Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 1. Involvement of FGF2-FGFR1 axisin Akt activation. (A) The effect of CAF-CM on proliferation of breast cancer (MCF-7, MDA-MB-231, and MDA-MB-468) cells was determined by the MTT assay. Cells were incubated with or without CAF-CM for 72 hours. ***Significantly different be- tween the groups compared (P < 0.001). (B) MDA-MB-231 cells were incubated with CAF-CM for the indicated time periods. Phosphorylation of Akt and STAT3 were detected by Western blot analysis. (C) MDA-MB-231 cells were exposed to CAF-CM with or without FGF-2-neutralizing antibody for 3 hours. Phosphorylation of Akt was detected by Western blot analysis. *,***Significantly different between the groups compared (*P < 0.05; ***P < 0.001). (D) MDA-MB-231 cells were treated with 20 ng/mL of FGF2 for the indicated time periods. The phosphorylation of FRS2α as well as Akt was analyzed by Western blot. (E) RNA-seq data set of TCGA breast invasive carcinoma was downloaded from XenaBrower (https://xenabrowser.net). mRNA expression levels of total 1,097 samples (Illumina HiSeq log [normalized counts + 1]) were prepared by quantile normalization. Pearson cor- relation coefficient was calculated to assess the relationship between FGF2 and FGFR1. (F, G) Correlation of FGFR1 protein expression with FGF2 (F) and Akt (G), based on 105 breast invasive carcinoma protein specimens (TCGA, Pan-Cancer Atlas) from the cBioportal database (www.cbiopor- tal.org). FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; CAFs, cancer-associated fibroblasts; NFs, normal fibroblasts; CM, conditioned medium; ns, not significantly different; FRS2, FGFR substrate 2; TCGA, The Cancer Genome Atlas; CPTAC, the Clinical Proteomic Tumor Analysis Consortium.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Activation Assay, MTT Assay, Incubation, Phospho-proteomics, Western Blot, RNA Sequencing, Expressing

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 2. Role of FGFR1 in Akt phosphorylation and breast cancer cell growth and progression. (A) MDA-MB-231 cells were transfected with scrambled or FGFR1 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 15 minutes to measure phosphorylated FRS2α. (B) Mice were subjected to xenograft co-injecting with fibroblasts and MDA-MB-231 breast cancer cells. A complex collagen network was detected in H&E-stained tumors by an intense pink and in Masson’s trichrome stain by a blue stain (arrows). Stromal compartment was also detected by α-SMA immunostaining. Magnification, x100. Bars, 100 μm. (C) Phosphorylated Akt in the xenograft tumors was determined by Western blot analysis. *Sig- nificantly different between the groups compared (P < 0.05). (D) Enrichment plots of hallmark gene sets in the high FGFR1-expressing group. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; FRS2, FGFR substrate 2; α-SMA, alpha-smooth muscle actin; CONT, control; EMT, epithelial- mesenchymal transition.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Phospho-proteomics, Transfection, Incubation, Staining, Immunostaining, Western Blot, Expressing, Control

