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: