Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: S1P reduced osteoclastogenesis of primary human monocytes. Human PBMC-isolated monocytes were treated with RANKL to induce osteoclastogenesis. ( A , B ) RANKL-treated cells were either (untreated, black bars, n = 10), further stimulated with S1P (green bars, n = 10). ( A ) Representative images of untreated or S1P-treated cells. ( B ) Osteoclast counts (TRAP-positive, multinucleated) in each condition and presented as number of osteoclasts per well. ( C , D ) RANKL-induced osteoclasts were cultured on dentine slices for 21 days in the presence of vehicle control (untreated, black, n = 6) or S1P (green, n = 3) prior to cell removal and staining of resorption pits. ( C ) Representative images of masks generated to show areas resorbed by osteoclasts. ( D ) The resorption area was recorded and displayed as a percentage of the total area. ( E – G ) Cells were untreated (grey, n = 5) or treated with RANKL only (black, n = 5) or treated with RANKL and S1P (green, n = 3 independent donors) for 8 days and RNA and gene expression of ( E ) ACP5 , ( F ) CTSK , and ( G ) MSR1 was normalised as 2 −ΔCt to β 2 M . Data are mean ± SEM from (B) n = 10 from 3 independent donors, ( D – G ) n = 3–5 independent donors. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 by paired t -test.

Article Snippet: Tubes were placed into the EasySep ‘The Big Easy’ magnet (cat: 18001, Stem Cell) for 5 min before all non-stuck cells (monocytes) were collected and 1 × 10 6 /mL were cultured overnight in human osteoclast differentiation media containing 50 ng/mL m-CSF (cat: 216-MC-025, R&D systems, Abingdon, UK).

Techniques: Isolation, Cell Culture, Control, Staining, Generated, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: S1P increased osteoclastogenesis of primary murine monocytes. Murine bone marrow-derived monocytes (BMDM) were treated with M-CSF for 24 h before RANKL treatment. ( A , B ) RANKL-treated cells were either (untreated, black bars, n = 4), further stimulated with S1P (green bars, n = 4). ( A ) Representative images of untreated or S1P-treated wells stained for TRAP. ( B ) Osteoclast counts (TRAP-positive, multinucleated) in each condition and presented as number of osteoclasts per well. ( C , D ) RANKL-induced osteoclasts were cultured on hydroxyapatite coated plates for 8 days in the presence of vehicle control (untreated, black, n = 4) or S1P (green, n = 4) prior to cell removal and staining of resorption pits. ( C ) Representative images of masks generated to show areas resorbed by osteoclasts. ( D ) The resorption area was displayed as a percentage of the total area. ( E , G ) Cells were untreated (black, n = 5) or treated with RANKL only (black, n = 5) or treated with RANKL and S1P (green, n = 3 independent donors) for 8 days and gene expression for ( E ) Acp5 , ( F ) Ctsk , and ( G ) Msr1 were analysed and normalised as 2 −ΔCt to β 2 m . Data are mean ± SEM from ( B ) n = 10 from 3 independent donors, ( D – G ) n = 4 independent donors. * = p < 0.05 by unpaired t -test.

Article Snippet: Tubes were placed into the EasySep ‘The Big Easy’ magnet (cat: 18001, Stem Cell) for 5 min before all non-stuck cells (monocytes) were collected and 1 × 10 6 /mL were cultured overnight in human osteoclast differentiation media containing 50 ng/mL m-CSF (cat: 216-MC-025, R&D systems, Abingdon, UK).

Techniques: Derivative Assay, Staining, Cell Culture, Control, Generated, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: S1P receptor expression changes over osteoclastogenesis but not between species. ( A , B ) Analysis of publicly available single cell sequencing datasets ( A ) GSE147174 (mouse) and ( E ) GSE162454 (human) and looking at S1P receptors (S1PR1–S1PR5). Dotplot of clustered ( A ) murine monocytes, macrophages, pre-osteoclasts, and osteoclasts or ( B ) human monocytes, macrophages, pre-osteoclasts, and osteoclasts, showing expression of S1PR1-5, where circle size represents percentage of cells expressing the gene and colour indicates average expression value. ( C – E ) BMDMs cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (Mono, blue, n = 3) or osteoclasts (Oc, black, n = 3), analysed for ( C ) S1pr1 , ( D ) S1pr3 , and ( E ) S1pr4 . ( F – H ) Human monocytes derived from PBMCs cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (Mono, blue, n = 5) or osteoclasts (Oc, black, n = 5), analysed for ( F ) S1PR1 , ( G ) S1PR3 , and ( H ) S1PR4 . Data are displayed as 2 −ΔCt , where ΔCt is the relative expression compared to β 2 m or β 2 M housekeeping gene. Data are mean ± SEM. *** = p < 0.001 by unpaired t -test.

Article Snippet: Tubes were placed into the EasySep ‘The Big Easy’ magnet (cat: 18001, Stem Cell) for 5 min before all non-stuck cells (monocytes) were collected and 1 × 10 6 /mL were cultured overnight in human osteoclast differentiation media containing 50 ng/mL m-CSF (cat: 216-MC-025, R&D systems, Abingdon, UK).

Techniques: Expressing, Sequencing, Cell Culture, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: Kinases involved in S1P production vary over osteoclast differentiation and species. ( A , B ) Analysis of publicly available single cell sequencing datasets GSE147174 (mouse) and GSE162454 (Human) and looking at S1P producer ( Sphk1 and Sphk2 ) and transporter (Spns2). ( A ) Dotplot of clustered murine monocytes, macrophages, pre-osteoclasts, and osteoclasts, showing expression of Sphk1 , Sphk2 , and Spns2 . ( B ) Dotplot of clustered human monocytes, macrophages, pre-osteoclasts, and osteoclasts, showing expression of SPHK1 , SPHK2 , and SPNS2 . ( C – F ) Primary mouse bone marrow-derived monocytes cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (mono, blue) or osteoclasts (Oc, black), analysed for ( C ) Sphk1 (n = 3) and ( D ) Sphk2 (n = 3) gene expression and ( E ) mSPHK1 and ( F ) mSPHK2 protein expression. ( G – J ) Human monocytes derived from PBMCs cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (blue) or osteoclasts (black), analysed for ( G ) SPHK1 (n = 5) and ( H ) SPHK2 (n = 5) gene expression and ( I ) hSPHK1 and ( J ) hSPHK2 protein expression. ( K , L ) Primary mouse bone marrow-derived osteoclasts ( K ) or primary human osteoclasts ( L ) were treated with or without S1P and analysed for ( K ) mSPHK2 and ( L ) hSPHK1 protein expression. Gene expression data are displayed as 2 −ΔCt normalised to β 2 m or β 2 M . Protein expression data normalised to human or mouse β-actin. Data are mean ± SEM. ** = p < 0.01 by unpaired t -test.

Article Snippet: Tubes were placed into the EasySep ‘The Big Easy’ magnet (cat: 18001, Stem Cell) for 5 min before all non-stuck cells (monocytes) were collected and 1 × 10 6 /mL were cultured overnight in human osteoclast differentiation media containing 50 ng/mL m-CSF (cat: 216-MC-025, R&D systems, Abingdon, UK).

Techniques: Sequencing, Expressing, Derivative Assay, Cell Culture, Gene Expression