Journal: The Journal of Endocrinology

Article Title: Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice

doi: 10.1530/JOE-18-0153

Figure Lengend Snippet: Porcupine expression. (A) Porcn mRNA levels in different tissues from n mice. (B) Porcn mRNA levels in calvarial osteoblasts cultured with or without BMP2 for 7 days ( n = 4 wells/condition and time point). Results are expressed as fold-change to the levels in control osteoblasts at day 2. As a positive control, we show Alpl mRNA levels at day 7. (C) Porcn mRNA levels in bone marrow cell-derived osteoclasts cultured with or without RANKL for 4 days ( n = 4 wells/condition and time point). Results are expressed as fold-change to the levels in control osteoclasts at day 1. As a positive control, we show Acp5 mRNA levels at day 4. Bars represent the mean and error bars represent the 95% CI of the mean. P values are indicated as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems), and real-time PCR analysis was performed using predesigned real-time PCR assays (primers from Applied Biosystems) for Porcupine ( Porcn , Mm00450403_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), cathepsin K ( Ctsk , Mm00484036_m1), Acp5 (encoding TRAP; Mm00475698_m1), osteoprotegerin ( Opg , Tnfrsf11b , Mm00435452_m1) and rank-ligand ( Rankl , Tnfrsf11 , Mm00441908-m1) on the StepOnePlus Real-Time PCR system (Applied Biosystems).

Techniques: Expressing, Cell Culture, Control, Positive Control, Derivative Assay

Journal: The Journal of Endocrinology

Article Title: Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice

doi: 10.1530/JOE-18-0153

Figure Lengend Snippet: Effects of Porcupine inhibition on cortical bone. (A) Cortical thickness (Ct.Th) of the femur assessed by µCT. (B) Maximal Failure load (F max) of the tibia from the 3-point bending test. (C) Bone histomorphometry at the periosteal surface of femur cortical bone showing periosteal bone formation rate (Ps BFR), mineralization apposition rate (Ps MAR), mineralizing surface over bone surface (Ps MS/BS) and osteoclast number per bone perimeter (Ps Oc.N). (D) Bone histomorphometry at the endocortical surface of femur showing endocortical bone formation rate (Ec BFR), mineralization apposition rate (Ec MAR), mineralising surface over bone surface (Ec MS/BS) and osteoclast number per bone perimeter (Ec Oc.N). (E) Alpl , Ctsk and Rankl/Opg ratio mRNA levels in cortical bone. For each graph, n = 10 animals per group. Bars represent the mean and error bars represent the 95% CI of the mean. P values are indicated as * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems), and real-time PCR analysis was performed using predesigned real-time PCR assays (primers from Applied Biosystems) for Porcupine ( Porcn , Mm00450403_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), cathepsin K ( Ctsk , Mm00484036_m1), Acp5 (encoding TRAP; Mm00475698_m1), osteoprotegerin ( Opg , Tnfrsf11b , Mm00435452_m1) and rank-ligand ( Rankl , Tnfrsf11 , Mm00441908-m1) on the StepOnePlus Real-Time PCR system (Applied Biosystems).

Techniques: Inhibition

Journal: The Journal of Endocrinology

Article Title: Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice

doi: 10.1530/JOE-18-0153

Figure Lengend Snippet: Effects of Porcupine inhibition on trabecular bone. (A) Trabecular bone volume fraction (BV/TV) in L5 vertebral body assessed by µCT. (B) 3D reconstruction of 200 axial sections from an elliptic region of interest within trabecular bone of the L5 vertebra body from a representative animal treated with vehicle or LKG974 at 6 mg/kg/day (Bar: 200 µm). (C) BV/TV in the distal metaphyseal region of the femur assessed by µCT. (D) Bone histomorphometry at the trabecular bone of L5 vertebral body showing bone formation rate (BFR), mineralization apposition rate (MAR), mineralising surface over bone surface (MS/BS) and osteoclast number per bone surface (Oc.N). (E) Microscopy images of the trabecular bone from L5 vertebra body stained with TRAP showing multinucleated osteoclasts at the bone surface (Bar: 50 µm). (F) Alpl , Ctsk and Rankl/Opg ratio mRNA levels in trabecular bone. (G) Summary diagram of the effects of Porcupine inhibition on cortical or trabecular bone formation and resorption. For each graph, n = 10 animals per group. Bars represent the mean and error bars represent the 95% CI of the mean. P values are indicated as * P < 0.05; ** P < 0.01; *** P < 0.001. Ps , periosteal; Ec , endocortical.

Article Snippet: The RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (#4368814, Applied Biosystems), and real-time PCR analysis was performed using predesigned real-time PCR assays (primers from Applied Biosystems) for Porcupine ( Porcn , Mm00450403_m1), alkaline phosphatase ( Alpl , Mm00475834_m1), cathepsin K ( Ctsk , Mm00484036_m1), Acp5 (encoding TRAP; Mm00475698_m1), osteoprotegerin ( Opg , Tnfrsf11b , Mm00435452_m1) and rank-ligand ( Rankl , Tnfrsf11 , Mm00441908-m1) on the StepOnePlus Real-Time PCR system (Applied Biosystems).

