Journal: JBMR Plus

Article Title: Paradoxical combination of osteosclerosis and osteopenia in an adult woman with biallelic TNFRSF11A loss-of-function variants escaping nonsense-mediated decay

doi: 10.1093/jbmrpl/ziae179

Figure Lengend Snippet: Delayed formation and impaired function of mutated osteoclasts. (A) Staining of control and patient osteoclast cultures demonstrated successful formation of osteoclasts on glass surfaces, but impaired formation on tissue-culture plastic. Nuclei are stained blue with DAPI, actin is stained green with phalloidin, and TRAP is shown in red through enzymatic staining. (B) Colorimetric analysis of TRAP activity in cell culture supernatants from bone resorption assays revealed no significant reduction in TRAP secretion ( n = 4 controls, n = 1 patient-derived osteoclasts; single experiment) each point represents an individual measurements. (C) Representative images of resorption pits (indicated by red arrowheads) and quantification of eroded surfaces show minimal residual bone resorption compared to control cells (combined results from 3 independent experiments). (D) The release of CTx-I into cell culture supernatants from the bone resorption experiments, measured by ELISA, was significantly reduced in patient samples ( n = 3 controls, n = 1 patient-derived osteoclasts, single experiment).

Article Snippet: Cells were cultured in osteoclast medium, composed of MEM Alpha Eagle modification (PAN Biotech), supplemented with 10% FBS Superior (Sigma Aldrich), 1% GlutaMax and 1% penicillin/streptomycin (both Gibco) and 30 ng/mL M-CSF (Bio-Techne).

Techniques: Staining, Control, Activity Assay, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: JBMR Plus

Article Title: Paradoxical combination of osteosclerosis and osteopenia in an adult woman with biallelic TNFRSF11A loss-of-function variants escaping nonsense-mediated decay

doi: 10.1093/jbmrpl/ziae179

Figure Lengend Snippet: The primary transcript escapes NMD. (A) the fold change in TNFRSF11A expression was determined by qRT-PCR, showing only a slight reduction compared to the average expression in control samples. (B) the expression levels of osteoclast differentiation markers CTSK, MMP9, and NFATC1, also quantified by qRT-PCR, exhibited reduced expression associated with differentiation. Each point represents 3 technical replicates from 2 different control samples compared to technical replicates from patient samples. (C) Gel electrophoresis of amplified cDNAs from both control and patient samples revealed no significant secondary transcripts or splice abnormalities. (D) Sanger sequencing of the amplified cDNA confirmed the presence of both mutated mRNAs in a heterozygous state, suggesting that relevant nonsense-mediated decay (NMD) does not occur.

Article Snippet: Cells were cultured in osteoclast medium, composed of MEM Alpha Eagle modification (PAN Biotech), supplemented with 10% FBS Superior (Sigma Aldrich), 1% GlutaMax and 1% penicillin/streptomycin (both Gibco) and 30 ng/mL M-CSF (Bio-Techne).

Techniques: Expressing, Quantitative RT-PCR, Control, Nucleic Acid Electrophoresis, Amplification, Sequencing

Journal: Biomarker Research

Article Title: GDF15 propeptide promotes bone metastasis of castration-resistant prostate cancer by augmenting the bone microenvironment

doi: 10.1186/s40364-024-00695-6

Figure Lengend Snippet: Intracellular and extracellular molecular dynamics of GDPP in HOB and OSC15C. ( A ) Western blot analysis of cell lysates revealed the expression of pro-GDF15 in osteoblasts (MC3T3-E1 and HOB), osteoclasts (OSC14C and OSC15C), and osteocytes (MLO-Y4). Furthermore, the images depict the differentiation of OSC15C cells into mature osteoclasts. Scale bar, 500 μm. ( B ) ELISA-based evaluation of the concentration of GDPP in the culture medium of HOB and OSC15C. ( C ) Immunohistochemical staining of pro-GDF15 in human PCa cells, as well as in osteoblasts, osteoclasts, and osteocytes within the BM of PCa patients. Black arrows indicate osteocytes. Scale bar, 250 μm

Article Snippet: MC3T3-E1 cells (RIKEN BRC Cell Bank) were maintained in basal culture medium (αMEM, Nacalai Tesque) with 10% FBS, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin, MLO-Y4 cells (Kerafast) were cultured on type I collagen-coated dishes (Corning) and maintained in basal culture medium (αMEM with 5% heat inactivated FBS, 5% calf serum, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin), and OSC14C cells (Cosmo Bio) were suspended in osteoclast culture medium (OSCMW and OSCMM, Cosmo Bio).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunohistochemical staining, Staining

Journal: Biomarker Research

Article Title: GDF15 propeptide promotes bone metastasis of castration-resistant prostate cancer by augmenting the bone microenvironment

doi: 10.1186/s40364-024-00695-6

Figure Lengend Snippet: Functional analysis of GDPP in HOB and OSC15C. ( A ) Western blot analysis of the expression of GDPP in HOB. HOB was transfected with negative control siRNA or siGDF15. ( B ) Cell growth curve of HOB lines transfected with siGDF15 or negative control siRNA that were seeded with or without rGDPP, and proliferation was examined by MTS assays. ( C ) Expression analysis of genes related to differentiation of HOB. Total RNA was isolated from HOB treated with or without rGDPP. mRNA expression of RUNX2 , OSX , ATF4 and ALP was evaluated using quantitative real-time PCR analysis. The expression of each gene was normalized to GAPDH expression. ( D ) Analysis of ALP activity in HOB. Representative images of ALP staining, OD values and ALP + area percentages of HOB with or without rGDPP are shown. ( E ) Bone mineralization analysis of HOB. Representative images of HOB mineralization detected by Alizarin Red S staining, with OD values and area percentages with or without GDPP are shown. ( F ) Expression analysis of genes related to differentiation of OSC15C. Total RNA was isolated from OSC15C treated with or without rGDPP after differentiation into mature osteoclasts. mRNA expression of NFATc1 , DC-STAMP , CTSK and TRAP was evaluated using quantitative real-time PCR analysis. The expression of each gene was normalized to GAPDH expression. ( G ) TRAP staining analysis of OSC15C. OSC15C was treated with or without rGDPP and stained for TRAP. Scale bar, 500 μm. ( H ) Pit formation analysis of OSC15C. OSC15C was seeded into bone resorption assay plates and treated with or without rGDPP. Representative images of resorption pit formation and the percentage of resorbed area (bright area) were quantified. Data are expressed as the mean ± SD, and statistical analyses were performed using the Tukey‒Kramer method (* p < 0.05, ** p < 0.01; n.s., not significant). Scale bar, 500 μm

Article Snippet: MC3T3-E1 cells (RIKEN BRC Cell Bank) were maintained in basal culture medium (αMEM, Nacalai Tesque) with 10% FBS, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin, MLO-Y4 cells (Kerafast) were cultured on type I collagen-coated dishes (Corning) and maintained in basal culture medium (αMEM with 5% heat inactivated FBS, 5% calf serum, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin), and OSC14C cells (Cosmo Bio) were suspended in osteoclast culture medium (OSCMW and OSCMM, Cosmo Bio).

Techniques: Functional Assay, Western Blot, Expressing, Transfection, Negative Control, Isolation, Real-time Polymerase Chain Reaction, Activity Assay, Staining

Journal: Biomarker Research

Article Title: GDF15 propeptide promotes bone metastasis of castration-resistant prostate cancer by augmenting the bone microenvironment

doi: 10.1186/s40364-024-00695-6

Figure Lengend Snippet: Functional analysis of GDPP in a xenograft model of human PCa cells within the bone microenvironment. ( A ) Schema of the experimental procedure. PC3-Luc2 cells were directly injected into the tibia of male NOD/SCID mice. The mice were subsequently subcutaneously administered rGDPP ( n = 10), while the control group received saline injections ( n = 10). Weekly imaging using an IVIS was performed. ( B ) Macroscopic image of a tibial bone tumor 50 days following the injection of PC3-Luc2 cells. ( C ) µCT and 3D modeling images demonstrated that the tumor cells had invaded the tibial bone. Scale bar, 500 μm. ( D ) Histological staining using hematoxylin and eosin revealed the infiltration of tumor cells into the tibial bone. Scale bar, 500 μm. ( E ) Weekly bioluminescence imaging captured changes in the tumor growth pattern of PC3-Luc2 cells within the tibia over time ( n = 20). ( F ) The comparison of bone tumor volume 50 days after intratibial injection ( n = 20). ( G ) Representative images of immunohistochemical staining for Ki67 in bone tumor and quantitative comparison of Ki67- positive cells (control: n = 10, GDPP: n = 10). Scale bar, 100 μm. ( H ) The number of OC-positive cells was quantified per unit trabecular bone surface ( n = 20). Black arrows indicate osteoblasts. Scale bar, 500 μm. ( I ) The number of TRAP-positive cells was quantified per unit trabecular bone surface ( n = 20). Black arrows indicate osteoclasts. Scale bar, 500 μm. Data are expressed as the mean ± SD, and statistical analyses were performed using a Mann–Whitney U test (* p < 0.05, ** p < 0.01)

Article Snippet: MC3T3-E1 cells (RIKEN BRC Cell Bank) were maintained in basal culture medium (αMEM, Nacalai Tesque) with 10% FBS, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin, MLO-Y4 cells (Kerafast) were cultured on type I collagen-coated dishes (Corning) and maintained in basal culture medium (αMEM with 5% heat inactivated FBS, 5% calf serum, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin), and OSC14C cells (Cosmo Bio) were suspended in osteoclast culture medium (OSCMW and OSCMM, Cosmo Bio).

Techniques: Functional Assay, Injection, Control, Saline, Imaging, Staining, Comparison, Immunohistochemical staining, MANN-WHITNEY

Journal: Biomarker Research

Article Title: GDF15 propeptide promotes bone metastasis of castration-resistant prostate cancer by augmenting the bone microenvironment

doi: 10.1186/s40364-024-00695-6

Figure Lengend Snippet: Graphical abstract showing the role of GDPP in promoting bone metastasis in CRPC patients. In CRPC patients, GDPP augments the tumor microenvironment of BM and is an accurate biomarker for BM. The illustration on the left shows the schema in which PCa cells, osteoblasts, and osteoclasts secrete GDPP, PCa progresses, and osteoblasts and osteoclasts also proliferate, each of which exacerbates BM. The illustration on the right shows that blood GDPP levels in CRPC patients with BM reflect BSI accurately and are a very useful biomarker

Article Snippet: MC3T3-E1 cells (RIKEN BRC Cell Bank) were maintained in basal culture medium (αMEM, Nacalai Tesque) with 10% FBS, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin, MLO-Y4 cells (Kerafast) were cultured on type I collagen-coated dishes (Corning) and maintained in basal culture medium (αMEM with 5% heat inactivated FBS, 5% calf serum, 100 U/mL penicillin G, and 0.1 μg/mL streptomycin), and OSC14C cells (Cosmo Bio) were suspended in osteoclast culture medium (OSCMW and OSCMM, Cosmo Bio).

Techniques: Biomarker Discovery