gdf15 growth differentiation factor 15 levels  (R&D Systems)

Journal: Molecular Metabolism

Article Title: Muscle weakness and mitochondrial stress occur before severe metastasis in a novel mouse model of ovarian cancer cachexia

doi: 10.1016/j.molmet.2024.101976

Figure Lengend Snippet: The effects of ID8 implantation on muscle mass, fat mass, spleen mass, GDF15 and gene expression of inflammation and atrogenes. Analysis of muscle mass at all time points in hindlimb muscles was completed (A, n = 22–24; soleus (SOL), extensor digitorum longus (EDL), plantaris (PLA), tibialis anterior (TA), gastrocnemius (GA) and quadriceps (QUAD)). Subcutaneous adipose mass in the inguinal fat depot (B, n = 9–12), serum GDF15 (C, n = 8–11) and spleen mass (D, n = 21–22) were also analyzed. mRNA content of inflammatory and atrophy markers interleukin-6 (IL-6), tumour necrosis factor – alpha (TNF-α), atrogin and muscle RING-finger protein-1 (MURF-1) were measured using quantitative PCR in the TA and diaphragm of all groups (E-H, n = 6–8). Results represent mean ± SD. Lettering denotes statistical significance when different from each other ( p < 0.05). C57BL/6J female mice ∼75 days post PBS injection as controls (CTRL); C57BL/6J female mice ∼45 days post ovarian cancer injection (45 Days); C57BL/6J female mice ∼75 days post ovarian cancer injection (75 Days); C57BL/6J female mice ∼90 days post ovarian cancer injection (90 Days). All data was analyzed using a one-way ANOVA or Kruskal–Wallis test when data did not fit normality. All ANOVAs were followed by a two-stage step-up method of Benjamini, Krieger and Yukutieli multiple comparisons test.

Article Snippet: GDF15 (Growth differentiation factor 15) levels were analyzed in serum using the mouse GDF-15 DuoSet ELISA kit according to the manufacturer's instructions (R&D Systems DY6385).

Techniques: Expressing, Muscles, Real-time Polymerase Chain Reaction, Injection

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). MLO-Y4 cells were treated with C4-2B conditioned-media (CM) (Left), VCaP CM (Right) or MLO-Y4 CM as control for 24 hours. CM and whole cell lysates were collected and subjected to cytokine array. (B). Spots were subjected to densitometry using Image J. Results are shown as change in optical density of the cells subjected to prostate cancer CM relative to Control CM. (C). Ovals indicate the differentially regulated proteins. The overlap indicates the cytokines regulated similarly in both VCaP and C4-2B cells. (D). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA was collected and subjected to RT-PCR for endoglin mRNA. (E). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA and proteins was collected and subjected to immunoblot for GDF15 protein (upper left figure) and RT-PCR for GDF15 mRNA (lower left graph). After 24 hours of cell culture, supernatants were collected and subjected to immunoblot (upper right figure) and ELISA (lower right graph) for GDF15 protein. Data are shown as the mean±SD of 3 independent experiments. ** P < 0.01.

Article Snippet: ELISA assays Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Migration (left figure) and invasion (right figure) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with the indicated concentrations of rGDF15 for 24 hours. The membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. (B). C4-2B cells (1.5×104 per well) were treated with the indicated concentrations of rGDF15 for 48 hours. Cell numbers were quantified using a hemocytometer. (C). Validation of GDF15 knockdown in MLO-Y4 cells: Total cell protein and RNA were collected and subject to immunoblot (left figure) and real-time PCR (middle graph), respectively. Supernatant was collected from the indicated cells after 24 hours of incubation and subjected to ELISA for GDF15 (right graph). (D). Upper Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM from the genetically-modified MLO-Y4 cells. In some cases, rGDF15 (500 pg/ml) was added as indicated. After 48 hours, cell numbers were quantified using a hemocytometer. Lower Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM with the addition of either anti-GDF15 antibody or isotype antibody from the genetically-modified MLO-Y4 cells. After 48 hours, cell numbers were quantified using a hemocytometer. (E). Migration (upper figures) and invasion (lower figures) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with CM from untreated or C4-2B CM pre-treated MLO-Y4 cells with knockdown (or control) of GDF15. In some cases, rGDF15 was added to the CM as indicated. After 24 hours, the membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. Data are shown as the mean±SD of 3 independent experiments. * P < 0.05; ** P < 0.01. (F). Total protein and RNA from the indicated cell lines were subjected to immunoblot (left figure) and real time PCR (right graph) for GFRAL. The PCR data are shown as the mean±SD of 3 independent experiments.

