Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Impact of Receptor Activator of Nuclear factor κB (RANK) over-expression in osteosarcoma cells on tumor growth and the number of lung metastases. No significant difference was observed concerning tumor growth regardless of the cell-line considered (K-HOS ( A ), MOS-J PG1 ( C , E )) or the immune status of the host mouse strain (Nude ( A , C ) or C57BL/6 ( E )). However, regarding the number of lung metastases, a significant increase was observed regardless of the RANK over-expressing cell-line considered, in immune-deficient Nude mice ( B , D ) but not in immune-competent C57BL/6 mice ( F ). Moreover, injections of a Receptor Activator of Nuclear factor κB Ligand (RANKL)-blocking antibody (IK22.5) in Nude mice made it possible to reduce the number of lung metastases obtained with RANK expressing PG1 ( D ). n: number of mice in each group. Growth curves ( A , C , E ) are shown as the mean ± SEM. All data analysis was performed with the Kruskal Wallis test. ns: not significant; **: p < 0.01; ****: p < 0.0001.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Over Expression, Expressing, Blocking Assay

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Consequences of RANK over-expression in osteosarcoma cells on cell viability ( A ) and migration ( B ). A moderate decrease (tendency) in the cell viability in response to the addition of RANKL to the culture medium was observed for both RANK over-expressing HOS cells and PG1 cells ( A ). For cell migration, which was evaluated with Boyden chambers, no significant impact of RANK over-expression and RANKL stimulation was observed, regardless of the cell-line considered ( B ).

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Over Expression, Migration, Expressing

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Impact of RANK over-expression in osteosarcoma cells on bone structure ( A ) and bone parameters ( B , C ). Whatever tumor cell-line or host mouse strain was considered, representative three dimension images did not make it possible to observe any difference concerning bone resorption and osteoid tissue formation in relationship with RANK over-expression ( A ). As expected, IK22-5 RANKL blocking antibody injections made it possible to protect the bone from resorption ( A ). The bone parameters BS/TV, BS/BV and BV/TV were measured on 450 sections whose positions are presented in ( B ). The results showed no difference between the cells over-expressing or not RANK, whatever parameter was considered ( C ). The IK22-5 RANKL blocking antibody made it possible to significantly reduce the BS/TV and BS/BV parameters, with no impact on the BV/TV parameter. A significant difference was observed concerning the BV/TV parameter only in C57BL/6 mice when comparing contra-lateral control tibias (CT) and tibias with tumors independently of RANK over-expression by tumor cells. The data in ( C ) are shown as the mean ± SD. Data analyses were performed using the Kruskal Wallis test. ns: no significant. *: p < 0.05. **: p < 0.01. ***: p < 0.001.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Over Expression, Blocking Assay, Expressing, Control

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: RT-qPCR analysis of the impact of adding RANKL to cultures of HOS cells that are native or over-expressing RANK.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Migration

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Consequences of T-cell specific invalidation of Rankl in recipient mice on RANK over-expressing PG1 tumor growth, number of metastases and bone parameters. Rankl depletion in mouse T-cells (LCK-CRE) was validated by PCR on gDNA extracted from tails and T cells ( A ). PCR data confirmed the effective recombination, specifically in T-cells. After injection of one million PG1 cells over-expressing RANK, tumor growth ( B ) and the number of lung metastases formed ( C ) were compared between mice invalidated for Rank in T-cells ( n = 4) and control mice ( n = 5) showing no difference. Representative three-dimensional images of tibias with tumors (and their controls, C ) did not make it possible to observe any differences regarding bone resorption or tumor osteoid tissue formation ( D ). Micro-CT analysis of the BS/TV (mm-1), BS/BV (mm-1) and BV/TV (%) parameters of the tibias revealed no differences. However, an increase in BV/TV was observed comparatively to contralateral safe tibias, independently of the mouse genotype ( E ). The data in ( E ) are shown as the mean ± SD. Data analyses were performed using the Kruskal Wallis test. ns: no significant. *: p < 0.05.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Expressing, Injection, Control, Micro-CT

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Tissue array analysis of RANK, RANKL and OPG expressions in a cohort of 50 biopsies of patients with ( n = 28) and without ( n = 22) metastases at time of diagnosis. Representative views of the different immuno-stainings are presented ( A ) with enlargement inset to clearly show the stained osteosarcoma cells. Statistical analyses (Student test) of the staining based on the percentage of stained osteosarcoma cells revealed a significantly lower number of positive cells for RANKL and OPG in the metastatic patient group, while no difference was observed between the two groups concerning the number of RANK-expressing cells ( B ). Statistical analysis of the three ratios between these factors, established individually for each patient, showed that only the OPG/RANK ratio was significantly different between the two groups with lower values in the metastatic patient group ( C ). ns: not significant. *: p < 0.05. p values are given for each test.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques: Biomarker Discovery, Staining, Expressing

Journal: Cancers

Article Title: The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

doi: 10.3390/cancers10110398

Figure Lengend Snippet: Schematic representation of the different stages of osteosarcoma from initiation to lung metastasis, and the implications of intrinsic and extrinsic RANKL and RANK in these different stages. +: implication revealed; 0: No implication; *: part of the vicious cycle; **: part of the escape bolstering; ∞: according to Chen et al., 2016.

Article Snippet: Immunohistochemistry was performed with antibodies against RANK (Goat polyclonal antibody AF683 from R&D Systems (Abingdon, UK), dilution 1/20), RANKL (Goat polyclonal antibody AF626 from R&D Systems dilution 1/20) and OPG (Rabbit polyclonal antibody Ab9986 from Abcam, Cambridge, UK, dilution 1/50) following the protocol previously described [ ].

Techniques:

Journal: PLoS ONE

Article Title: Differences in PGE 2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

doi: 10.1371/journal.pone.0098517

Figure Lengend Snippet: U373-CD14 cells were transfected with control siRNA or with siRNA targeting TRIF or IRF3 (A, C), or TRAM (B, D). Transfected cells were left untreated (solid bars), or were incubated with LPS at 1 ng/ml (shaded bars), with rIL-1β at 100 ng/ml for 18 h (striped bars), or were primed for 1 h with LPS and incubated with rIL-1β for additional 18 h (open bars). Cells were collected and COX-2 mRNA (A, B) and mPGES-1 mRNA (C, D) expressions were assayed by qPCR. The data are shown as fold increase over control PCR with primers specific to β-actin , run in the same samples. PCR was performed using triplicate wells and the data are shown as mean fold increase ± STDEV. This is representative of three experiments.

Article Snippet: For the Western Blotting of the cell lysates prepared from transfected cells and resolved in SDS-PAGE, the following antibodies were used: goat anti-human TRIF Ab (Cat AF6216, R&D Systems), rabbit anti-human TRAM Ab (Cat ab17221, Abcam), and rabbit anti-human IRF3 Ab (Cat 4302, Cell Signaling).

Techniques: Transfection, Control, Incubation