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 3. The involvement of FGF2-induced ROS generation in nuclear localization of FGFR1. (A) MDA-MB-231 cells were co-cultured with NFs or CAFs for 24 hours. MDA-MB-231 (5 x 10 3 cells) and NFs or CAFs (5 x 10 3 cells) were mixed prior to seeding and incubated for 24 hours. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with DAPI for detection of nuclei. Magnification, x100. Bars, 200 μm. (B) MDA-MB-231 cells were incubated with FGF2 for 1 hour. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with PI for detection of nuclei. Magnification, x100. Bars, 200 μm. (C) MDA-MB-231 cells were treated with 20 ng/ mL of FGF2 for 1 hour, followed by Western blot analysis of FGFR1 in cytosolic and nuclear extracts. Lamin B was used as a nuclear marker. *Sig- nificantly different between the groups compared (P < 0.05). (D, E) MDA-MD-231 cells were incubated with CAF-CM or FGF2 for 3 hours and 1 hour, respectively. After staining with DCF-DA for 30 minutes, fluorescent microscopic (D) or flow cytometric (E) analysis was performed to detect intracellu- lar ROS accumulation. Magnification, x40. (F) After pretreatment with NAC for 3 hours, cells were exposed to FGF2 for additional 1 hour. Nuclear ex- tracts were subjected to Western blot analysis to detect the presence of FGFR1 and Nrf2 in the nucleus. **Significantly different between the groups compared (P < 0.01). (G) MDA-MB-231 cells were exposed to FGF2 (20 ng/mL) for 1 hour. Cell lysates were subjected to immunoprecipitation using CBP antibody for 16 hours followed by immunoblotting with. FGFR1 or Nrf2 antibody. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; ROS, reactive oxygen species; CAFs, cancer-associated fibroblasts; CM, conditioned medium; NFs, normal fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; CONT, cotrol; DCF-DA, 2’,7’-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Cell Culture, Incubation, Staining, Western Blot, Marker, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 4. Possible association between nuclear FGFR1 and Nrf2. (A) TNBC patient cohorts were validated based on the mean expression value of the indicated single genes (FGFR1 or NFE2L2) or as a signature of two genes together and patient survival was analyzed (n = 255). (B, C) MDA- MB-231 cells were transfected with scrambled or Nrf2 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 3 hours. The mRNA (B) and protein (C) expression of cyclin D1 was assessed by RT-PCR and Western blot analyses, respectively. The expression of cyclin D1 was mea- sured by RT-PCR (B) and Western blot (C) analyses. (D) In tumor microenvironment, fibroblasts are activated to form CAFs, which secrete FGF2. CAF-derived FGF2 could induces nuclear translocation as well as de novo synthesis of FGFR1, ultimately contributing to cancer cell proliferation, mi- gration and tumor growth. While membrane bound FGFR1 may translocate to nucleus as a complex with FGF2 which has nuclear localization signal (NLS), the complex is likely rather to stimulate the intracellular signaling via FRS2α, which induces transcription of FGFR-1 gene. On the other hand, newly synthesized FGFR-1 is speculated to enter the nucleus as a complex with a cargo protein harboring NLS. FGFR-1 is translocated to the inner nuclear membrane through the nuclear pore complexes (NPCs), which is regulated by importin β. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; TNBC, triple negative breast cancer; HR, hazard ratio; CAFs, cancer-associated fibroblasts; ER, endoplasmic reticulum; FRS2, FGFR substrate 2; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Translocation Assay, Membrane, Synthesized, Binding Assay

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of primers for transcript analysis

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques:

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: List of antibodies for WB, IF, and flow cytometry analysis

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Flow Cytometry, Recombinant

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by late-onset AD. A Scheme illustrating the sample collection of NSCs from two iPSC clones of the same donor. Quantification of B transcript and D protein amounts of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare three healthy iPSCs (WISCi004-B, WAi001-B, and MLUi009-A) versus four AD iPSCs (MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; healthy: n = 9, AD: n = 12). E Representative image of WB membranes. C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to health status ( N = 3 differentiations; healthy: n = 9, AD: n = 12). Results are shown as mean ± SEM (* p = 0.05)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Clone Assay, Multiplex Assay, Quantitative RT-PCR

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Detection of marker proteins for cellular aging in NSCs through immunofluorescence (IF). IF analysis showed cellular localization of APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Protein of interest is shown in green versus DNA staining in blue (scale bar, 100 μm)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Marker, Immunofluorescence, Staining

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Aging markers in NSCs are affected by APOE genotype. A Transcript and B protein expression analysis of the aging markers APOE, ATG7, FGF2, p21, PTEN, SIRT1, and STAT3. Results are shown as mean ± SEM (* p = 0.05). The bar charts compare two iPSCs carrying APOE3 (WISCi004-B and MLUi009-A) versus five iPSCs carrying APOE4 (WAi001-B, MLUi007-H/J and MLUi008-B/F) on 7 d, which were differentiated three times into NSCs ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). C Telomere length measured by multiplex QRT-PCR in iPSCs on 0 d and in NSCs on 7 d, shown as the ratio NSCs/iPSCs grouped according to APOE status ( N = 3 differentiations; APOE3: n = 6, APOE4: n = 15). Results are shown as mean ± SEM (* p = 0.05). D Manipulation of APOE gene expression by APOE siRNAs and APOE3 plasmids in NSCs generated from reference cell line WISCi004-B on 7 d. No morphological changes are shown by phase contrast imaging up to 4 d after transfection. WB analysis showed the strongest APOE protein repression by APOE siRNAs and strongest APOE induction by APOE3 plasmids on 4 d. Representative images of stained WB membranes are shown (scale bar, 100 μm). Transcript analysis of aging markers in NSCs on 4 d after E APOE inhibition and F APOE induction. Results are shown as mean ± SEM (* p = 0.05). For APOE inhibition, MLUi009-A (healthy matched control, APOE3 carrier) was differentiated into NSCs and transfected with siRNAs on 7 d ( N = 3 differentiations; mock: n = 3, siRNA: n = 3). For APOE induction, the MLUi007-H from (AD, APOE4 carrier) was differentiated into NSCs and transfected with APOE3 plasmids at 7 d ( N = 3 differentiations; mock: n = 3, APOE3 plasmids: n = 3)

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Expressing, Multiplex Assay, Quantitative RT-PCR, Generated, Imaging, Transfection, Staining, Inhibition

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Significant mRNA expression changes observed in aging markers

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques: Expressing, Plasmid Preparation

Journal: Molecular Neurobiology

Article Title: Neuronal Stem Cells from Late-Onset Alzheimer Patients Show Altered Regulation of Sirtuin 1 Depending on Apolipoprotein E Indicating Disturbed Stem Cell Plasticity

doi: 10.1007/s12035-023-03633-z

Figure Lengend Snippet: Proposed mechanisms for APOE and its impact on aging markers for regulating NSC plasticity. Observed effects on PTEN, ATG7, FGF2, and SIRT1 may reflect the impact of APOE on RTK signaling including FGF2 as key signaling molecule, SIRT1 as transcriptional regulator, and PTEN and ATG7 as target genes. RTKs: receptor tyrosine kinases; JAKs: Janus kinases; AKT: protein kinases B; gray: Golgi apparatus; green arrows: APOE signaling; violet arrows: aging markers signaling via JAKs, RTKs, and Notch

Article Snippet: Mouse IgG anti-human FGF2 , IF: 1:100 , Bio-Techne , NBP1-47749.

Techniques:

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: High pY levels might affect the proliferation, migration and differentiation of lung fibroblasts. A , Changes in cell proliferation between hnLFs and hfLFs determined by CCK-8 kit (n = 7). *** P < 0.001. B-C , Assessing cell proliferation by EdU assay between hnLFs and hfLFs (n = 6). *** P < 0.001. D-G , Comparison of migration between hnLFs and hfLFs (n = 6). *** P < 0.001. H , Representative images of hnLFs and hfLFs under TEM. Arrows pointed to the lipid droplets in cytoplasm. I , Representative fluorescence images of Nile Red in hnLFs and hfLFs. J-K , The contractility of hnLFs and hfLFs in 3D collagen matrices (n = 6). *** P < 0.001. L , Phospho-RTK array analysis with 200 μg of lysates from hnLFs and hfLFs, which were serum-starved for 6 h. M , Quantification of phospho-RTK levels (average of two drops fold average of positive control). N-O , Western blot and quantification of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001. P-Q , Western blot and quantification of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001. R-S , Western blot and quantification of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs (n = 6). ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Migration, CCK-8 Assay, EdU Assay, Comparison, Fluorescence, Positive Control, Western Blot

Journal: Theranostics

Article Title: Blockade of phosphotyrosine pathways suggesting SH2 superbinder as a novel therapy for pulmonary fibrosis

doi: 10.7150/thno.72269

Figure Lengend Snippet: SH2 superbinder blocked multiple fibrosis associated pathways through interrupting pY-SH2 combination in pY mediated signal transmission. A-B , Phospho-RTK array analysis with 200 μg of lysates from hfLFs treated with 1 μM GST-SH2 WT or GST-SH2 TrM for 24 h. C , Western blot of p-VEGR2, p-PDGFRβ, p-EGFR and p-FGFR1 in hnLFs and hfLFs after GST, GST-SH2 WT and GST-SH2 TrM incubation. D , Western blot of p-PLCγ1, p-GAB1, p-SHC and p-SRC in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. E , Western blot of p-AKT, p-ERK1/2 and p-STAT3 in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM incubation. F , hnLFs and hfLFs were incubated with 1 μM GST, GST-SH2 WT or GST-SH2 TrM for 24 h. The immunoprecipitation of EGFR and SHC was detected. G , Representative fluorescence images of EGFR and SHC colocalization in hnLFs and hfLFs after GST, GST-SH2 WT or GST-SH2 TrM treatment. H , Immunoprecipitations of GRB2 with GAB1, SHC, EGFR and PDGFRβ. I , GST pull down assay showed the binding capacity of SH2 superbinder with pY in hnLFs and hfLFs. J , Representative fluorescence images of GST tag and pY colocalization in hnLFs and hfLFs after incubation with GST, GST-SH2 WT or GST-SH2 TrM. K-L , GST pull down assay showed the binding capacity of SH2 superbinder with RTKs (such as VEGFR2, PDGFRβ, EGFR and FGFR1) and adaptor proteins (such as GAB1, SHC and SRC) in hfLFs and hfLFs.

