in size and cell complexity. Representative figure of unlabeled hCVCs and hAFCs (<b>a,b</b>): when they were mixed, a single cell population was detected (<b>c</b>); different labeling of hCVCs (<b>d,g</b>) and hAFCs (<b>e,h</b>), before mixing the cells, allowed us to identify simultaneously both the cell population confirming their comparable size and complexity (<b>f</b>).</p

Evaluation of intra-cellular ROS during internalization and subsequent removal of SPIOn.

<p><b>a</b> hAFCs and <b>b</b> hCVCs were plated and labeled with DAF-FM to visualize intra-cellular ROS, before being incubated with SPIOn (35 or 100 µg/ml). ROS measurement was performed every hour (from 1^st to 5^th) during SPIOn internalization (see experimental scheme). Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). No statistically significant differences were observed between CTR and SPIOn labeled cells. ***p<0.001 versus CTR; °°°p<0.001 versus SPIOn (35 µg/ml). Two way analysis of variance (ANOVA); <b>c</b> hAFCs and <b>d</b> hCVCs were plated and labeled with DAF-FM and then were incubated with SPIOn (35 µg/ml). ROS evaluation was done at different time (6, 12, 24, 48 and 72 hours) after SPIOn addition. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). ***p<0.001 versus respective 6 hours; °°°p<0.001 respective 12 hours; +++p<0.001 versus respective 24 hours. Two way analysis of variance (ANOVA) as specified in the Statistical section. To better visualize the significant difference in intra-cellular ROS formation between hAFCs and hCVCs, we compared the two cellular groups at <b>e</b> 6, <b>f</b> 12, <b>g</b> 24, <b>h</b> 48 and <b>i</b> 72 hours after SPIOn removal. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). **p<0.01 and ***p<0.001 versus hAFCs. Two way analysis of variance (ANOVA), as specified in the statistical section.</p

Intra-cellular ferric ions measurement.

<p>Cells were incubated for 24, 48 or 72 hours with 35 µg/ml of SPIOn. Iron content expressed as Fe and Fe₃O₄ in control-samples of AFC, CVC and physiological solution along with a Fe₃O₄ standard at 35 ppm are reported. Unlabeled cells represent control condition (CTR). Data (mg/L) are expressed as mean ± SD and analyzed with two way analysis of variance (ANOVA).</p

Role of ongoing degeneration on cell migration.

<p><b>a</b> SH-SY5Y cells were plated and treated with 6-OHDA 100 µM. After 1, 4 and 6 hours viability was measured by MTS assay. Data are expressed as a percentage of viable cells versus CTR (no 6-OHDA treated cells). ***p<0.001 versus CTR. One way analysis of variance (ANOVA) as specified in the Statistical section; <b>b</b> hAFCs and <b>c</b> hCVCs were incubated for 72 hours with SPIOn (35 µg/ml) and then were treated with 6-OHDA (100 µM). MTS assay was performed 1, 4 and 6 hours after 6-OHDA/toxin addition. Data are expressed as a percentage of viable cells versus respective CTR (no 6-OHDA treated cells). Two way analysis of variance (ANOVA) as specified in the Statistical section. <b>d</b> timeline of migration experiments; <b>e</b> hAFCs and <b>f</b> hCVCs were labeled with SPIOn 35 µg/ml (72 hours of incubation) followed by the migration assay. In these experiments, SH-SY5Y cells were plated in the bottom chamber and then treated with/exposed to 6-OHDA (100 µM). Migration was evaluated 1, 4 and 6 hours after treatment. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). ***p<0.001 versus not treated (NT) SH-SY5Y cells; °p<0.05, °°p<0.01 and °°°p<0.001 versus 1 hour treatment; +++p<0.001 versus 4 hours treatment. Two way analysis of variance (ANOVA) as specified in the statistical section.</p

Effect of serum concentration on the migration of SPIOn labeled hAFCs and hCVCs.

<p><b>a</b> hAFCs and <b>b</b> hCVCs were incubated for 72 hours with different concentration of SPIOn and then migration assay was performed. In these experimental setting, medium with 10% FBS was added in the bottom chamber. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). **p<0.01 and ***p<0.001 versus serum free medium. Two way analysis of variance (ANOVA) as specified in the Statistical section; <b>c</b> hAFCs and <b>d</b> hCVCs were labeled with SPIOn 35 µg/ml (72 hours of incubation) and migration assay was performed. In these experiments medium plus 10, 20 or 30% FBS was added in the bottom chamber. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). *p<0.05 and ***p<0.001 versus serum free medium. °p<0.05 and °°°p<0.001 versus 10% FBS; +p<0.05 and +++p<0.001 versus 20% FBS. Two way analysis of variance (ANOVA) as specified in the statistical section.</p

TBSS results: areas of decreased fractional anisotropy (FA, red-yellow), and increased mean (MD, green) and radial diffusivity (radD, blue) in PLS patients with cognitive deficits (PLS-cd) <i>vs.</i> healthy controls are displayed on a FA template in the Montreal Neurological Institute space.

<p>FWE =  family wise error.</p

Neuropsychological and behavioral data of PLS patients.

<p>Scores are corrected for age, gender and education. P = differences between patient groups; values refer to the Mann-Whitney U-test. WCST, global score calculation = [N used cards-(completed categories*10)], higher scores mean worse performances <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082017#pone.0082017-Laiacona1" target="_blank">[23]</a>. Fluency indices calculation = for each letter (P, F, L) or category (animals, fruits, cars) a partial index was calculated in order to correct for motor disabilities as following: [(60-seconds for reading words previously reported in 1’)/N words previously reported in 1’]; the total semantic or phonemic index was obtained by averaging the 3 semantic or phonemic partial indices, respectively. Higher scores mean worse performances <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082017#pone.0082017-Abrahams1" target="_blank">[21]</a>. Abbreviations: BADA =  “Batteria per l'Analisi del Deficit Afasico”; co =  cut-off; FBI =  frontal behavioral battery; HDRS =  Hamilton depression rating scale; PLS-cd =  primary lateral sclerosis with cognitive deficits; PLS-cu =  cognitively unimpaired primary lateral sclerosis; WCST =  Wisconsin card sorting test.</p

TBSS results: areas of decreased fractional anisotropy (FA, red-yellow), and increased mean (MD, green) and radial diffusivity (radD, blue) in PLS patients with cognitive deficits (PLS-cd) <i>vs.</i> PLS patients without cognitive impairment (PLS-cu) are displayed on a FA template in the Montreal Neurological Institute space.

<p>FWE =  family wise error.</p