Techniques: Inhibition, Microscopy, Staining

Journal: Cytokine

Article Title: Short-term RANKL Exposure Initiates a Neoplastic Transcriptional Program in the Basal Epithelium of the Murine Salivary Gland

doi: 10.1016/j.cyto.2019.154745

Figure Lengend Snippet: List of murine Taqman expression assays used in these studies.

Article Snippet: Detailed information concerning the TaqMan gene expression assays used in these experiments is described in ; 18S ribosomal RNA served as the internal control. table ft1 table-wrap mode="anchored" t5 Table 1 │ caption a7 Gene ID Catalog number Aqp5 11830 Mm00437578_m1 Amy1 11722 Mm00651524_m1 Ccl8 20307 Mm01297183_m1 Ccl9 20308 Mm00441260_m1 Ccr1 12768 Mm00438260_s1 Cldn22 75677 Mm04209227_sH Clec4n 56620 Mm00490934_m1 Ctsk 13088 Mm00484039_m1 Cxcl11 56066 Mm00444662_m1 Dcpp1 13184 Mm03019597_gH Elf5 13711 Mm00468732_m1 Esp8 100126778 Mm04243104_m1 Esp18 100126774 Mm04279607_m1 Fscn1 14086 Mm00456046_m1 Krt17 1667 Mm00495207_m1 Mmp12 17381 Mm00500554_m1 Muc19 239611 Mm01306462_m1 Nfkb2 18034 Mm00479807_m1 Postn 50706 Mm01284919_m1 Prom2 192212 Mm00617472_m1 Pthrp/Pthlh 19227 Mm00436057_m1 Relb 19698 Mm00485664_m1 Relt 320100 Mm00723872_m1 Scgb2b26 110187 Mm01254729_m1 Smr3a 20599 Mm01964237_s1 Sox8 20681 Mm00803422_m1 Spp1 20750 Mm00436767_m1 Tfec 22797 Mm01161234_m1 Tnfaip2 21928 Mm00447578_m1 Tnfaip3 21929 Mm00437121_m1 Tnfrsf4 22163 Mm00442039_m1 Tnfrsf8 21936 Mm00437140_m1 Tnfrsf1b 21938 Mm00441889_m1 Tnfrsf11 (Rankl) 21943 Mm00441906_m1 Tnfrsf11a (Rank) 21934 Mm00437132_m1 Traf1 22029 Mm00493827_m1 18S rRNA Thermo Fisher Scientific Inc.: 4352930E Open in a separate window List of murine Taqman expression assays used in these studies.

Techniques: Expressing

Journal: Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA

Article Title: The skeletal impact of the chemotherapeutic agent etoposide

doi: 10.1007/s00198-017-4032-1

Figure Lengend Snippet: Bone formation and resorption indices after etoposide treatment. a–h Six-week-old female C57B6/J mice were treated with etoposide (20 mg/kg) for 7 days. RNA was harvested from bone marrow (a–c) or whole bone (marrow + bone tissue) (d, e) and real-time qPCR performed. Data was analyzed using the delta delta CT method and the y-axis represents the relative change of leptin receptor (Lepr) (a), C-X-C motif chemokine ligand 12 (Cxcl12) (b), receptor activator of NFκB ligand (Rank;Tnfsf11) and osteoprotegerin (Opg;Tnfrsf11b) (c), runt-related transcription factor 2 (Runx2) (d), and osteocalcin (Ocn;Bglap3) (e). The ratio of RANKL/OPG (Tnfsf11/Tnfrsf11b) mRNA expression is also indicated in c. Serum analyses of P1NP (f). TRAP staining was performed on tibial sections and analyzed in the proximal tibiae beginning 150 μm from the growth plate and extending 900 μm distally, up to but excluding cortical to determine osteoclast numbers per linear bone (OC#/mm) (g). Serum analyses of TRAcP5b (h). Sixteen-week-old female C57B6/J mice were treated with etoposide (20 mg/kg) daily for 5 days, then 3×/week for a total of 6 weeks. Serum analyses of P1NP (i) and TRAcP5b (j). TRAP staining was performed on tibial sections and analyzed immediately distal to the growth plate (200 μm) to determine k osteoclast numbers per linear bone (OC#/mm). Data is mean ± SEM; n = 7–11/gp; *p < 0.05, **p < 0.01, ***p < 0.0001 vs. vehicle (VEH) controls

