Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A-D) HeLa cells not expressing (A) or transiently expressing 3xHA-S1PR1-eGFP (B-D) were incubated with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (Carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. HA antibody was used to follow the uptake of S1P1R. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin or HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) Effect of clathrin or AP2 depletion in the uptake of S1PR1 in Hela cells transiently expressing 3xHA-S1P1-eGFP. Cells were depleted of clathrin or of its endocytic adaptor AP2 by transduction with lentiviruses encoding shRNA specific for clathrin heavy chain (CLC) or the μ2 subunit of AP2 (AP2); scrambled shRNA was used as a negative control. The cells were incubated with anti-HA antibody to follow the uptake of S1PR1 upon addition of DMSO only (Carrier) or the ligands (S1P or FTY720P). Transferrin-Alexa Fluor 647 was used to monitor its clathrin-based receptor mediated endocytosis. The representative images correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove surface bound transferrin and anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Transduction, shRNA, Negative Control, Incubation

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) MEF cells from mice expressing (β-Arrestin1/2+/+) or not (β-Arrestin1/2−/−) β-arrestin1 and β-arrestin2 and stably expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the presence of DMSO only (Carrier) or the ligands S1P or FTY720P. Images for each condition correspond to a center plane obtained using spinning disc confocal microscopy. An acid wash step at the end of the experiment was used to remove most of the surface bound anti-HA antibodies. Scale bar 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Stable Transfection, Incubation, Confocal Microscopy

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: HEK 293A cells stably expressing 3xHA-S1PR1-mCherry were incubated or not with anti-HA antibody to label receptor at the cell surface and then follow their internalization upon incubation with DMSO only (carrier) or the S1PR1 ligands (S1P or FTY720P). Fluorescent transferrin-Alexa Fluor 647 was used to monitor the clathrin-based receptor mediated endocytosis of transferrin. Data obtained using flow cytometry was from more than 10,000 cells analyzed per condition.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Stable Transfection, Expressing, Incubation, Flow Cytometry

Journal: Traffic (Copenhagen, Denmark)

Article Title: ENDOCYTOSIS OF LIGAND ACTIVATED SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 MEDIATED BY THE CLATHRIN-PATHWAY

doi: 10.1111/tra.12343

Figure Lengend Snippet: (A) HeLa cells transiently expressing 3xHA-S1PR1-eGFP were incubated with anti-HA antibody in the absence (− dynasore-OH) or presence (+ dynasore-OH) of the modified more potent cell permeable inhibitor of dynamin, dynasore-OH. Following brief 15 min incubation with dynasore-OH, the media was replaced for 30 min with a new solution only containing DMSO, S1P or FTY720P. Fluorescent transferrin-Alexa Fluor 647 was used to follow the efficiency of the clathrin-based endocytic pathway. Images obtained using spinning disc confocal microscopy correspond to a center plane from which quantification data was obtained. An acid wash step at the end of the experiment was used to remove the surface bound transferrin and anti-HA antibodies. Scale bar, 10 μm.

Article Snippet: Reagents Sphingosine-1-phosphate (S1P) and FTY720-phosphate (FTY720P) were from Echelon Bioscience (Salt Lake City, UT), poly-d-lysine (Sigma) and collagen (BD Biosciences).

Techniques: Expressing, Incubation, Modification, Confocal Microscopy

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 2. S1P and FTY720P induce lipid accumulation in HepG2 cells. Lipid accumulation in the presence of (a) Sphk1 inhibitor PF543; (b) blockers of all S1PRs. Values are means ± SEM. N = 4. Bars not sharing a common letter are considered significantly different from each other at P < 0.05; c) various concentrations of the S1P analog FTY720P. Values are means ± SEM; N = 3. *Significantly different from the control at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques: Control

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 3. Effect of FTY720P on lipid accumulation in the presence of (a) S1PR1 blocker W146. (b) S1PR2 blocker JTE 013. (c) S1PR4 blocker CYM50358. Values are means ± SEM; N = 5. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 4. S1PR3 and Gq mediate the FTY720P effect on lipid accumulation. (a) Effect of FTY720P on lipid accumulation in the presence of S1PR3 blocker CAY10444. (b) Effect of FTY720P on lipid accumulation in the presence of the Gq inhibitor YM254890. (c) Protein expression of S1PR3. The blot is representative of an experiment repeated 3 times. Values were normalized to GAPDH and reported as arbitrary densitometry units. All values are means ± SEM; N = 3. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques: Expressing

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 5. Effect of FTY720P on lipid accumulation in the presence of (a) the PI3K inhibitor wortmannin. (b) the mTOR inhibitor rapamycin. All values are means ± SEM; N = 4. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 6. Effect of FTY720P on lipid accumulation in the presence of (a) the SREBP inhibitor fatostatin. (b) The PPARγ inhibitor GW9662. Values are means ± SEM; N = 5. Bars not sharing a common letter are considered significantly different from each other at P < 0.05.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: Scientific reports

Article Title: Effect of FTY720P on lipid accumulation in HEPG2 cells.

doi: 10.1038/s41598-023-46011-4

Figure Lengend Snippet: Figure 13. The signaling pathway activated by FTY720P.

Article Snippet: FTY720P (Cat # sc-205332A), rosiglitazone (Cat # sc-202795), anti-SREBP1 (Cat# sc-365513, Lot#B1821), anti-p-mTOR (Cat# sc-293132, Lot#K2717), anti-mTOR (Cat# sc-8319, Lot#K2415), anti-GAPDH (Cat# sc-47724, Lot# H0917) mouse primary antibodies as well as anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies (Cat# sc-2005, Lot# C2011) were purchased all from Santa Cruz Biotechnology, CA, USA.

Techniques:

Journal: PLoS ONE

Article Title: Sphingosine 1-Phosphate Modulates Antigen Capture by Murine Langerhans Cells via the S1P 2 Receptor Subtype

doi: 10.1371/journal.pone.0049427

Figure Lengend Snippet: XS52 cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM), FTY720-P (1 µM), VPC24191 (10 µM), and SEW2871 (1 µM) for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P < 0.01 indicate a statistically significant difference vs. control experiments (A). Cells were treated with similar concentrations of S1P, FTY720-P, VPC24191, and SEW2871 for 15 min followed by the detection of Akt activity (B). Values of the densitometric analysis are expressed as x-fold decrease of p-Akt formation compared to untreated cells ± SEM from three experiments. **P<0.01 indicates a statistically significant difference versus control (B).

Article Snippet: SEW2871 and FTY720-phosphate (FTY720-P) were obtained from Cayman Chemical (Michigan, USA).

Techniques: Incubation, Labeling, Fluorescence, Flow Cytometry, Control, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) modulates neuronal morphology in mature hippocampal neurons. FeGFP expressing 21DIV primary hippocampal neurons from healthy wild type BL/6J mice were treated with 10nM FTY720-P for 24h. ( A ) Representative images (left) of feGFP expressing neurons treated with DMSO (above, black) and 10nM FTY720-P (below, magenta) and the relative Neurolucida tracing used to perform the Sholl analysis (right). Scale bar: 100μm. ( B ) Sholl analysis displaying dendritic complexity plotted against distance from the cell soma. F value refers to the DMSO v/s FTY720-P comparison. The graphs show ( C ) the total dendritic complexity and ( D ) total lengths of dendrites for the DMSO (black) and FTY720-P (magenta) treated neurons. ( E ) Representative segments of secondary dendrites from feGFP transfected neurons used to analyze dendritic spine density. Scale bar: 5μm ( F ) Graphical representation of the spine density for neurons treated with 10 (magenta solid) or 100nM (magenta open) FTY720-P their respective DMSO controls (10nM black solid; 100nM black open). ( G ) the graph shows the distribution of different dendritic spine types across all treatment groups. The four different types of spines– mushroom (M), thin (T), stubby (S) and filopodia (F) as represented in the schematic, were quantified and represented as a fraction of total spines for neurons treated with FYT720P (10nM magenta solid; 100nM magenta open) or DMSO (10nM black solid; 100nM black open). ( H ) Images of fields of view (FOV) of DMSO (left) and FTY720-P (right) treated primary hippocampal cultures immunostained for c-Fos (inset: above) and MAP2 (inset: below) with their respective merged images (right). The arrows indicate c-Fos expressing MAP2 positive neurons in the merged picture. Scale bar: 100μm. ( I ) Graph comparing the normalized number of c-Fos + cells over total MAP2 + neurons between DMSO (black) and FTY720-P (magenta) treated cultures. All plots represent data as mean + SEM. Numbers in the bars indicate total number of neurons or of FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ) and ( G ). For ( C,D ) and ( I ) unpaired Student’s t -test and for ( F ), one-way ANOVA with Bonferroni post-hoc was used. Significance is denoted as ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Fingolimod hydrochloride: FTY720 (USBiological, Swampscott, MA, USA) and Fingolimod-Phosphate: FTY720-P (Cayman chemical, Ann Arbor, MI, USA), were dissolved in Dimethyl sulfoxide (DMSO, anhydrous; ThermoFisher Scientific,Waltham, MA, USA).

