Effect of lipid extracts from LDL and OxLDL on CerS activity.

<p>(A) Oxidation of LDL was performed as described under “Experimental Procedure”. RAW 264.7 cells were stimulated with lipid extracts from native LDL and OxLDL (50 µg protein/mL respectively) for 24 h. CerS activity in cell homogenates was measured using C₁₆-CoA and C₂₂-CoA substrates. Results are means ± S.D., *p < 0.05, of a typical experiment repeated four times with similar results. (B) Cells were treated with intact LDL and OxLDL (50 µg protein/mL respectively) as described above for 24 h. CerS activity in cell homogenates was measured using C₁₆-CoA and C₂₂-CoA substrates. Results are means ± S.D. *p < 0.05, of a typical experiment repeated four times with similar results. (C) Cells were treated as described above and lipids were extracted and analyzed for ceramide content as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (D) Ceramide species were analyzed after treatment with lipid extracts from native LDL and OxLDL as described earlier. The data are means ± S.E., *p < 0.05, n = 4.</p

Effect of FB1 on OxPL induced CerS activation and ceramide levels.

<p>(A) Raw 264.7 cells were pre-incubated with FB1 (20 µM) for 2 h prior to the 24 h OxPL treatment. Lipids were extracted and ceramide levels were analyzed as described under “Experimental Procedure”. No probability values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide species were analyzed as earlier. The data are means ± S.E., *p < 0.05, **p < 0.01 compared with control, n = 4.</p

The oxidized phospholipids POVPC and PGPC are cytotoxic and induce apoptosis.

<p>(A) Chemical structures of POVPC and PGPC. Dotted circle represents the functional group at <i>sn-2</i> position. (B) RAW 264.7 cells were incubated with 50 µM POVPC and PGPC for indicated periods. Control cells were treated with 1% ethanol. Cell viability was determined by Vybrant® MTT assay kit. Results are expressed as a percentage of viable cell number in treated cells compared with that of untreated control cells. Data are means ± S.D., n = 8 in each group. (C) Cells were incubated with the stated concentrations of POVPC and PGPC for 4 h. The cells were analyzed for Alexa Fluor₄₈₈-annexin V and propidium iodide fluorescence staining by Flow cytometry as described under “Experimental Procedures”. Results are represented as means ± S.D. Probabilities compared to control were determined by Student’s t-test (two-tailed, unpaired); ***p < 0.001,(n = 8 in each group).</p

Influence of POVPC and PGPC on ceramide levels and CerS activation in RAW 264.7 cells.

<p>(A) Both POVPC and PGPC elevate ceramide generation in RAW 264.7 cells. Cells were stimulated for 24 h with respective OxPLs (50 µM) in parallel to ethanol treated control cells. Lipids were extracted and analyzed for ceramide levels by LC/MS-MS as described under “Experimental Procedure”. No probability. values are given for total ceramide levels because these levels are the sum of ceramide species with different acyl chain lengths. (B) Ceramide speciation was performed after OxPL treatment as above. The data are means ± S.E., *p<u><</u>0.05, **p < 0.01 compared with control, n = 4. (C) After OxPL treatment cells were harvested and CerS activity in cell homogenates was measured as described earlier. Results are means ± S.E., *p < 0.01, **p < 0.05, of a typical experiment repeated four times with similar results. (D) CerS mRNA levels were measured by RT-qPCR after 24 h incubation with 50 µM POVPC or PGPC as described under “Experimental Procedures”. The data are normalized to GAPDH mRNA expression and data are means ± S.E. for four independent experiments performed in triplicate. (E) nSMase activity was measured in cell homogenates after exposure to OxPL as described under “Experimental Procedures”. The data are represented as means ± S.E., n = 4.</p

Elevation of GPNMB levels in CSF and brain of nGD patients.

<p>(A) Levels of GPNMB determined by LC-MS/MS in CSF of four type 3 GD patients. Results are means ± SEM. ** <i>p</i>< 0.01. (B) Levels of GPNMB in CSF of four type 3 GD patients determined by ELISA. Results are means ± SEM (n = 4). ** <i>p</i><0.01. (C) Western blot of GPNMB in CSF of control and a type 3 GD patient (sample designation 4). Results are from a typical experiment repeated 3 times. (D) Levels of GPNMB in nGD brain determined by ELISA(n = 3 for control, n = 6 for nGD (type 2 and type 3 patients). Results are means ± SEM, ** <i>p</i>< 0.01</p

GPNMB peptides identified by LC-MS/MS.

<p>The GPNMB sequence is shown (UniProtKB/Swiss-Prot Q14956), with the site of cleavage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120194#pone.0120194.ref020" target="_blank">20</a>] indicated in red and the two peptides identified by LC-MS/MS indicated in green and brown.</p

Clinical information and correlation with GPNMB levels in the CSF of type 3 GD patients.

<p>* Full scale IQ</p><p>^# Arbitrary units</p><p>Clinical information and correlation with GPNMB levels in the CSF of type 3 GD patients.</p

Hyperphosphorylation of Tau in nGD samples.

<p>(A) Western blot of Tau and P-Tau (using two different anti-P-Tau antibodies) in brains of 21 day-old Gba^flox/flox; nestin-Cre mice and (B) P-Tau in the brain of one type 2 GD patient. Results are from a typical experiment repeated 3 times which gave similar results. GAPDH was used as a loading control. A molecular weight marker is shown (Mr = 55 kDa)</p

GPNMB levels in brain and serum of Gba^flox/flox; nestin-Cre mice.

<p>Levels of GPNMB in (A) brain (n = 3) at different days post-natal (p) and (B) serum (n = 4,n = 5) of 21-day old Gba^flox/flox; nestin-Cre mice determined by ELISA. Results are means ± SEM *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Up-regulated proteins in the CSF of type 3 GD patients.

<p>CSF from type 3 GD patients and age matched controls (n = 4) was digested with trypsin and subjected to label-free quantitative global proteomic analysis using liquid chromatography and tandem mass spectrometry (LC-MS/MS).</p><p>Up-regulated proteins in the CSF of type 3 GD patients.</p