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: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: Endothelial cells (BME-1) were cultured on Matrigel®-coated surfaces and incubated either without (A, C and E) or with the addition of FGF2 and VEGF-165 (B, D and F) for 3 hrs. Panels A and B show phase contrast images, panels C and D show computer-generated projections of panels A and B used for quantification and panels E and F show the superimposed images. Panel G shows quantification of tube length (mean ± SEM of triplicate determinations in each case) as indicated. *** p < .001 relative to cells receiving no growth factors (left four bars) or to cells receiving growth factors without HIP/RPL29 (right three bars).

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Cell Culture, Incubation, Generated

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: Mouse aortic outgrowth assays were performed and quantified by computer-based morphometric analyses as described in Materials and Methods. Panels A–F show 8 day outgrowths from cultures grown in the presence of EBM with the following additions: A, none (control); B. 40 ng/ml FGF2; C, 40 ng/ml VEGF-165; D, 40 μg/ml HIP/RPL29; E, 40 ng/ml FGF2 plus 40 μg/ml HIP/RPL29; F, 40 ng/ml VEGF-165 plus 40 μg/ml HIP/RPL29. Panel G shows the quantitation of results of these types of assays and demonstrates near complete inhibition of outgrowth in the presence of HIP/RPL29 in all cases. * p < 0.001 vs. corresponding growth factor treatment in the absence of HIP/RPL29.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Control, Quantitation Assay, Inhibition

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 (A) or VEGF-165 (B) were preincubated with perlecan domain I in a solid phase assay, unbound growth factor rinsed off and the substrates subsequently incubated with the indicated concentrations of HIP/RPL29 (HIP; filled circles) or lysozyme (LYS; open circles) for 2 hr. The surface was rinsed again and bound growth factor determined by ELISA as described in Materials and Methods. The points indicate the means ± SEM of triplicate determinations from a representative experiment.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 was bound to a solid phase perlecan domain I substrate as described in Materials and Methods and Figure 5. In panel A, complexes subsequently were incubated with HPSE at the indicated concentrations for 24 hr, rinsed to remove unbound/released FGF2 and bound FGF2 measured by ELISA as described in Materials and Methods. The negative control was the perlecan domain I substrate not exposed to FGF2. In panel B, 5 μg/ml HPSE was incubated in the presence of the FGF2 bound to perlecan domain I for 24 hr at pH 5 or pH 7.2 as indicated and FGF2 release assayed by ELISA as described in Materials and Methods. FGF2 that remained bound at either pH 5.0 or 7.2 in the absence of HPSE served as a buffer only control. The bars represent the means ± SEM of triplicate determinations in each case.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control, Control

Journal: Journal of cellular biochemistry

Article Title: HIP/RPL29 Antagonizes VEGF and FGF2 Stimulated Angiogenesis by Interfering with HS-dependent Responses

doi: 10.1002/jcb.21899

Figure Lengend Snippet: FGF2 was bound to a solid phase perlecan domain I substrate as described in the legend to Figure 5. This complex subsequently was incubated with HPSE (5 μg/ml) alone or in the presence of 0.5, 5 or 40 μg/ml HIP/RPL29 as indicated on the figure. After 24 hr, the surfaces were rinsed to remove unbound/released FGF2 and bound FGF2 measured by ELISA as described in Materials and Methods. The negative control was the perlecan domain I substrate not exposed to FGF2. The bars represent the means ± SEM of triplicate determinations from a representative experiment in each case.

Article Snippet: After washing three times with 0.05% (v/v) Tween 20 in PBS, the bound FGF2 and VEGF-165 were identified with 2 μg/ml biotinylated anti-recombinant human FGF2 antibody (R&D Systems, BAM-233) and 3 μg/ml biotinylated anti-recombinant human VEGF antibody (R&D Systems, BAF-293), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Negative Control

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

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 3 Neurotrophin secretion from PBMC cultures from 15 patients before and 12 months after alemtuzumab treatment. Cultures were either unstimulated (unstim) or stimulated with the myelin antigen, MBP or polyclonally stimulated with anti-CD3/anti-CD28 antibodies (CD3/28). Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) FGF, and (E) insulin-like growth factor-1. Error bars represent 95% confidence intervals. (*P50.05, **P50.01, ***P50.001).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Derivative Assay

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 4 Peripheral blood mononuclear cell neurotrophin secretion from three patients 12 months after alemtuzumab in response to a wide variety of antigens. PBMCs were cultured either unstimulated (unstim) or stimulated with; MBP, tetanus toxoid (TT), myelin oligodendrocyte glycoprotein (MOG), collagen type II fragment aa245–270 (Collagen), myelin basic peptide aa87–99 (MBP-P), keyhole limpet haemocyanin (KLH) or recombinant human insulin (Insulin). Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) insulin-like growth factor-1 and (E) FGF (*P50.05).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Cell Culture, Recombinant, Derivative Assay

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 5 Peripheral blood mononuclear cell neurotrophin secretion, induced by MBP stimulation. PBMCs from 15 healthy controls (HC), 15 patients before (pre) and at three time points (6, 9 and 12 months) after alemtuzumab and from 10 patients treated with interferon b-1a (rebif), were cultured with MBP. Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) insulin-like growth factor-1 and (E) FGF. Error bars represent 95% confidence intervals (**P50.01, ***P50.001).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Cell Culture, Derivative Assay

Journal: General physiology and biophysics

Article Title: Tripterine emerges as a potential anti-scarring agent in NIH/3T3 cells by repressing ANRIL.

doi: 10.4149/gpb_2019050

Figure Lengend Snippet: Figure 2. Effect of tripterine treatment on growth of NIH/3T3 cells. A. Cell viability of NIH/3T3 cells after tripterine (2–10 μM, treatment for 4 h) administration by cell counting kit-8 assay. B. Migratory NIH/3T3 cells after tripterine (6 μM, treatment for 4 h) by 24-Transwell assay. Protein expression of Cyclin D1 (C), MMP-2 and MMP-9 (D) as well as VEGF and bFGF (E) in NIH/3T3 cells treated with 6 μM tripterine for 4 h assayed by Western blot assay. Data represented means ± SD. ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. TPR, tripterine; CTRL, control; MMP, matrix metallopeptidase; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor.

Article Snippet: The polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) carrying protein imprints were blocked with bovine serum albumin (BSA) (Thermo Fisher Scientific) for 1 h at room temperature, followed by co-incubation with primary antibodies against cyclin D1 (ab16663; Abcam, Cambridge, MA, USA; 1:100), MMP2 (87809; Cell Signaling Technology, CST, Danvers, MA, USA; 1:1,000), MMP-9 (ab38898; Abcam; 1:1,000), vascular endothelial growth factor (VEGF) (MAB4931; R&D Systems, Abingdon, UK; 1 μg/ml), basic fibroblast growth factor (bFGF) (NBP1-18579; Novus Biologicals, Colombia, USA; 1:1,000), β-actin (4967; CST; 1:1,000), total-p65 (8242; CST; 1:1,000), phospho (p) Ser536-p65 (3033; CST; 1:1,000), total-inhibitor of nuclear factor kappa-B alpha (IκBα) (4812; CST; 1:1,000), pSer32-IκBα (2859; CST; 1:1,000).

Techniques: Cell Counting, Transwell Assay, Expressing, Western Blot, Control

Journal: General physiology and biophysics

Article Title: Tripterine emerges as a potential anti-scarring agent in NIH/3T3 cells by repressing ANRIL.

doi: 10.4149/gpb_2019050

Figure Lengend Snippet: Figure 4. Effect of tripterine on the growth of ANRIL-overexpressed NIH/3T3 cells. NIH/3T3 cells were transfected with or without ANRIL, empty vector as a negative control, and then treated with 6 μM tripterine for 4 h. A. ANRIL level assayed by qRT-PCR. B. Cell viability evaluated by cell counting kit-8 assay. C. Cyclin D1 protein expression quantified by Western blot method. D. Migration behav iors assessed by 24-Transwell assay. MMPs (MMP-2 and MMP-9; E) as well as VEGF and bFGF (F) examined by Western blot analysis. Data represented means ± SD. * p < 0.05, ** p < 0.01. MMP, matrix metallopeptidase; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor. For more abbreviations, see Fig. 3.

Article Snippet: The polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) carrying protein imprints were blocked with bovine serum albumin (BSA) (Thermo Fisher Scientific) for 1 h at room temperature, followed by co-incubation with primary antibodies against cyclin D1 (ab16663; Abcam, Cambridge, MA, USA; 1:100), MMP2 (87809; Cell Signaling Technology, CST, Danvers, MA, USA; 1:1,000), MMP-9 (ab38898; Abcam; 1:1,000), vascular endothelial growth factor (VEGF) (MAB4931; R&D Systems, Abingdon, UK; 1 μg/ml), basic fibroblast growth factor (bFGF) (NBP1-18579; Novus Biologicals, Colombia, USA; 1:1,000), β-actin (4967; CST; 1:1,000), total-p65 (8242; CST; 1:1,000), phospho (p) Ser536-p65 (3033; CST; 1:1,000), total-inhibitor of nuclear factor kappa-B alpha (IκBα) (4812; CST; 1:1,000), pSer32-IκBα (2859; CST; 1:1,000).

Techniques: Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Cell Counting, Expressing, Western Blot, Migration, Transwell Assay