Article Snippet: Antibodies against FN (Cat#15613-1-AP), COL1A1 (Cat#67288-1-lg), α-SMA (Cat#55135-1-AP, Cat#67735-1-AP), FGFR1 (Cat#60325-1-lg), CD31 (Cat#60287-1-lg), CD45 (Cat#11265-1-lg), EpCAM (Cat#60287-1-lg), Vimentin (Cat#10366-1-AP) and GAPDH (Cat#60004-1-lg) were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Transmission Assay, Western Blot, Incubation, Immunoprecipitation, Fluorescence, Pull Down Assay, Binding Assay

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: Diagrammatic representation of FGFR1 mutation from www.cbioportal.org .

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Mutagenesis

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: FGFR1 protein expression by immunohistochemistry in SCLC and their correlations with prognosis. (A ) shows FGFR1 protein positive expression; ( B ) shows FGFR1 protein negative expression. Kaplan-Meier Survival analysis of FGFR1 protein-positive vs. FGFR1 protein-negative ( C and D ).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: Clinicopathological data of the patients with SCLC and FGFR1 status

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Amplification

Journal: Journal of Cancer

Article Title: mRNA Expression of FGFR1 as Potential Marker for Predicting Prognosis of Surgical Resection of Small Cell Lung Cancer may be better than Protein Expression and Gene Amplification

doi: 10.7150/jca.44476

Figure Lengend Snippet: FGFR1 amplification by fluorescence in situ hybridization in SCLC and their correlations with prognosis. ( A ) shows FGFR1 amplification ( B ) shows FGFR1 non-amplification. Kaplan-Meier Survival analysis of FGFR1 amplified vs. non-amplified tumors ( C and D ).

Article Snippet: IHC for FGFR1 was performed using FGFR1 antibody (Cat, #BA0485, Boster Biological Technology Co. Ltd).

Techniques: Amplification, Fluorescence, In Situ Hybridization

Journal: Cancers

Article Title: Endothelial Progenitor Cells Promote Osteosarcoma Progression and Invasiveness via AKT/PI3K Signaling.

doi: 10.3390/cancers15061818

Figure Lengend Snippet: Figure 2. VEGF-A and FGF2 in the EPC-CM influence OS migration. (A) EPC-CM protein content. EPC-CM displays an angiogenic profile. CCKR, cholecystokinin receptors; EGF, epidermal growth factor; FGF, fibroblast growth factor; PI3 kinase, phosphoinositide 3 kinase; VEGF, vascular endothe- lial growth factor. (B) Migration rate of U2-OS cells cultured with EPC-CM, EPC-CM+ BV (2 mg/mL), EPC-CM+ anti FGF2 (0.08 µg/mL), and EPC-CM + BV (2 mg/mL) + anti FGF2 (0.08 µg/mL).

Article Snippet: Histological sections from human specimens were blocked with Block Buster (Background Buster, Innovex bioscience, Richmond, CA, USA) for 30 min, rinsed twice with PBS for 5 min, and immunolabeled with the following Cancers 2023, 15, 1818 7 of 20 primary antibodies: anti-CD31 antibody (1:70, primary: N = 7, Metastasis: N = 18, MA513188, Thermo Fisher Scientific, Waltham, MA, USA), anti VEGF-A (1:200, primary: N = 7, Metastasis: N = 8, ABS82, MERCK, Millipore, Tullagreen, Ireland), and anti FGF2 antibodies (1:20, primary: N = 6, Metastasis: N = 14, NB600-1536-0.025 mL, Novus biologicals, Englewood, CO, USA) for 1 h at room temperature (see Supplementary Materials Table S1).

Techniques: Migration, Cell Culture

Journal: Cancers

Article Title: Endothelial Progenitor Cells Promote Osteosarcoma Progression and Invasiveness via AKT/PI3K Signaling.

doi: 10.3390/cancers15061818

Figure Lengend Snippet: Figure 4. High VEGF-A and FGF2 labeling levels in an orthotopic OS mouse model and in human lung metastasis specimens: (A) U2-OS tumor image at 5 weeks and (B) anti-VEGF-A immunolabeling. Representative images of primary OS tumors in mice and nearby bone. The labeling pattern was mainly cytoplasmic; however, nuclear labeling was also noticed. Images were obtained under a ×40 magnification. Scale bar denotes 50 µm. (C) Quantitative analysis of immunolabeled area (tumor/bone ratio) of primary OS tumors in mice normalized to nearby bone. Primary OS tumors exhibit significantly higher VEGF-A levels compared to control bone; *** p < 0.001. (D) FGF2 immunolabeling. Representative images of primary OS tumors in mice and nearby bone. Images were obtained under ×40 magnification. Scale bar denotes 50 µm. (E) Quantitative analysis of immunolabeled area (tumor/bone ratio) of primary OS tumors in mice normalized to nearby bone. Primary OS tumors exhibit significantly higher FGF2 levels compared to control bone; ** p < 0.01. (F) Representative anti-VEGF-A and anti-FGF2 immunolabeling of human metastatic OS and non- metastatic OS patient specimens. Images showing nuclear labeling pattern. Microscope images were obtained under ×40 magnification. Scale bar denotes 50 µm. (G) Quantitative analysis of immunolabeled area (1/µm2) in metastatic vs. non-metastatic human OS specimens. Higher levels of VEGF-A (primary, N = 7; metastasis, N = 8) and FGF2 (primary, N = 6; metastasis, N = 14) were obtained in metastatic samples compared to primary tumors; * p < 0.05, and **** p < 0.0001.

Article Snippet: Histological sections from human specimens were blocked with Block Buster (Background Buster, Innovex bioscience, Richmond, CA, USA) for 30 min, rinsed twice with PBS for 5 min, and immunolabeled with the following Cancers 2023, 15, 1818 7 of 20 primary antibodies: anti-CD31 antibody (1:70, primary: N = 7, Metastasis: N = 18, MA513188, Thermo Fisher Scientific, Waltham, MA, USA), anti VEGF-A (1:200, primary: N = 7, Metastasis: N = 8, ABS82, MERCK, Millipore, Tullagreen, Ireland), and anti FGF2 antibodies (1:20, primary: N = 6, Metastasis: N = 14, NB600-1536-0.025 mL, Novus biologicals, Englewood, CO, USA) for 1 h at room temperature (see Supplementary Materials Table S1).

Techniques: Labeling, Immunolabeling, Control, Microscopy

Journal: Cancers

Article Title: Endothelial Progenitor Cells Promote Osteosarcoma Progression and Invasiveness via AKT/PI3K Signaling.

doi: 10.3390/cancers15061818

Figure Lengend Snippet: Figure 5. Proposed schematic illustration of the molecular mechanisms of the OS-EPC interaction: EPCs promote OS migration and invasion via the PI3K/AKT signaling pathway. EPCs secrete VEGF-A and FGF2 that activate, in a paracrine manner, PI3K/AKT signaling in OS cells, leading to upregulation of metastasis-related genes, including MMP9. OS cells secrete VEGF-A, which activates the pathway in an autocrine manner. PI3K inhibitor, FGF2 antibody, and Bevacizumab attenuate OS cell migration by inhibiting this pathway. The illustration was created with BioRender.com.

Article Snippet: Histological sections from human specimens were blocked with Block Buster (Background Buster, Innovex bioscience, Richmond, CA, USA) for 30 min, rinsed twice with PBS for 5 min, and immunolabeled with the following Cancers 2023, 15, 1818 7 of 20 primary antibodies: anti-CD31 antibody (1:70, primary: N = 7, Metastasis: N = 18, MA513188, Thermo Fisher Scientific, Waltham, MA, USA), anti VEGF-A (1:200, primary: N = 7, Metastasis: N = 8, ABS82, MERCK, Millipore, Tullagreen, Ireland), and anti FGF2 antibodies (1:20, primary: N = 6, Metastasis: N = 14, NB600-1536-0.025 mL, Novus biologicals, Englewood, CO, USA) for 1 h at room temperature (see Supplementary Materials Table S1).

Techniques: Migration