Article Snippet: ELISA assays Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Membrane, Staining, Light Microscopy, Biomarker Discovery, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Genetically Modified, Control

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). SCID mice injected with mixture of prostate cancer PC3-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left graph and middle figure) and radiography (right figure). The tumor-laden tissue area is delineated by all 4 arrows within each radiograph. Results are shown as mean±SEM. * P < 0.05. (B). SCID mice injected with mixture of prostate cancer C4-2B-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left and middle) and radiography (right). Results are shown as mean±SEM. * P < 0.05. (C). One set of mice for each tumor type and treatmen (n=5 per tumor and treatment) were sacrificed at day 14-post initial injection and the intratibial tumors were subjected to immunohistochemistry for Ki67, Caspase 3 and EGR1. Images (left) and quantification (right) of Ki67, Caspase 3 and EGR1 staining. * P < 0.05, ** P < 0.01. (D). One set of mice (n=5) were sacrificed at day 14 -post initial injection, the tibiae flash frozen, total RNA collected and subjected to qPCR for murine GDF15 and human EGR1 mRNA expression. ** P < 0.01. (E). A proposed model system for secreted GDF15 promoting prostate cancer progression. Prostate cancer cells disseminate to bone, interact with osteocytes to induce production and release of GDF15 into the bone microenvironment which enhances prostate cancer proliferation, migration and invasion resulting in progression of bone metastasis.. Furthermore, our finding of the GDF15 receptor, GFRAL, on the prostate cancer cells, suggests that GDF15 mediates its activity through GFRAL in the prostate cancer cells. Finally, GDF15 induced EGR1 signaling in the prostate cancer cells, suggesting that EGR1 may contribute to the pro-metastatic effects of GDF15. *The data for EGR1 activation was supported by our in vitro findings and consistent with our in vivo studies; however, it has not been evaluated critically in vivo as of yet.

Article Snippet: ELISA assays Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Injection, Imaging, Immunohistochemistry, Staining, Expressing, Migration, Activity Assay, Activation Assay, In Vitro, In Vivo

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Confirmation of siRNA-mediated GDF15 protein expression knockdown in mouse primary osteocytes cells by immunoblot (left figure) and ELISA (right graph). (B). SCID mice were subcutaneously injected with a C4-2B cells alone or a mixture of C4-2B cells and mouse primary osteocytes that had been pre-treated with siGDF15 or siControl. The ratio of cells injected was 10 C4-2B cells to 1 osteocyte. Subcutaneous tumor volume was measured using caliper. Results are reported as mean±SEM. (C). Subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl were stained using Ki67, Caspase 3 and EGR1. (D). Quantification of Ki67, Caspase 3 and EGR1 in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl. ** P < 0.01. (E). EGR1 protein expression in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl.

Article Snippet: ELISA assays Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Expressing, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). MLO-Y4 cells were treated with C4-2B conditioned-media (CM) (Left), VCaP CM (Right) or MLO-Y4 CM as control for 24 hours. CM and whole cell lysates were collected and subjected to cytokine array. (B). Spots were subjected to densitometry using Image J. Results are shown as change in optical density of the cells subjected to prostate cancer CM relative to Control CM. (C). Ovals indicate the differentially regulated proteins. The overlap indicates the cytokines regulated similarly in both VCaP and C4-2B cells. (D). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA was collected and subjected to RT-PCR for endoglin mRNA. (E). MLO-Y4 cells treated with MLO-Y4 CM, VCaP CM, C4-2B CM or PC3 CM. After 24 hours, total RNA and proteins was collected and subjected to immunoblot for GDF15 protein (upper left figure) and RT-PCR for GDF15 mRNA (lower left graph). After 24 hours of cell culture, supernatants were collected and subjected to immunoblot (upper right figure) and ELISA (lower right graph) for GDF15 protein. Data are shown as the mean±SD of 3 independent experiments. ** P < 0.01.

