pmx205 ref  (Tocris)

Journal: Bone Research

Article Title: Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

doi: 10.1038/s41413-023-00312-6

Figure Lengend Snippet: C5AR1, SSTR2 and GPR120/FFAR4 activation modulates osteoclastogenesis and/or resorptive activity of mature osteoclasts. a Box plot of RNA-seq-based gene expression levels of complement 5A receptor 1 ( C5AR1 ). b UMAP plot of C5AR1 gene expression in differentiated human osteoclasts at the single-cell level. c Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). d Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). e Box plot of the RNA-seq-based gene expression levels of free fatty acid receptor 4 ( FFAR4 ). f Box plot quantifying IP-1 levels in an HTRF assay, showing that the specific FFAR4 agonist TUG-891 increases IP-1 levels in mature osteoclasts (Day 9) after 30 min ( n = 6). g Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 4). h Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 5). i Box plot of RNA-seq-based gene expression levels of somatostatin receptor 2 ( SSTR2 ). The inlet highlights the expression at Days 5 and 9. Lines represent individual donors. j Box plot quantifying the cAMP-Glo assay. k Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the SSTR2 agonist somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 4). l Box plot quantifying osteoclast resorptive activity as a percentage of eroded surface area per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the SSTR2 agonists somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 6). m Box plot of real-time PCR-based gene expression levels of SSTR2 relative to TBP in osteoclasts on Day 9 of differentiation ( n = 6). n Box plot quantifying osteoclast resorptive activity after 72 h on bovine bone slices in the presence of the SSTR2 agonist somatostatin (100 nmol/L) as a percentage of the eroded surface area per bone surface ( n = 6). o Scatter plot quantifying the percentage change in bone resorption following SSTR2 knockdown (vehicle values from Fig. : (siCTR – siSSTR2)/siCTR) relative to changes in SSTR2 expression (values from Fig. : siCTR – siSSTR2) ( n = 6). p Scatter plot quantifying the percentage change in bone resorption in response to somatostatin (values from Fig. : (Veh – somatostatin)/Veh) over relative SSTR2 expression (values from Fig. ) ( n = 6). The small scatterplot was used to quantify the delta of the x and y values of siCTR and si SSTR2 , and the big scatterplot P value was calculated based on a linear regression model

Article Snippet: The following compounds were used at the following concentrations according to the literature and purchased from the following companies: BM221 (ref. ) (0.2 µg/mL, 1 µg/mL and 10 µg/mL; Product No. GLXC-24851; Glixx Laboratories, Inc., MA, USA); PMX205 (ref. ) (5 µg/mL and 15 µg/mL; Product No. 5196; Bio-Techne Tocris, MN, USA); somatostatin-14 (ref. ) (1, 10 and 100 nmol/L; Product No. S9129; Merck, London, UK); octreotide (1 and 10 nmol/L; Product No. O1014; Merck, London, UK); BIM-23627 (ref. ) (1, 10 and 100 nmol/L; Product No. 4041642; Bachem, Bubendorf, CH); and TUG-891 (ref. , ) (0.1, 1 and 10 μmol/L; Product No. SML1914; Merck, London, UK).

Techniques: Activation Assay, Activity Assay, RNA Sequencing Assay, Expressing, HTRF Assay, Glo Assay, Real-time Polymerase Chain Reaction

Journal: Bone Research

Article Title: Transcriptional reprogramming during human osteoclast differentiation identifies regulators of osteoclast activity

doi: 10.1038/s41413-023-00312-6

Figure Lengend Snippet: C5AR1, SSTR2 and GPR120/FFAR4 activation modulates osteoclastogenesis and/or resorptive activity of mature osteoclasts. a Box plot of RNA-seq-based gene expression levels of complement 5A receptor 1 ( C5AR1 ). b UMAP plot of C5AR1 gene expression in differentiated human osteoclasts at the single-cell level. c Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). d Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the C5AR1 agonist BM221 (1 µg/mL) and/or antagonist PMX205 (5 µg/mL) ( n = 6). e Box plot of the RNA-seq-based gene expression levels of free fatty acid receptor 4 ( FFAR4 ). f Box plot quantifying IP-1 levels in an HTRF assay, showing that the specific FFAR4 agonist TUG-891 increases IP-1 levels in mature osteoclasts (Day 9) after 30 min ( n = 6). g Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 4). h Box plot quantifying osteoclast resorptive activity as the percentage of eroded surface per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the FFAR4 agonist TUG-891 (10 nmol/L) ( n = 5). i Box plot of RNA-seq-based gene expression levels of somatostatin receptor 2 ( SSTR2 ). The inlet highlights the expression at Days 5 and 9. Lines represent individual donors. j Box plot quantifying the cAMP-Glo assay. k Box plot quantifying osteoclast numbers and nuclei per osteoclast after 9 days of osteoclast differentiation in the presence of the SSTR2 agonist somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 4). l Box plot quantifying osteoclast resorptive activity as a percentage of eroded surface area per bone surface for mature osteoclasts (Day 9) after 72 h on bovine bone slices in the presence of the SSTR2 agonists somatostatin (100 nmol/L) and octreotide (10 nmol/L) and antagonism by BIM-23627 (100 nmol/L) ( n = 6). m Box plot of real-time PCR-based gene expression levels of SSTR2 relative to TBP in osteoclasts on Day 9 of differentiation ( n = 6). n Box plot quantifying osteoclast resorptive activity after 72 h on bovine bone slices in the presence of the SSTR2 agonist somatostatin (100 nmol/L) as a percentage of the eroded surface area per bone surface ( n = 6). o Scatter plot quantifying the percentage change in bone resorption following SSTR2 knockdown (vehicle values from Fig. : (siCTR – siSSTR2)/siCTR) relative to changes in SSTR2 expression (values from Fig. : siCTR – siSSTR2) ( n = 6). p Scatter plot quantifying the percentage change in bone resorption in response to somatostatin (values from Fig. : (Veh – somatostatin)/Veh) over relative SSTR2 expression (values from Fig. ) ( n = 6). The small scatterplot was used to quantify the delta of the x and y values of siCTR and si SSTR2 , and the big scatterplot P value was calculated based on a linear regression model

Article Snippet: The following compounds were used at the following concentrations according to the literature and purchased from the following companies: BM221 (ref. ) (0.2 µg/mL, 1 µg/mL and 10 µg/mL; Product No. GLXC-24851; Glixx Laboratories, Inc., MA, USA); PMX205 (ref. ) (5 µg/mL and 15 µg/mL; Product No. 5196; Bio-Techne Tocris, MN, USA); somatostatin-14 (ref. ) (1, 10 and 100 nmol/L; Product No. S9129; Merck, London, UK); octreotide (1 and 10 nmol/L; Product No. O1014; Merck, London, UK); BIM-23627 (ref. ) (1, 10 and 100 nmol/L; Product No. 4041642; Bachem, Bubendorf, CH); and TUG-891 (ref. , ) (0.1, 1 and 10 μmol/L; Product No. SML1914; Merck, London, UK).

Techniques: Activation Assay, Activity Assay, RNA Sequencing Assay, Expressing, HTRF Assay, Glo Assay, Real-time Polymerase Chain Reaction, Knockdown