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rabbit polyclonal anti human gdf15  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti human gdf15
    Correlations between <t> GDF15 </t> IHC score and clinical characteristics in patients with breast cancer
    Rabbit Polyclonal Anti Human Gdf15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti human gdf15/product/Cell Signaling Technology Inc
    Average 91 stars, based on 15 article reviews
    rabbit polyclonal anti human gdf15 - by Bioz Stars, 2026-03
    91/100 stars

    Images

    1) Product Images from "Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling"

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21765

    Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer
    Figure Legend Snippet: Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer

    Techniques Used:

    (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).
    Figure Legend Snippet: (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Control, Clone Assay, Staining, Flow Cytometry

    (A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.
    Figure Legend Snippet: (A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Techniques Used: Plasmid Preparation, Control, Clone Assay, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Stable Transfection, Membrane, Staining

    (A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.
    Figure Legend Snippet: (A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.

    Techniques Used: Plasmid Preparation, Control, Clone Assay, Western Blot, Imaging, Software, Stable Transfection, Standard Deviation, Staining

    (A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.
    Figure Legend Snippet: (A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Clone Assay, Standard Deviation, Staining

    (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.
    Figure Legend Snippet: (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

    Techniques Used: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

    (A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.
    Figure Legend Snippet: (A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.

    Techniques Used: Recombinant, Western Blot, Control, Imaging, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.
    Figure Legend Snippet: (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

    Techniques Used: Transfection, Control, Recombinant, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Staining

    (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.
    Figure Legend Snippet: (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

    Techniques Used: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Knockdown, Standard Deviation

    GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.
    Figure Legend Snippet: GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.

    Techniques Used: Expressing



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    Image Search Results


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    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: Induction of GDF15 and an activation of its downstream signaling molecule during the differentiation of hESCs into BAs. ( a ) Results of microarray analyses regarding TGFB/BMP/GDF family are shown. The vertical and horizontal axes indicate the signal intensity and the time duration after an induction of differentiation, respectively. The cubic polynomial approximation curve is shown on the graph. ( b ) Western blotting studies for GDF15 protein and beta-tubulin (TUBB) proteins using the differentiating hESCs or human umbilical vein endothelial cells (HUVEC) as a positive control ( upper ), Western blotting for phosphorylated SMAD2 (pSMAD2), total SMAD2, and TUBB proteins at indicated time points ( lower ) are shown. ( c ) qRT-PCR for PAI mRNA. The vertical axis indicates “fold increments” when compared to the data of the samples at 0 h. Data are shown as mean ± SD ( n = 3). ( d ) Western blotting for PAI precursor protein and mature PAI protein. ( e ) qRT-PCR for GDF15 mRNA. The vertical axis indicates the fold increments compared to the data of the samples at Day 3. Data are shown as mean ± SD ( n = 3). ( f ) Immunostaining studies for GDF15 protein (green) using the terminally differentiated samples at Day 10. Nuclei were counterstained by DAPI (blue).

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Activation Assay, Microarray, Western Blot, Positive Control, Quantitative RT-PCR, Immunostaining

    GDF15 - /- mice have normal brown adipose tissue (BAT) and are metabolically healthy. Results of the PCR-based genotyping of the offspring ( a ). GDF15 mRNA expressions ( b , left ) and macroscopic features ( b , right ) of the iBAT of wild-type (WT) or GDF15 -/- mice are shown. There were no significant differences in body weight ( c ) or glucose tolerance, which was evaluated by an oral glucose tolerance test (OGTT) ( d ), between WT and GDF15 - /- mice (WT: n = 8; GDF15 -/- : n = 15) after an 8-week high-fat diet. Data are shown as mean ± SD.

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: GDF15 - /- mice have normal brown adipose tissue (BAT) and are metabolically healthy. Results of the PCR-based genotyping of the offspring ( a ). GDF15 mRNA expressions ( b , left ) and macroscopic features ( b , right ) of the iBAT of wild-type (WT) or GDF15 -/- mice are shown. There were no significant differences in body weight ( c ) or glucose tolerance, which was evaluated by an oral glucose tolerance test (OGTT) ( d ), between WT and GDF15 - /- mice (WT: n = 8; GDF15 -/- : n = 15) after an 8-week high-fat diet. Data are shown as mean ± SD.

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Metabolic Labelling

    GDF15 - /- hESCs normally differentiated into BAs. GDF15 -/ - hESCs were subjected to differentiation into BAs. The expressions of the two major BA-selective genes, PRDM16 and PPARG , were properly induced in GDF15 -/- hESCs despite the lack of GDF15 expression ( n = 3 experiments) ( a ). There were no differences in the activities of the BA-SUP to enhance insulin secretion from murine pancreatic beta cells between WT and GDF15 -/- hESCs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: GDF15 - /- hESCs normally differentiated into BAs. GDF15 -/ - hESCs were subjected to differentiation into BAs. The expressions of the two major BA-selective genes, PRDM16 and PPARG , were properly induced in GDF15 -/- hESCs despite the lack of GDF15 expression ( n = 3 experiments) ( a ). There were no differences in the activities of the BA-SUP to enhance insulin secretion from murine pancreatic beta cells between WT and GDF15 -/- hESCs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Expressing

    IL6 expressions were reduced by the absence of GDF15 . The expression of IL6 mRNA was measured in the iBATs of WT and GDF15 -/- mice ( n = 3 mice) ( a ) or in the hESCs that were subjected to the differentiation into BAs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: IL6 expressions were reduced by the absence of GDF15 . The expression of IL6 mRNA was measured in the iBATs of WT and GDF15 -/- mice ( n = 3 mice) ( a ) or in the hESCs that were subjected to the differentiation into BAs ( n = 3 experiments) ( b ). Data are shown as mean ± SD.

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Expressing

    GDF15 expressions were blunted by the absence of IL6. Using three clones of IL6 - /- hESCs, PRDM16 and GDF15 expressions were examined in the course of differentiation into BAs. Despite the induction of PRDM16 ( upper panels ), GDF15 mRNA was markedly repressed ( middle panels ) ( n = 3 experiments) by the absence of IL6 ( lower panels ). Data are shown as mean ± SD.

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: GDF15 expressions were blunted by the absence of IL6. Using three clones of IL6 - /- hESCs, PRDM16 and GDF15 expressions were examined in the course of differentiation into BAs. Despite the induction of PRDM16 ( upper panels ), GDF15 mRNA was markedly repressed ( middle panels ) ( n = 3 experiments) by the absence of IL6 ( lower panels ). Data are shown as mean ± SD.

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Clone Assay

    GDF15 is required for long-term survival of BAT grafts. The iBATs of either GDF15 -/- or WT mice were intraperitoneally transplanted into GDF15 - /- mice according to the method presented by Stanford et al. . After 12 weeks, the grafts were removed. ( a ) Macroscopic observations. The BAT grafts of GDF15 -/- mice were shrunk and no angiogenesis was detected ( upper ), whereas those of WT mice retained their sizes and neo-angiogenesis was detected in the soft tissue around the grafts ( lower ). ( b ) Histological observations. Tissue slices of BAT grafts derived from GDF15 - /- mice ( left ) and those of WT mice ( right ) were subjected to HE staining. Structures of the elastic membranes of the arteries were well preserved even in GDF15 -/- -derived BAT grafts ( left , arrows).

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: GDF15 is required for long-term survival of BAT grafts. The iBATs of either GDF15 -/- or WT mice were intraperitoneally transplanted into GDF15 - /- mice according to the method presented by Stanford et al. . After 12 weeks, the grafts were removed. ( a ) Macroscopic observations. The BAT grafts of GDF15 -/- mice were shrunk and no angiogenesis was detected ( upper ), whereas those of WT mice retained their sizes and neo-angiogenesis was detected in the soft tissue around the grafts ( lower ). ( b ) Histological observations. Tissue slices of BAT grafts derived from GDF15 - /- mice ( left ) and those of WT mice ( right ) were subjected to HE staining. Structures of the elastic membranes of the arteries were well preserved even in GDF15 -/- -derived BAT grafts ( left , arrows).

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques: Derivative Assay, Staining

    Model. IL6 induces GDF15 in the former phase of the differentiation of hESCs into BAs, whereas GDF15 and IL6 create a mutually inducing loop in the later phase. The GDF15, which may be secreted as a component of large-sized EVs, and IL6 potentiate and stabilize neovascularization, guaranteeing long-term survival of BAT grafts.

    Journal: Cells

    Article Title: New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6

    doi: 10.3390/cells9061365

    Figure Lengend Snippet: Model. IL6 induces GDF15 in the former phase of the differentiation of hESCs into BAs, whereas GDF15 and IL6 create a mutually inducing loop in the later phase. The GDF15, which may be secreted as a component of large-sized EVs, and IL6 potentiate and stabilize neovascularization, guaranteeing long-term survival of BAT grafts.

    Article Snippet: The 1st antibody reaction was performed by using either a rabbit polyclonal anti-human GDF15 antibody (sc66904; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a normal IgG (sc-2027; Santa Cruz Biotechnology, Dallas, TX, USA), and the 2nd antibody reaction was performed by using an Alexa Fluor ® 594-conjugated goat anti-rabbit IgG antibody (A11036; Thermo Fisher Scientific Inc., Waltham, MA, USA).

    Techniques:

    Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: Correlations between GDF15 IHC score and clinical characteristics in patients with breast cancer

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques:

    (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) Western blotting ( above ) and real-time PCR ( graph ) for GDF15 in BT474, JIMT1 and MDA-MB-231 (MDA231) breast cancer cell lines. PCR values reflect fold change in GDF15 transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (B) Real-time PCR for GDF15 in BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in GDF15 transcript level normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C-D) BT474 parental, pCMV empty vector control clone (pCMV), and GDF15 stable clones 2 and 3 (C2, C3) were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells in each cell cycle phase is shown per cell line (C) (white, G0/G1; gray, S; black, G2/M). Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. Representative cell cycle histograms are shown per line (D).

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Plasmid Preparation, Control, Clone Assay, Staining, Flow Cytometry

    (A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) Total protein whole-cell lysates were collected from BT474 pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for E-Cadherin, N-Cadherin, vimentin, and FoxM1; actin was measured as a loading control. Blots were repeated at least three times, and representative blots are shown. (B) Real-time PCR for Snail, Zeb-1 and Slug in BT474 parental, pCMV stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Values reflect fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated at least 3 times. (C) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones 3 and 5 (C3 and C5) were imaged at 10× magnification to evaluate changes in morphology. (D) Representative images of spheroid cultures are shown for BT474 parental and GDF15 stable clone 2 (C2). (E) BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5) were plated in basement membrane matrix mimic (Matrigel)-coated Boyden chambers in serum-free media; 10% FBS was added to the well below each chamber as a chemo-attractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Plasmid Preparation, Control, Clone Assay, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Stable Transfection, Membrane, Staining

    (A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) Total protein lysates were collected from BT474 stable empty vector control clone (pCMV) and GDF15 stable clones 2, 3, and 5 (C2, C3, and C5). Western blots were performed for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) BT474 GDF15 stable clone 2 (C2) cells were treated with normal mouse IgG control or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 48 hours. Western blots of total protein lysates were performed for total IGF-1R, vimentin, FoxM1, and E-Cadherin. Bar graphs show quantification relative to actin loading control, and was performed using Odyssey Li-Cor imaging software. Error bars represent standard deviation between triplicates; experiments were performed at least 3 times. (C) BT474 stable empty vector control clone (pCMV) and BT474 GDF15 stable clone 5 (C5) cells were pre-treated with normal mouse IgG or 0.25 μg/mL alpha IR3 (aIR3) IGF-1R monoclonal antibody for 24 hours. Cells were then seeded in Matrigel-coated Boyden chambers in serum-free media plus control IgG or aIR3; 10% FBS was added to the well as a chemo-attractant. After 24 hours of invasion, cells in chambers were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Plasmid Preparation, Control, Clone Assay, Western Blot, Imaging, Software, Stable Transfection, Standard Deviation, Staining

    (A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) Real-time PCR of total RNA from BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3, and C5) for MMP2 ( left graph ) and MMP9 ( right graph ). Values reflect average fold in transcript normalized to internal control RPLPO relative to pCMV group. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (B) BT474 pCMV empty vector control clone (pCMV) and GDF15 stable clones (C2, C3 and C5) were plated in serum-free media in Matrigel-coated Boyden chambers and treated with vehicle control or 1 μg pan-MMP inhibitor GM6001 for 24 hours, after which cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Clone Assay, Standard Deviation, Staining

    (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV) and GDF15 stable clone 5 (C5) were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 48 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line; student’s t-test, ** p<0.005. (B) BT474 GDF15 stable clone 5 (C5) cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (FoxM1) for 48 hours. Real-time PCR was performed for FoxM1, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times, ** p<0.005.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Standard Deviation

    (A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) JIMT1 cells were serum starved for 24 hours, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for p-Tyr1131 IGF-1R and total IGF-1R; actin was probed as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (B) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total IGF-1R with actin as loading control. Experiments were repeated 3 times; representative blots are shown. Quantification (shown beneath each band) was normalized to actin and performed using Odyssey Li-Cor imaging software. (C) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then stimulated with 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for SNAIL, SLUG, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (D) JIMT-1 cells were pretreated for 24 hours with control IgG or 0.25 μg/ml alpha IR3 (aIR3) in serum-free media, and then seeded in Matrigel-coated Boyden chambers in serum-free media with 10% FBS in the lower chamber as chemoattractant. Drug treatment was continued (in indicated samples), and 2 or 20 ng/mL of recombinant human GDF15 (rhGDF15) was added to the chambers of treatment groups where indicated. After 24 hours of invasion, photos were taken at 20× magnification; representative photos are shown. The number of invaded cells is shown per group; error bars represent standard deviation between triplicate samples, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Recombinant, Western Blot, Control, Imaging, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Western blots of total protein lysates are shown for total FoxM1 with actin as loading control. (B) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then stimulated with 2 or 20 ng/mL recombinant human GDF15 (rhGDF15) for another 24 hours. Real-time PCR was performed for Snail, Slug, MMP2, and MMP9. Values reflect average fold in transcript normalized to internal control RPLPO. Error bars represent standard deviation between triplicate samples; experiments were repeated 3 times. (C) JIMT-1 cells were transfected with 100 nM control siRNA (siCtrl) or FoxM1 siRNA (siFoxM1) for 24 hours, and then plated in serum-free media in Matrigel-coated Boyden chambers. Recombinant human GDF15 (rhGDF15; 2 or 20 ng/mL) was added to lower chambers where indicated. After 24 hours, cells were fixed and stained; representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, ** p<0.005, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Transfection, Control, Recombinant, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Staining

    (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: (A) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15). After 24 hours, transfected cells were plated in serum-free media in Matrigel-coated Boyden chambers with 10% FBS in the wells as a chemoattractant. After 24 hours, cells were fixed and stained. Representative photos of invading cells are shown at 20× magnification. The total number of invading cells was counted in 10 random fields; the average number of invading cells is shown for triplicate cultures per cell line, * p<0.05. (B) BT474 stable empty vector control clone (pCMV), BT474 GDF15 stable clone 5 (C5), and MDA-MB-231 cells were transfected with 100 nM siRNA control (siCtrl) or GDF15 siRNA (siGDF15) for 48 hours. Real-time PCR was performed to confirm GDF15 knockdown. Values reflect the fold change in transcript normalized to RPLPO housekeeping gene. Error bars represent standard deviation between triplicate samples; experiments were repeated twice; ** p<0.005, * p<0.05.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Plasmid Preparation, Control, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Knockdown, Standard Deviation

    GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.

    Journal: Oncotarget

    Article Title: Growth differentiation factor 15 mediates epithelial mesenchymal transition and invasion of breast cancers through IGF-1R-FoxM1 signaling

    doi: 10.18632/oncotarget.21765

    Figure Lengend Snippet: GDF15 activates IGF-1R signaling and induces expression of FoxM1, which upregulates expression of EMT transcription factors (TFs), including Snail and Slug, and induces expression of matrix metalloproteinases (MMPs), stimulating EMT and breast cancer cell invasion.

    Article Snippet: TMAs were then stained with rabbit polyclonal anti-human GDF15 (also known as MIC-1; antibody 3249; dilution 1:100; Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923) overnight at 4°C.

    Techniques: Expressing

    Serum Growth differentiation factor 15 (GDF15 ) levels in patients with various hematological malignancies. Serum concentrations of GDF15 were measured by ELISA in 14 normal subjects and 128 patients with various hematopoietic malignancies. Each individual sample was assayed in duplicate. The box and central bars show interquartile ranges and medians, whereas whiskers on bars above and below the boxes indicate the 90th and 10th percentiles. * P < 0.05; ** P < 0.01. Asterisks above the bars indicate significant increases in comparison with normal subjects. AML, acute myelogenous leukemia; MDS, myelodysplastic syndromes; CML, chronic myelogenous leukemia; PMF, primary myelofibrosis; PV, polycythemia vera; ET, essential thrombocythemia; MM, multiple myeloma; ML, malignant lymphoma; ALL, acute lymphoblastic leukemia; ATL, adult T-cell leukemia/lymphoma.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Serum Growth differentiation factor 15 (GDF15 ) levels in patients with various hematological malignancies. Serum concentrations of GDF15 were measured by ELISA in 14 normal subjects and 128 patients with various hematopoietic malignancies. Each individual sample was assayed in duplicate. The box and central bars show interquartile ranges and medians, whereas whiskers on bars above and below the boxes indicate the 90th and 10th percentiles. * P < 0.05; ** P < 0.01. Asterisks above the bars indicate significant increases in comparison with normal subjects. AML, acute myelogenous leukemia; MDS, myelodysplastic syndromes; CML, chronic myelogenous leukemia; PMF, primary myelofibrosis; PV, polycythemia vera; ET, essential thrombocythemia; MM, multiple myeloma; ML, malignant lymphoma; ALL, acute lymphoblastic leukemia; ATL, adult T-cell leukemia/lymphoma.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Enzyme-linked Immunosorbent Assay, Comparison

    Histological examination of Growth differentiation factor 15 (GDF15) expression in bone marrow (BM) specimens. (A) Hematoxylin and eosin staining and (B–F) immunohistochemical staining for GDF15. (A–D) BM biopsy specimens from patients with primary meylofibrosis and (E) essential thrombocythemia. (F) A BM biopsy specimen with normal morphology obtained from a patient with diffuse large B-cell lymphoma without BM involvement. Original magnification, (A and B) ×100; (C, E, and F) ×400; (D) ×1000.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Histological examination of Growth differentiation factor 15 (GDF15) expression in bone marrow (BM) specimens. (A) Hematoxylin and eosin staining and (B–F) immunohistochemical staining for GDF15. (A–D) BM biopsy specimens from patients with primary meylofibrosis and (E) essential thrombocythemia. (F) A BM biopsy specimen with normal morphology obtained from a patient with diffuse large B-cell lymphoma without BM involvement. Original magnification, (A and B) ×100; (C, E, and F) ×400; (D) ×1000.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Expressing, Staining, Immunohistochemical staining

    Expression of Growth differentiation factor 15 (GDF15) during megakaryocytic differentiation of HEL cells. (A−D) May-Grunwald-Giemsa staining (original magnification, ×400). HEL cells were cultured in the presence of (A) control solvent, (B) 10 μ mol/L diosgenin, (C) 10 nmol/L PMA, or (D) 50 μ mol/L hemin for 96 h. (E) Expression of GDF15 mRNA in HEL cells during megakaryocytic differentiation by diosgenin. Cells were treated with 10 μ mol/L diosgenin for 48, 72, 96, 120, and 144 h, and GDF15 mRNA levels were measured by quantitative real-time PCR. The relative expression level of GDF15 was normalized to that of β-actin and is shown relative to the expression levels of the control. (F–H) GDF15 protein secretion levels in the culture supernatant determined by ELISA. HEL cells were cultured in the presence of (F) 10 μ mol/L diosgenin for 24, 48, 72, 96, 120, and 144 h, (G) 10 nmol/L PMA for 96 h, or (H) 50 μ mol/L hemin for 96 h. Values represent the mean fold change compared to the respective control from triplicate experiments ± SE ( n = 3). * P < 0.05 versus control; ** P < 0.01 versus control.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Expression of Growth differentiation factor 15 (GDF15) during megakaryocytic differentiation of HEL cells. (A−D) May-Grunwald-Giemsa staining (original magnification, ×400). HEL cells were cultured in the presence of (A) control solvent, (B) 10 μ mol/L diosgenin, (C) 10 nmol/L PMA, or (D) 50 μ mol/L hemin for 96 h. (E) Expression of GDF15 mRNA in HEL cells during megakaryocytic differentiation by diosgenin. Cells were treated with 10 μ mol/L diosgenin for 48, 72, 96, 120, and 144 h, and GDF15 mRNA levels were measured by quantitative real-time PCR. The relative expression level of GDF15 was normalized to that of β-actin and is shown relative to the expression levels of the control. (F–H) GDF15 protein secretion levels in the culture supernatant determined by ELISA. HEL cells were cultured in the presence of (F) 10 μ mol/L diosgenin for 24, 48, 72, 96, 120, and 144 h, (G) 10 nmol/L PMA for 96 h, or (H) 50 μ mol/L hemin for 96 h. Values represent the mean fold change compared to the respective control from triplicate experiments ± SE ( n = 3). * P < 0.05 versus control; ** P < 0.01 versus control.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Expressing, Staining, Cell Culture, Control, Solvent, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effect of Growth differentiation factor 15 (GDF15) treatment of human BM-MSCs on expansion of HPCs. (A) Schematic representation of the experimental procedure used to analyze the effect of GDF15 on the proliferation of CD34 + HPCs cultured in the absence of human BM-MSCs as feeders (upper) and on the ability of human BM-MSCs to facilitate the expansion of CD34 + HPCs (lower). (B) Number of HPCs in the absence of feeder cells. HPCs were cultured in the absence or presence of 0.5, 5, and 50 ng/mL rhGDF15 for 10 days in the absence of feeder cells, and the cells were then counted in a hemocytometer by using Trypan blue exclusion. The results are presented relative to those of the untreated control. Each point signifies the mean from five replicates; bars indicate ± SE. (C) Number of total CD45 + hematopoietic cells expanded after 10 days of co-culture with either nonstimulated or rhGDF15 (30 ng/mL)-stimulated BM-MSCs, as determined by flow cytometry and a hemocytometer ( n = 5). (D) Percentage of total CD45 + CD34 + hematopoietic cells (left panel), and the ratios of CD38 – (middle panel) and CD38 + (right panel) cells among them, expanded after 10 d of co-culture with either nonstimulated or rhGDF15-stimulated BM-MSCs. SFEM, serum-free expansion medium. * P < 0.05 versus control; ** P < 0.01 versus control; n.s., not significant.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Effect of Growth differentiation factor 15 (GDF15) treatment of human BM-MSCs on expansion of HPCs. (A) Schematic representation of the experimental procedure used to analyze the effect of GDF15 on the proliferation of CD34 + HPCs cultured in the absence of human BM-MSCs as feeders (upper) and on the ability of human BM-MSCs to facilitate the expansion of CD34 + HPCs (lower). (B) Number of HPCs in the absence of feeder cells. HPCs were cultured in the absence or presence of 0.5, 5, and 50 ng/mL rhGDF15 for 10 days in the absence of feeder cells, and the cells were then counted in a hemocytometer by using Trypan blue exclusion. The results are presented relative to those of the untreated control. Each point signifies the mean from five replicates; bars indicate ± SE. (C) Number of total CD45 + hematopoietic cells expanded after 10 days of co-culture with either nonstimulated or rhGDF15 (30 ng/mL)-stimulated BM-MSCs, as determined by flow cytometry and a hemocytometer ( n = 5). (D) Percentage of total CD45 + CD34 + hematopoietic cells (left panel), and the ratios of CD38 – (middle panel) and CD38 + (right panel) cells among them, expanded after 10 d of co-culture with either nonstimulated or rhGDF15-stimulated BM-MSCs. SFEM, serum-free expansion medium. * P < 0.05 versus control; ** P < 0.01 versus control; n.s., not significant.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Cell Culture, Control, Co-Culture Assay, Flow Cytometry

    Effects of Growth differentiation factor 15 (GDF15) on normal human dermal fibroblasts (NHDFs). (A and B) Effects of rhGDF15 and rhTGF- β 1 on proliferation of NHDFs. NHDFs were cultured in the absence or presence of (A) 30 ng/mL rhGDF15 or (B) 2 ng/mL rhTGF- β 1 for 24, 48, 72, and 96 h, and the cells were counted with a hemocytometer using Trypan blue exclusion. Each point signifies the mean from five replicates; bars indicate ± SE (left panels). (A, right panels) NHDF were visualized using light microscopy after 72 h of rhGDF15 exposure (original magnification, ×400). (C) Effect of an anti-GDF15 neutralizing antibody on GDF15-induced NHDF proliferation. NHDFs were treated with rhGDF15 in the absence or presence of anti-GDF15 neutralizing antibodies (1 μ g/mL) or control isotype-matched mouse IgG for 96 h, and the cells were counted with a hemocytometer. (D) Immunoblot analysis of ERK1/2 phosphorylation in NHDFs treated with either rhGDF15 (left panels) or rhTGF- β 1 (right panels) for 5, 15, and 30 min. The relative phosphorylation levels of ERK1/2 were normalized to the levels of total ERK1/2 protein and quantified using ImageJ. (E) Quantitative real-time polymerase chain reaction analysis examining extracellular matrix (ECM)-related mRNA expression in NHDFs treated with rhGDF15 or rhTGF- β 1 for 3 days. The fold of increase in each mRNA in the treated group was obtained by normalization to untreated controls. COL1A1, collagen type 1 alpha 1; COL3A1, collagen type 3 alpha 1; COL4A1, collagen type 4 alpha 1; COL7A1, collagen type 7 alpha 1; ASMA, alpha-smooth muscle actin; CTGF, connective tissue growth factor; MMP-1, matrix metalloproteinase-1; TIMP-1, tissue inhibitor of metalloproteinase-1. Values represents the mean ± SE ( n = 3). * P < 0.05 versus control; ** P < 0.01 versus control.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Effects of Growth differentiation factor 15 (GDF15) on normal human dermal fibroblasts (NHDFs). (A and B) Effects of rhGDF15 and rhTGF- β 1 on proliferation of NHDFs. NHDFs were cultured in the absence or presence of (A) 30 ng/mL rhGDF15 or (B) 2 ng/mL rhTGF- β 1 for 24, 48, 72, and 96 h, and the cells were counted with a hemocytometer using Trypan blue exclusion. Each point signifies the mean from five replicates; bars indicate ± SE (left panels). (A, right panels) NHDF were visualized using light microscopy after 72 h of rhGDF15 exposure (original magnification, ×400). (C) Effect of an anti-GDF15 neutralizing antibody on GDF15-induced NHDF proliferation. NHDFs were treated with rhGDF15 in the absence or presence of anti-GDF15 neutralizing antibodies (1 μ g/mL) or control isotype-matched mouse IgG for 96 h, and the cells were counted with a hemocytometer. (D) Immunoblot analysis of ERK1/2 phosphorylation in NHDFs treated with either rhGDF15 (left panels) or rhTGF- β 1 (right panels) for 5, 15, and 30 min. The relative phosphorylation levels of ERK1/2 were normalized to the levels of total ERK1/2 protein and quantified using ImageJ. (E) Quantitative real-time polymerase chain reaction analysis examining extracellular matrix (ECM)-related mRNA expression in NHDFs treated with rhGDF15 or rhTGF- β 1 for 3 days. The fold of increase in each mRNA in the treated group was obtained by normalization to untreated controls. COL1A1, collagen type 1 alpha 1; COL3A1, collagen type 3 alpha 1; COL4A1, collagen type 4 alpha 1; COL7A1, collagen type 7 alpha 1; ASMA, alpha-smooth muscle actin; CTGF, connective tissue growth factor; MMP-1, matrix metalloproteinase-1; TIMP-1, tissue inhibitor of metalloproteinase-1. Values represents the mean ± SE ( n = 3). * P < 0.05 versus control; ** P < 0.01 versus control.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Cell Culture, Light Microscopy, Control, Western Blot, Phospho-proteomics, Real-time Polymerase Chain Reaction, Expressing

    Effects of GDF15 on proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stromal cells (BM-MSC). (A) Human BM-MSCs were cultured in the absence or presence of 30 ng/mL rhGDF15 for 4, 6, and 8 days, and the cells were counted in a hemocytometer. Each point signifies the mean from five replicates; bars indicate ± SE. (B) Human BM-MSCs cultured in the presence of 0, 1, and 30 ng/mL rhGDF15 for 8 days were counted in a hemocytometer. The results are presented relative to those of untreated controls (left panel), as visualized using light microscopy (right panels, original magnification ×400). Bars indicate ± SE. (C and D) GDF15-induced osteoblastic differentiation of BM-MSCs. (C) Human BM-MSCs were treated with rhGDF15 (30 ng/mL) for 4 weeks. Mineralized deposits indicated by Alizarin Red S staining were quantified using ImageJ in five different fields viewed at ×100 magnification. The results are presented relative to those of untreated controls (left panel), as visualized using light microscopy (right panels; magnification, ×100). (D) RUNX2 and osterix (OSX) protein expression in GDF15-treated BM-MSCs were analyzed by immunoblotting. The expression levels were normalized against β -actin protein levels and quantified using ImageJ. The results are presented relative to those of untreated controls. (E) In vivo bone formation assays. (Upper panel) Schematic representation of the experimental procedure used to analyze the effects of GDF15 on bone formation in a xenograft mouse model. Human BM-MSCs treated with rhGDF15 (30 ng/mL) for 3 days were subcutaneously transplanted into NOD/SCID mice ( n = 4) using a hydroxyapatite-poly d , l -lactic-co-glycolic acid composite block type scaffold (HA scaffold) as a carrier and harvested 10 weeks later. (Lower right and left panels) Representative microscopic images and quantitative measurement of bone formation areas in sections of human BM-MSC implants stained with hematoxylin and eosin. HA, hydroxyapatite; B, bone. Bar = 50 μ m. The results are expressed as the ratio of the total bone area to the total implant area, quantified using ImageJ in five different fields viewed at ×100 magnification (lower middle panel). Bars indicate ± SE. * P < 0.05; ** P < 0.01.

    Journal: Cancer Medicine

    Article Title: The role of growth differentiation factor 15 in the pathogenesis of primary myelofibrosis

    doi: 10.1002/cam4.502

    Figure Lengend Snippet: Effects of GDF15 on proliferation and osteoblastic differentiation of human bone marrow-derived mesenchymal stromal cells (BM-MSC). (A) Human BM-MSCs were cultured in the absence or presence of 30 ng/mL rhGDF15 for 4, 6, and 8 days, and the cells were counted in a hemocytometer. Each point signifies the mean from five replicates; bars indicate ± SE. (B) Human BM-MSCs cultured in the presence of 0, 1, and 30 ng/mL rhGDF15 for 8 days were counted in a hemocytometer. The results are presented relative to those of untreated controls (left panel), as visualized using light microscopy (right panels, original magnification ×400). Bars indicate ± SE. (C and D) GDF15-induced osteoblastic differentiation of BM-MSCs. (C) Human BM-MSCs were treated with rhGDF15 (30 ng/mL) for 4 weeks. Mineralized deposits indicated by Alizarin Red S staining were quantified using ImageJ in five different fields viewed at ×100 magnification. The results are presented relative to those of untreated controls (left panel), as visualized using light microscopy (right panels; magnification, ×100). (D) RUNX2 and osterix (OSX) protein expression in GDF15-treated BM-MSCs were analyzed by immunoblotting. The expression levels were normalized against β -actin protein levels and quantified using ImageJ. The results are presented relative to those of untreated controls. (E) In vivo bone formation assays. (Upper panel) Schematic representation of the experimental procedure used to analyze the effects of GDF15 on bone formation in a xenograft mouse model. Human BM-MSCs treated with rhGDF15 (30 ng/mL) for 3 days were subcutaneously transplanted into NOD/SCID mice ( n = 4) using a hydroxyapatite-poly d , l -lactic-co-glycolic acid composite block type scaffold (HA scaffold) as a carrier and harvested 10 weeks later. (Lower right and left panels) Representative microscopic images and quantitative measurement of bone formation areas in sections of human BM-MSC implants stained with hematoxylin and eosin. HA, hydroxyapatite; B, bone. Bar = 50 μ m. The results are expressed as the ratio of the total bone area to the total implant area, quantified using ImageJ in five different fields viewed at ×100 magnification (lower middle panel). Bars indicate ± SE. * P < 0.05; ** P < 0.01.

    Article Snippet: Immunohistochemical staining for GDF15 was performed using rabbit polyclonal antibodies against human GDF15 (1:100, anti-human HPA011191; Atlas Antibodies, Stockholm, Sweden) on an automated slide stainer (Ventana XT System Benchmark; Ventana Medical Systems, Tucson, AZ).

    Techniques: Derivative Assay, Cell Culture, Light Microscopy, Staining, Expressing, Western Blot, In Vivo, Blocking Assay