Article Snippet: Genes of interest were amplified using TaqMan PCR Master Mix and TaqMan primer/probe sets, including receptor activator of nuclear factor-κB ligand ( Rankl ; Tnfsf11 ; Mm00441906_m1), osteoprotegerin ( Opg ; Tnfrsf11 ; Mm00435452_m1), leptin receptor ( Lepr ; Mm00440181_m1), C-X-C motif chemokine 12 ( Cxcl12 ; Mm00445553_m1), Runt-related transcription factor 2 ( Runx2 ; Mm00501584_m1), and osteocalcin ( Ocn ; Bglap3 ; Mm03413826_mH) (Applied Biosystems).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Osteoprotegerin Regulates Pancreatic β-Cell Homeostasis upon Microbial Invasion

doi: 10.1371/journal.pone.0146544

Figure Lengend Snippet: (A, B) Immunofluorescence analysis showing localization of OPG protein (red) with glucagon (green) (A) or insulin (green) (B) in pancreatic islets. OPG is predominantly expressed in β-cells based on co-localization with insulin. Scale bars, 20 μm. (C) Expression of Opg , Rank , and Rankl transcripts in isolated islets treated without or with LPS as measured by qPCR (n = 3–6). (D) Effect of LPS treatment on Opg , Rank and Rankl expression in MIN6 cells (n = 3). (E) Insulin secretion by MIN6 cells. Cells were untreated (n = 6) or treated with 100 ng/ml soluble RANKL (sRANKL, n = 6), 100 ng/ml recombinant OPG (rOPG, n = 6), 10 μg/ml LPS (n = 6), both LPS and sRANKL (n = 6), or both LPS and rOPG (n = 6), and then stimulated with 3, 9.8, or 20 mM glucose. Levels of secreted insulin were normalized to total cell protein. Shown are means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: TaqMan probes specific for Tnfrsf11b (Mm00435452_m1), Tnfsf11 (Mm00441908_m1), Tnfrsf11 (Mm00437132_m1), and Gapdh (Mm03302249_g1, Mm99999915_g1) were purchased from TaqMan Assays-on-Demand Gene Expression Products (Life Technologies). mRNA levels were quantified using a standard curve generated with serially-diluted plasmids containing the PCR amplicon and normalized to Gapdh expression.

Techniques: Immunofluorescence, Expressing, Isolation, Recombinant

Journal: PLoS ONE

Article Title: Osteoprotegerin Regulates Pancreatic β-Cell Homeostasis upon Microbial Invasion

doi: 10.1371/journal.pone.0146544

Figure Lengend Snippet: When β-cells are exposed to inflammatory stimuli, they secrete OPG, which blocks RANKL-RANK signaling. Both osteoblast- and β-cell-derived OPG negatively regulates insulin secretion. Lower panel was adopted from Wei and Karsenty (2015) . Glu, undercarboxylated. Gla, carboxylated.

Article Snippet: TaqMan probes specific for Tnfrsf11b (Mm00435452_m1), Tnfsf11 (Mm00441908_m1), Tnfrsf11 (Mm00437132_m1), and Gapdh (Mm03302249_g1, Mm99999915_g1) were purchased from TaqMan Assays-on-Demand Gene Expression Products (Life Technologies). mRNA levels were quantified using a standard curve generated with serially-diluted plasmids containing the PCR amplicon and normalized to Gapdh expression.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

doi: 10.1371/journal.pone.0194847

Figure Lengend Snippet: (A) Cells were cultured in an osteogenic medium for 7, 16, or 21 days to RANKL gene. RANKL genes expression was the highest at 16 days post osteoinduction as well as the osteogenic markers Osterix, Runx2, Collagen3A1, Osteopontin and BSP (Fig 6B-6F). Mesenchymal stem cells derived from human adipose tissue (hASCs). Osteoblasts cultured for 7 days with osteoinduction medium (Ost. 7 days). Osteoblasts cultured for 16 days with osteoinduction medium (Ost. 16 days). Osteoblasts cultured for 21 days with osteoinduction medium (Ost. 21 days). An ANOVA followed by Tukey's test was used to analyze the data. P <0.005.

Article Snippet: The expression levels of RANKL (Applied Biosystems TNFRSF11-Hs00243522_m1 assay), was performed using the TaqMan commercial qPCR kit (Invitrogen) according to the manufacturer's instructions.

Techniques: Cell Culture, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells

doi: 10.1371/journal.pone.0194847

Figure Lengend Snippet: (A) CD45RO. (B) CD105. (C) STRO-1. (D) The arrows indicate the perinuclear location of Nanog. (E) The arrows indicate the location of RANKL. (F) Levels of expression of markers. Cell images showing the expression of the markers CD 45RO, RANKL, Nanog, CD105, and STRO-1 in osteoblasts cultured in osteogenic medium for 16 days. Data are expressed as mean ± standard deviation. An ANOVA followed by Tukey’s test was used to analyze the data. P < 0.005.

Article Snippet: The expression levels of RANKL (Applied Biosystems TNFRSF11-Hs00243522_m1 assay), was performed using the TaqMan commercial qPCR kit (Invitrogen) according to the manufacturer's instructions.

Techniques: Expressing, Cell Culture, Standard Deviation