Techniques: Expressing, Comparison, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) regulates neuronal architecture in a BDNF-dependent manner. ( A ) Representative Neurolucida tracings, used to perform the Sholl analysis of dendritic complexity for feGFP expressing neurons treated for 24h with: DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). Scale bar: 100 μm. ( B ) Sholl analysis displayed as number of dendritic intersections against distance from the cell body for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) and ( C ) total dendritic complexity of all treatment groups. F value in ( B ) refers to comparison between all the 4 treatment groups. ( D ) Dendritic spine densities for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) calculated using segments of secondary dendritic branches as shown in ( E ) feGFP panel. The Syn panel displays the corresponding staining of the pre-synaptic marker SynapsinI/II and the merge panel shows the images with overlapping SynapsinI/II puncta (red, pre-synapse) to its matching feGFP dendrite segment (green, post-synapse). The arrows point to coinciding puncta, indicative of mature synapse between the post and pre-synaptic compartments. Scale bar: 5μm. ( F ) The graph compares the fraction of SynapsinI/II positive feGFP labelled spines for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). ( G ) Representative fields of view (FOV) of the DMSO, 2nM FTY720-P, DMSO + TrkBFc and 2nM FTY720-P + TrkBFc treated hippocampal cultures stained for c-fo s . Scale bar: 100μm. ( H ) Quantification of the proportion of c-Fos expressing neurons represented as normalized fraction for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) treated cultures. All graphs represent data as mean + SEM. Numbers in the bars show either total number of neurons or of FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( C , D , F ) and ( H ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Fingolimod hydrochloride: FTY720 (USBiological, Swampscott, MA, USA) and Fingolimod-Phosphate: FTY720-P (Cayman chemical, Ann Arbor, MI, USA), were dissolved in Dimethyl sulfoxide (DMSO, anhydrous; ThermoFisher Scientific,Waltham, MA, USA).

Techniques: Expressing, Comparison, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Treatment with the non-phosphorylated Fingolimod (FTY720) modulates neuronal architecture. ( A ) | Representative Neurolucida tracings from feGFP positive hippocampal neurons used for the Sholl analysis from cultures treated either with DMSO or 10nM FTY20 for 24 h. Scale bar: 100μm. ( B ) | Dendritic complexity shown by the number of dendritic intersections plotted against the distance from the soma for DMSO (black) and FTY720 (blue) treated neurons. The F value shows the statistical comparison between the two groups. The inset graph represents total dendritic complexity upon treatment with DMSO (black) and FTY720 (blue). ( C ) | Total dendritic length for DMSO (black) and FTY720 (blue) treated neurons. ( D ) | Representative stretches from dendrites of eGFP transfected hippocampal neurons showing dendritic spine protrusions, treated either with DMSO or FTY720 for 24h. Scale bar: 5μm. ( E ) | The graph shows dendritic spine density for DMSO (black) and FTY720 (blue) treated neurons . ( F ) | Representative images of fields of view (FOV) from primary hippocampal cultures stained with anti phospho-ERK1/2 antibody, 30 min post-application of one of the following: DMSO, 10nM FTY720, DMSO_100, 100nM FTY720, DMSO_100 + TrkB-Fc, 100nM FTY720 + TrkB-Fc or 40ng recombinant BDNF protein as a positive control. Scale bar: 100μm. ( G ) | The graph displays the fraction of pERK1/2 expressing neurons relative to the total number of MAP2 + neurons. The data is normalized to the respective controls and compared between the different treatment groups: DMSO (black), 10nM FTY720 (light blue solid), DMSO100 (gray), 100nM FTY720 (dark blue solid), 40ng recombinant BDNF (magenta), DMSO100 + TrkB-Fc (gray open), 100nM FTY720 + TrkB-Fc (dark blue open). All data is plotted as mean + SEM. Numbers in the bars show total number of neurons or FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( B ) total intersections, ( C ) and ( E ) unpaired Student’s t-test and for ( G ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Fingolimod hydrochloride: FTY720 (USBiological, Swampscott, MA, USA) and Fingolimod-Phosphate: FTY720-P (Cayman chemical, Ann Arbor, MI, USA), were dissolved in Dimethyl sulfoxide (DMSO, anhydrous; ThermoFisher Scientific,Waltham, MA, USA).

Techniques: Comparison, Transfection, Staining, Recombinant, Positive Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) completely rescues the neuronal architecture in developing Mecp -/y cortical neurons. Young DIV7 cortical cultures from Mecp2 -/y mice or littermate wild type (WT) controls were allowed to develop for one week with either DMSO alone or with 10nM FTY720-P in the growth medium. ( A ) The diagram shows the scheme of treatment from culture preparation on DIV0, treatment from DIV1 to fixation on DIV7. ( B ) Micrographs showing representative fluorescent images of DIV7 WT and Mecp2 -/y cortical neurons stained for MAP2 and treated with either DMSO or FTY720-P. Scale bar: 50 μm. Neurite complexity plotted as number of neurite intersections against the distance from the soma for: ( C ) all four test groups- WT (black), WT neurons treated with 10nM FTY720-P (gray), Mecp2 -/y (dark green) and Mecp2 -/y neurons treated with 10nM FTY720-P (light green). F value for comparison between all sets is described with the graph. For better visualization of differences between different pairs, Sholl curves are also plotted separately: ( D ) WT (black) and Mecp2 -/y (dark green) neurons ( E ) Mecp2 -/y neurons treated with 10nM FTY720-P (light green) v/s DMSO controls (dark green) and ( F ) WT neurons treated with 10nM FTY720-P (gray) v/s DMSO controls (black). ( G ) Total intersections, ( H ) number of neurites and ( I ) total length of neurites as computed for WT neurons treated with DMSO (black) or FTY720-P (gray) and Mecp2 -/y treated with DMSO (dark green) or FTY720-P (light green). Data in graphs is plotted as mean + SEM. Numbers in the bars show total number of neurons analyzed for each genotype and treatment, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in A. For G,H and I one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are *** p < 0.001, **** p < 0.0001.

Article Snippet: Fingolimod hydrochloride: FTY720 (USBiological, Swampscott, MA, USA) and Fingolimod-Phosphate: FTY720-P (Cayman chemical, Ann Arbor, MI, USA), were dissolved in Dimethyl sulfoxide (DMSO, anhydrous; ThermoFisher Scientific,Waltham, MA, USA).

Techniques: Staining, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) shows mild rescue in neuronal morphology of young cortical neurons from Cdkl5 -/y mice. Young DIV7 cortical cultures from mice carrying a Cdkl5 -/y mutation or littermate wild type (WT) controls, were grown in presence of DMSO alone or 10 nM FTY720-P in the growth medium. ( A ) The diagram shows the scheme of treatment from culture preparation on DIV0, treatment from DIV1 to fixation on DIV7. ( B ) Micrographs display representative DIV7 MAP2 positive cortical neurons. The WT and Cdkl5 -/y neurons were both treated either with DMSO or FTY720-P. Scale bar: 50 μm. ( C ) Neurite complexity for both genotypes treated with DMSO and FTY720-P was analyzed and plotted as number of neurite intersections against the distance from the soma. F value for comparison between the four sets is described in the graph. The Sholl curve for following genotype and treatment sets are also displayed separately ( D ) WT (black) v/s Cdkl5 -/y (dark red), ( E ) Cdkl5 -/y neurons treated with 10nM FTY720-P (rosa) v/s DMSO controls (dark red), ( F ) WT neurons treated with 10nM FTY720-P (gray) v/s DMSO controls (black). ( G ) Total intersections, ( H ) number of neurites and ( I ) total length of neurites as computed for WT neurons treated with DMSO (black) or FTY720-P (gray) and Cdkl5 -/y neurons treated with DMSO (dark red) or FTY720-P (rosa). Data in graphs is represented as mean + SEM. Numbers in the bars show total number of neurons analyzed for each genotype and treatment, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( A ). For ( G , H ) and ( I ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, **** p < 0.0001.

Article Snippet: Fingolimod hydrochloride: FTY720 (USBiological, Swampscott, MA, USA) and Fingolimod-Phosphate: FTY720-P (Cayman chemical, Ann Arbor, MI, USA), were dissolved in Dimethyl sulfoxide (DMSO, anhydrous; ThermoFisher Scientific,Waltham, MA, USA).

Techniques: Mutagenesis, Comparison