Article Snippet: Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Migration (left figure) and invasion (right figure) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with the indicated concentrations of rGDF15 for 24 hours. The membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. (B). C4-2B cells (1.5×104 per well) were treated with the indicated concentrations of rGDF15 for 48 hours. Cell numbers were quantified using a hemocytometer. (C). Validation of GDF15 knockdown in MLO-Y4 cells: Total cell protein and RNA were collected and subject to immunoblot (left figure) and real-time PCR (middle graph), respectively. Supernatant was collected from the indicated cells after 24 hours of incubation and subjected to ELISA for GDF15 (right graph). (D). Upper Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM from the genetically-modified MLO-Y4 cells. In some cases, rGDF15 (500 pg/ml) was added as indicated. After 48 hours, cell numbers were quantified using a hemocytometer. Lower Figure: C4-2B cells (1.5×104 per well) were treated with the indicated CM with the addition of either anti-GDF15 antibody or isotype antibody from the genetically-modified MLO-Y4 cells. After 48 hours, cell numbers were quantified using a hemocytometer. (E). Migration (upper figures) and invasion (lower figures) were assessed using a transwell assay. C4-2B cells (1.5×105 per cells) were treated with CM from untreated or C4-2B CM pre-treated MLO-Y4 cells with knockdown (or control) of GDF15. In some cases, rGDF15 was added to the CM as indicated. After 24 hours, the membrane was stained using differential Quick staining kit and photographed under light microscopy (20x). The numbers of migrating (no Matrigel on membrane) and invading (Matrigel present on membrane) cells were counted in five random fields for each insert. Data are shown as the mean±SD of 3 independent experiments. * P < 0.05; ** P < 0.01. (F). Total protein and RNA from the indicated cell lines were subjected to immunoblot (left figure) and real time PCR (right graph) for GFRAL. The PCR data are shown as the mean±SD of 3 independent experiments.

Article Snippet: Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Migration, Transwell Assay, Membrane, Staining, Light Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Genetically Modified

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). SCID mice injected with mixture of prostate cancer PC3-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left graph and middle figure) and radiography (right figure). The tumor-laden tissue area is delineated by all 4 arrows within each radiograph. Results are shown as mean±SEM. * P < 0.05. (B). SCID mice injected with mixture of prostate cancer C4-2B-luc cells and siRNA (siGDF15 or siControl) into right tibia followed by an additional intratibial injection of siRNA at day 7. Tumor burden was evaluated using bioluminescent imaging (left and middle) and radiography (right). Results are shown as mean±SEM. * P < 0.05. (C). One set of mice for each tumor type and treatmen (n=5 per tumor and treatment) were sacrificed at day 14-post initial injection and the intratibial tumors were subjected to immunohistochemistry for Ki67, Caspase 3 and EGR1. Images (left) and quantification (right) of Ki67, Caspase 3 and EGR1 staining. * P < 0.05, ** P < 0.01. (D). One set of mice (n=5) were sacrificed at day 14 -post initial injection, the tibiae flash frozen, total RNA collected and subjected to qPCR for murine GDF15 and human EGR1 mRNA expression. ** P < 0.01. (E). A proposed model system for secreted GDF15 promoting prostate cancer progression. Prostate cancer cells disseminate to bone, interact with osteocytes to induce production and release of GDF15 into the bone microenvironment which enhances prostate cancer proliferation, migration and invasion resulting in progression of bone metastasis.. Furthermore, our finding of the GDF15 receptor, GFRAL, on the prostate cancer cells, suggests that GDF15 mediates its activity through GFRAL in the prostate cancer cells. Finally, GDF15 induced EGR1 signaling in the prostate cancer cells, suggesting that EGR1 may contribute to the pro-metastatic effects of GDF15. *The data for EGR1 activation was supported by our in vitro findings and consistent with our in vivo studies; however, it has not been evaluated critically in vivo as of yet.

Article Snippet: Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Injection, Imaging, Immunohistochemistry, Staining, Expressing, Migration, Activity Assay, Activation Assay, In Vitro, In Vivo

Journal: Oncogene

Article Title: Prostate cancer promotes a vicious cycle of bone metastasis progression through inducing osteocytes to secrete GDF15 that stimulates prostate cancer growth and invasion

doi: 10.1038/s41388-019-0736-3

Figure Lengend Snippet: (A). Confirmation of siRNA-mediated GDF15 protein expression knockdown in mouse primary osteocytes cells by immunoblot (left figure) and ELISA (right graph). (B). SCID mice were subcutaneously injected with a C4-2B cells alone or a mixture of C4-2B cells and mouse primary osteocytes that had been pre-treated with siGDF15 or siControl. The ratio of cells injected was 10 C4-2B cells to 1 osteocyte. Subcutaneous tumor volume was measured using caliper. Results are reported as mean±SEM. (C). Subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl were stained using Ki67, Caspase 3 and EGR1. (D). Quantification of Ki67, Caspase 3 and EGR1 in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl. ** P < 0.01. (E). EGR1 protein expression in subcutaneous tumors from SCID mice implanted with C4-2B cells and mouse primary osteocytes pre-treated with siGDF15 or siControl.

Article Snippet: Mouse GDF15 level in supernatants of MLO-Y4 cells and mouse primary osteocytes, and mouse TGF-β level in recombinant mouse GDF15 were analyzed by antibody sandwich ELISA (R&D Systems, Minneapolis, MN) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining