recombinant hgfl  (R&D Systems)

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: RON and HGFL overexpression co-occur in the murine MMTV-PyMT model of breast cancer and global HGFL deletion impairs tumor growth and metastasis and improves immune cell recruitment. ( A ) Representative western blot for RON and HGFL on lysates from normal mammary glands or PyMT tumor-bearing mammary glands of FVB mice (n = 2). ( B ) Immunohistochemistry staining for RON or HGFL on PyMT tumor-bearing mammary glands or mammary glands of non-transgenic (normal) FVB mice at specified ages (n = 4). ( C ) Tumor growth kinetics of HGFL proficient (+/+; n = 12) or HGFL deficient (−/−; n = 10) PyMT mice and ( D ) respective metastatic incidence and number of metastases in the lungs taken at 90 days of age. ( E ) Representative images and quantitation of specified staining for BrdU and TUNEL, ( F ) F4/80, iNOS, and Arginase-1, and ( G ) CD8a and CD4 in the mammary glands of PyMT (HGFL +/+ or HGFL −/− ) mice at 90 days. Comparisons of histological samples between groups were analyzed using Student’s t -tests. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Over Expression, Western Blot, Immunohistochemistry, Staining, Transgenic Assay, Quantitation Assay, TUNEL Assay

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: Conditional RON deletion in the myeloid compartment phenocopies global HGFL loss in a RON-driven mammary tumorigenesis model. Kaplan-Meier analysis with Log-Rank statistics of mice for ( A ) time to onset of mammary ductal hyperplasia (MMTV-RON, n = 9; ΔMyeloid, n = 8) and ( B ) time to tumor palpability of MMTV-RON+ mice with or without conditional deletion of RON in the myeloid compartment (ΔMyeloid; MMTV-RON, n = 16; ΔMyeloid, n = 10). ( C ) Tumor growth kinetics of MMTV-RON+ or MMTV-RON ΔMyeloid mice (MMTV-RON, n = 9; ΔMyeloid, n = 9) and ( D ) respective gross metastatic incidence at 90 days of age. ( E ) Micro-metastatic area measured on H&E stained sections from mice of both genotypes taken from mammary tumors with comparable tumor volume (MMTV-RON, n = 6; ΔMyeloid, n = 5). ( F ) Quantitated histological analysis of MMTV-RON and ΔMyeloid mammary tumor sections stained with Ki67, CC3, ( G ) F4/80, iNOS, Arginase-1, ( H ) CD8a, or CD4. Comparisons of histological samples between groups were analyzed using Student’s t -tests. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Staining

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: Loss of tumor cell-produced HGFL or systemic (physiologic) HGFL alters mammary tumorigenesis and immune cell recruitment. ( A ) Western blot analysis of lysates from R7 shNT and R7 shHGFL cells for HGFL and Actin (loading control). ( B ) Mammary tumor growth kinetics of R7 shNT and R7 shHGFL cells following orthotopic implantation into syngeneic HGFL deficient (FVB HGFL −/− ) or HGFL proficient FVB (FVB WT; HGFL +/+ ) mice (n = 5 mice/group) and results were compared with a repeated-measures two-way ANOVA with Holm-Šídák post hoc tests. ( C ) Tumor characterization through histological analyses for Ki67, CC3, ( D ) F4/80, iNOS, Arginase-1, and ( E ) CD8a and CD4. ( F ) Tumor growth kinetics of mammary tumors generated from R7 cells in either WT or HGFL −/− mice with or without liposomal clodronate treatment to deplete macrophages (n = 6 mice/group). Groups were compared using a two-way ANOVA with Holm-Šídák post hoc tests. ** p < 0.01; **** p < 0.0001; ns, not significant.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Produced, Western Blot, Control, Generated

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: Loss of HGFL in breast cancer cells impairs mammosphere formation, macrophage migration, and alters the secretome of both cell types. ( A ) Macrophage migration towards denoted stimulus including serum-free media (SFM), SFM + recombinant HGFL (rHGFL, 50ng/mL), conditioned media (CM) from R7 control or R7shHGFL cells, or cultured R7 control or R7shHGFL cells. Groups were compared using one-way ANOVA with Holm-Šídák post hoc tests. ( B ) Macrophage migration as in ( A ) toward R7 cells pretreated with vehicle, an Akt inhibitor (AKTi), a MAPK inhibitor (MAPKi), a Stat3 inhibitor (STAT3i), or an NFkB inhibitor (NFKBi). Following inhibitor treatment for 6 h, R7 cells were washed, placed in SFM, and used as a chemoattractant. ( C ) Mammosphere formation of R7 cells with and without HGFL knockdown (shHGFL), monoculture or co-culture with RON TK +/+ BMDM, RON TK −/− BMDM, and with and without supplementation with 50 ng/mL recombinant HGFL (rHGFL). Groups were compared using a one-way ANOVA with Holm-Šídák post hoc tests. ( D , E ) Heatmaps of secreted proteins detected by Mouse XL Cytokine Arrays incubated with conditioned media from ( D ) R7 cells and R7 sgRON cells (R7 cells with RON loss via CRISPR-Cas9 technology, R7 KD cells from ) or ( E ) with conditioned media from WT (RON TK +/+ ) or RON deficient TK −/− BMDM. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Migration, Recombinant, Control, Cell Culture, Knockdown, Co-Culture Assay, Incubation, CRISPR

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: Pharmacological inhibition of HGFL-RON signaling via BMS777607 treatment abrogates PyMT-driven tumorigenesis and metastasis. ( A ) Tumor growth kinetics of mammary tumors in PyMT+ mice treated with 50 mg/kg/day BMS777607 versus vehicle control (Vehicle: n = 6; BMS777607: n = 7). Data was analyzed with a two-way ANOVA with Holm-Šídák post hoc tests. ( B ) Metastatic incidence in PyMT+ mice from ( A ) with BMS777607 treatment versus vehicle control. Data was analyzed using Fisher’s exact test. ( C ) Histological analysis of BMS777607 treated and vehicle-treated PyMT tumor sections stained with Ki67, CC3, ( D ) F4/80, iNOS, Arginase 1, ( E ) CD8a, or CD4. ( F ) Working model of our experimental findings summarized (generated using BioRender). *** p < 0.001; **** p < 0.0001; ns, not significant.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Inhibition, Control, Staining, Generated

Journal: Cancers

Article Title: RON ( MST1R ) and HGFL ( MST1 ) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk

doi: 10.3390/cancers14102493

Figure Lengend Snippet: RON ( MST1R ) and HGFL ( MST1 ) expression are correlated in virtually all tumor types and expression is not associated with recurrent breast cancer driver mutations. ( A ) MST1R and MST1 gene expression are coordinately upregulated across specified cancer types from The Cancer Genome Atlas (TCGA) evaluated with Spearman’s correlation. ( B ) MST1R and MST1 RNA expression from TCGA showing coordinate expression in heatmap form. Volcano plots of ( C ) MST1R and ( D ) MST1 RNA expression stratified between Altered and Unaltered samples of listed recurrent breast cancer driver mutations from TCGA Pan-Cancer and METABRIC datasets were evaluated using Multiple t -tests on log-transformed expression values. Significant correlations of specified immune cell infiltration signatures with ( E ) MST1R and ( F ) MST1 gene expression using the tumor immune microenvironment estimation resource (TIMER) in breast cancer data from TCGA.

Article Snippet: Recombinant HGFL (rHGFL; Cat# 4306-MS/CF R&D Systems) supplementation was performed by daily addition of 50 ng rHGFL to respective cultures.

Techniques: Expressing, Gene Expression, RNA Expression, Transformation Assay

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 1. RT-PCR analysis of Ron expression in various adult mouse tis- sues. The RNA from virgin uterus (lane 1), E12 placenta (lane 2), E16 placenta (lane 3), virgin cervix (lane 4), liver (5), colon (lane 6), epidid- ymis (lane 7), and testes (lane 8) were analyzed by RT-PCR using primers specific for mouse Ron (A), HGFL (B), or beta actin (C). The Ron amplified product was 404 bp and the HGFL amplified product 178 bp, whereas the beta actin product was 218 bp. As a negative control, water only was included in a PCR reaction (lane 9). A 100-bp ladder was included as a standard.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 4. RT-PCR analysis of Ron expression in murine trophoblast cell lines. The RNA from SM9-1 (lane 1), SM9-2 (lane 2), SM-10 (lane 3), and MEL (lane 4) cell lines were analyzed by RT-PCR using primers specific for Ron (A), HGFL (B), and beta actin (C) expression. The amplified Ron expression product was 404 bp. The amplified HGFL product was 178 bp, and the beta actin product was 218 bp. Water only was included as a negative control (lane 5). A 100-bp ladder was included as a standard.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 6. HGFL stimulates invasion of SM9-1 cells through Matrigel. In- vasion of SM9-1 cells through Matrigel-coated transwell membranes fol- lowing incubation for 24 and 48 h in F12 medium only (Serum Free), kallikrein-treated CHO conditioned medium (CHO/mock), or kallikrein- treated conditioned medium from CHO cells expressing HGFL (CHO/ HGFL) is shown. At 48 h, cellular invasion was significantly increased in the presence of HGFL compared to the mock-treated cells (*). Data shown are the average numbers of cells that had invaded through the Matrigel in 10 different visual fields with the standard deviation shown and P , 0.001. Experiments were performed in duplicate.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Incubation, Expressing, Standard Deviation

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 7. SM-10 cell survival following incubation in RPMI media con- taining 10% FBS, recombinant HGFL, or RPMI media only (Serum Free). The percentage of surviving cells was calculated for each time-point by counting the number of attached cells and dividing by the total number of cells counted (floating and attached). The absence of HGFL or 10% FBS produced a significant decrease in the percentage of surviving cells over 48 h (compare triangle- to square- and/or diamond-denoted lines). The experiment was performed in triplicate with P , 0.001 for those points indicated by the asterisks. To test whether HGFL could block SM-10 cells from undergoing apoptosis or necrosis, a TUNEL assay was per- formed (Fig. 8, A–D). SM-10 cells incubated for 24 h in the presence of serum or recombinant HGFL exhibited few- er TUNEL-positive cells compared to those cells incubated in serum-free medium (Fig. 8E).

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Incubation, Recombinant, Produced, Blocking Assay, TUNEL Assay

Journal: Biology of reproduction

Article Title: Receptor tyrosine kinase Ron is expressed in mouse reproductive tissues during embryo implantation and is important in trophoblast cell function.

doi: 10.1095/biolreprod.102.009928

Figure Lengend Snippet: FIG. 8. TUNEL staining of SM-10 cells following incubation in the presence or absence of HGFL. TUNEL analysis of fixed SM-10 cells following incubation for 24 h in RPMI with 10% FBS (B), RPMI with HGFL (C), or RPMI only (D) is shown. The negative (2) control (A) was incubated without terminal transferase. Percentage of TUNEL-positive cells per treatment group (A–D) is indicated in E. Percentages were calculated based on determining the num- ber of TUNEL-positive cells per four high- power areas, counting at least 100 cells per area. Standard error bars are indicated. *P , 0.003 compared to the negative con- trol in A, **P , 0.0001 compared to all other samples.

Article Snippet: Recombinant human HGFL was purchased under the name of recombinant human macrophage-stimulating protein from R&D Systems, Inc. (Minneapolis, MN).

Techniques: TUNEL Assay, Staining, Incubation, Control