Journal: Oxidative medicine and cellular longevity

Article Title: The Interaction of Apelin and FGFR1 Ameliorated the Kidney Fibrosis through Suppression of TGF β -Induced Endothelial-to-Mesenchymal Transition.

doi: 10.1155/2023/5012474

Figure Lengend Snippet: Figure 1: Proximity between apelin and FGFR1 suppresses the TGFβ/Smad signaling pathway in HMVECs. (a) HMVECs were treated with TGFβ2 (5 ng/mL) or N-FGFR1 (1.5 μg/mL) for 48 h with or without preincubation with apelin (100 nM) for 2 h. The proximity between apelin and FGFR1 was then analyzed by the Duolink In Situ Assay. For each slide, images at a ×400 original magnification were obtained from six different areas. The scale bar is 60 μm in each panel. (b) HMVECs were treated with TGFβ2 (5 ng/mL) or with apelin (100 nM) for 48 h, and the p-Smad3, TGFβR1, TGFβR2, and FGFR1 levels were analyzed by western blot. Densitometric analysis of the p-Smad3/Smad3, TGFβR1/β-actin, TGFβR2/β-actin, and FGFR1/β-actin levels from each group (n = 5) was analyzed. (c) HMVECs were treated with or without apelin (100 nM) for 48 h, and the FGFR1 levels were analyzed by western blot. Densitometric analysis of the FGFR1/GADPH levels from each group (n = 5) was analyzed.

Article Snippet: Human neutralizing FGFR1 (MAB765) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: In Situ, Western Blot

Journal: Oxidative medicine and cellular longevity

Article Title: The Interaction of Apelin and FGFR1 Ameliorated the Kidney Fibrosis through Suppression of TGF β -Induced Endothelial-to-Mesenchymal Transition.

doi: 10.1155/2023/5012474

Figure Lengend Snippet: Figure 4: CEBPA knockdown promotes TGFβ-mediated EndMT. (a) HMVECs were transfected with or without CEBPA siRNA for 48 h in the presence or absence of TGFβ2, and the VE-cadherin, α-SMA, vimentin, and SM22α levels were analyzed by western blot. Densitometric analysis of the VE-cadherin/GADPH, α-SMA/GADPH, vimentin/GADPH, and SM22α/GADPH levels from each group (n = 5) was analyzed. (b) HMVECs were transfected with CEBPA siRNA for 48 h in the presence or absence of TGFβ2 and apelin, and the VE- cadherin, α-SMA, vimentin, and SM22α levels were analyzed by western blot. Densitometric analysis of the VE-cadherin/GADPH, α- SMA/GADPH, vimentin/GADPH, and SM22α/GADPH levels from each group (n = 5) was analyzed. (c) HMVECs were treated with TGFβ2 for 15 min or 48 h with or without preincubation with apelin for 2 h, and the CEBPA levels were analyzed by western blot. Densitometric analysis of the CEBPA/GADPH level from each group (n = 5) was analyzed. (d) HMVECs were treated with N-FGFR1 for 48 h or 15 min in the presence or absence of apelin, and the CEBPA levels was analyzed by western blot. Densitometric analysis of the CEBPA/GADPH level from each group (n = 5) was analyzed. Immunofluorescence analysis of CD31 (e) and α-SMA (f) coexpression in HUVECs following TGFβ2 or/and apelin or/and CEBPA siRNA treatment. For each slide, images of six different fields of view at ×200 magnification were evaluated. The scale bar is 50 μm in each panel.

Article Snippet: Human neutralizing FGFR1 (MAB765) was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Knockdown, Transfection, Western Blot

Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Intact processing and biological function of GFP-tagged neurotrophins. GFP-tagged NTs were expressed in COS7 cells, and cell lysates and supernatants were analyzed 2 d after transfection. A, Anti-GFP Western blot reveals dominant presence of the NT precursors in the cell lysates (top, arrows, NT-4 precursor with lower MW than the other NTs) and of the correctly processed mature NTs in the COS cell supernatants (bottom, arrow). The horizontal marks at the left indicate the position of GFP. B, NT-induced fiber outgrowth of PC12 cells via overexpressed TrkB and endogenously expressed TrkA receptors, respectively. PC12 cells were cotransfected at 1 DIV with TrkB and GFP plasmids and were incubated for 3 d with supernatants from COS cells overexpressing (for 2 d) GFP-tagged BDNF, NGF, NT-3, NT-4, or wt GFP, respectively, or were incubated with rhBDNF. C, Quantification of NT-induced fiber outgrowth of PC12 cells after 3 d of incubation with COS cell supernatants and recombinant (rec.) neurotrophins. **Significantly different from all NTs with p < 0.01. D, Anti-BDNF Western blot of COS cells expressing wt BDNF and BDNF-GFP, respectively, and of human recombinant BDNF. The blot indicates comparable amounts of wt BDNF and BDNF-GFP in the supernatants used in E. E, PC12 cell fiber outgrowth assay of supernatants and recombinant BDNF analyzed in D. *Significantly different from EGFP with p < 0.01. Data in C and E are from >100 cells per experiment and from more than two independent preparations for each condition. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Transfection, Western Blot, Incubation, Recombinant, Expressing

Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Neutralization of pH in secretory granules by monensin before depolarization speeds up the release of BDNF. Hippocampal neurons were transfected with GFP-tagged NTs at 8 DIV and used for secretion 2 d after transfection A, BDNF-GFP-expressing neuron. B, Time series of the marked box in A at higher magnification. C, Analysis of fluorescence intensity of regions marked in B. Note the pronounced increase in fluorescence intensity after application of monensin (4 μm), which neutralizes pH in previously acidic granules. The letters a- e refer to the pictures shown in B. Note the faster time course of BDNF secretion (compare Fig. 11) and the lack of GFP unquenching after K+-induced depolarization. rel., Relative; stimul., stimulation. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Neutralization, Transfection, Expressing, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Differential Vesicular Targeting and Time Course of Synaptic Secretion of the Mammalian Neurotrophins

doi: 10.1523/JNEUROSCI.1776-05.2005

Figure Lengend Snippet: Average time course of NT secretion after neutralization of intragranular pH with monensin. A-D, Hippocampal neurons expressing the different GFP-tagged NTs were treated as in Figure 9. Monensin (4 μm)-induced fluorescence increase and acceleration of the time course of release during subsequent depolarization was observed for all NTs (compare Fig. 11). The τ values represent the results of monoexponential fit curves for n ≥ 4 cells for each NT. E, Normalized average depolarization induced (50 mm K+) fluorescence decrease for indicated NTs after preincubation with monensin and for FM4-64 destaining of synaptic vesicles, respectively. The vertical line indicates start of depolarization. Average over n ≥ 4 cells for each condition. F, The boxed area in E at an expanded time scale. Note the similar half decay times (gray circles) for all NTs under these conditions but the still markedly faster speed of FM4-64 release from synaptic vesicles. Stimul., Stimulation. Error bars represent SEs.

Article Snippet: Recombinant human NTs were purchased from Alamone Labs. Monensin was obtained from Sigma.

Techniques: Neutralization, Expressing, Fluorescence

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Osteoblast-Derived ECM1 Promotes Anti-Androgen Resistance in Bone Metastatic Prostate Cancer.

doi: 10.1002/advs.202407662

Figure Lengend Snippet: Figure 3. ECM1 activates the MAPK signaling pathway in PCa cells. A) Waterfall plot showing differentially expressed genes (p < 0.05, log2 fold change>1.5) in C4-2B cells treated with ENZ (10 μM, 48 h) combined with ECM1 protein (200 ng mL−1, 48 h) or ENZ (10 μM, 48 h) alone. The highlighted genes were related to proliferation and apoptosis. B) KEGG analysis of pathways enriched in ENZ combined with ECM1 protein treatment group compared to the ENZ treatment group. C) WB analysis of MEK, p-MEK, ERK1/2, and p-ERK1/2 expression in the indicated groups of C4-2B cells. D) IHC staining and quantification of p-ERK1/2 expression in mice intratibial and subcutaneous tumors grouped as indicated (Scale bars, 500 μm and 100 μm, n = 6 per group). E) WB analysis and quantification of MEK, p-MEK, ERK1/2, and p-ERK1/2 expression in C4-2B cells stimulated with ENZ (10 μM) combined with ECM1 (200 ng mL−1) in the presence of inhibitors for either RAS (MCP110, 10 μM), MEK (U0126, 10 μM), ERK1/2 (Ulixertinib, 10 μM), EGFR (Lapatinib, 10 μM), FGFR1 (Fexagratinib, 10 μM), IGF1R (Linsitinib, 10 μM) or Veh (DMSO), compared to ENZ-treated alone or untreated C4-2B cells. F) C4-2B cell proliferation on day 7 of groups as shown in E. G) WB analysis and quantification of EGFR, p-EGFR, FGFR1, p-FGFR1, IGF1R and p-IGF1R expression in groups as indicated. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: GAPDH was used as the loading control. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. nology; #4695, 1:1000), Phospho-ERK1/2 (Thr202/Tyr204) Rabbit mAb (Cell Signaling Technology; #4370, 1:2000), EGFR Rabbit mAb (Cell Signaling Technology; #4267, 1:1000), Phospho-EGFR (Tyr978) Rabbit mAb (Cell Signaling Technology; #3790, 1:1000), IGF1R Rabbit mAb (Cell Signaling Technology; #9750, 1:1000), Phospho-IGF1R Rabbit mAb (Cell Signaling Technology; #3918, 1:1000), FGFR1 Mouse mAb (Proteintech; 60325-1-Ig, 1:1000), Phospho-FGFR1 (Tyr653/Tyr654) Rabbit pAb (Sigma Aldrich; 06– 1433, 1:1000) and Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Cell Signaling Technology; #9411, 1:2000).

Techniques: Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: Sorsby Fundus Dystrophy Mutation in Tissue Inhibitor of Metalloproteinase 3 (TIMP3) promotes Choroidal Neovascularization via a Fibroblast Growth Factor-dependent Mechanism

doi: 10.1038/s41598-019-53433-6

Figure Lengend Snippet: Increased bFGF in the CM of RPE cells expressing mutant TIMP3 is mediated by MMPs. ELISA was used to quantitate bFGF levels in the ( a ) CM and ( b ) ECM of RPE cells expressing control empty vector (V), wild-type TIMP3 (W1) or mutant TIMP3 (M1,M5) ( c ) Effect of an MMP2/9 inhibitor (SB-3CT, 5 μg/ml for16 hours) on bFGF secretion was evaluated in RPE cells expressing wild-type or mutant TIMP3. VEGF levels were quantitated in the ( d ) CM and ( e ) ECM of RPE cells expressing control empty vector (V), wild-type TIMP3 (W1) or mutant TIMP3 (M1,M5). Effect of MMP2/9 inhibitor (SB-3CT, 5 μg/ml for16 hours) on VEGF secretion was evaluated in RPE cells. Data are expressed as means ± SD (n = 3). **P ≤ 0.01 vs Vector.

Article Snippet: Antibodies: Anti-bFGF antibody (neutralizing) (EMD Millipore/Calbiochem, Darmstadt Germany); Monoclonal anti-TIMP3 antibody (EMD Millipore/Chemicon, Temecula CA); FGFR1 rabbit polyclonal antibody and phosphor FGFR1 rabbit monoclonal antibody (Epitomics, Inc. Burlingame, CA); Anti-MAP kinase and anti-MAP kinase, phospho-specific rabbit polyclonal antibodies (EMD Millipore/Calbiochem, Darmstadt Germany).

Techniques: Expressing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Scientific Reports

Article Title: Sorsby Fundus Dystrophy Mutation in Tissue Inhibitor of Metalloproteinase 3 (TIMP3) promotes Choroidal Neovascularization via a Fibroblast Growth Factor-dependent Mechanism

doi: 10.1038/s41598-019-53433-6

Figure Lengend Snippet: Decreased TEER and increased apical secretion of bFGF in RPE cells expressing mutant TIMP3. Box and whisker plots depict comparisons between RPE cells expressing empty vector (Vector), wild-type TIMP3 (WT) or mutant S179C-TIMP3 (Mutant) of ( a ) TEER ( b ) FGF2 concentration in the apical conditioned medium ( c ) FGF2 concentration in the basal conditioned medium ( d ) ratio of apical/basal FGF2 (n = 6–8). **p ≤ 0.01 vs Vector.

Article Snippet: Antibodies: Anti-bFGF antibody (neutralizing) (EMD Millipore/Calbiochem, Darmstadt Germany); Monoclonal anti-TIMP3 antibody (EMD Millipore/Chemicon, Temecula CA); FGFR1 rabbit polyclonal antibody and phosphor FGFR1 rabbit monoclonal antibody (Epitomics, Inc. Burlingame, CA); Anti-MAP kinase and anti-MAP kinase, phospho-specific rabbit polyclonal antibodies (EMD Millipore/Calbiochem, Darmstadt Germany).

Techniques: Expressing, Mutagenesis, Whisker Assay, Plasmid Preparation, Concentration Assay

Journal: Scientific Reports

Article Title: Sorsby Fundus Dystrophy Mutation in Tissue Inhibitor of Metalloproteinase 3 (TIMP3) promotes Choroidal Neovascularization via a Fibroblast Growth Factor-dependent Mechanism

doi: 10.1038/s41598-019-53433-6

Figure Lengend Snippet: Increased induction of endothelial cell tube formation by CM of RPE cells expressing S179C-TIMP3 is mediated by bFGF. ( a ) Parental porcine aortic endothelial cells (Parental PAE, top panel) and PAE expressing FGF receptor-1 (PAE/FGFR-1, 2 nd panel) were embedded in three-dimension (3D) collagen gels and incubated with conditioned medium (CM) of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5) in the presence of 10 μg/ml anti-bFGF neutralizing antibody (third panel) or a MMP2/9 inhibitor (SB-3CT, 5 μg/ml) for 48 hours. Differentiated cells were photographed using an inverted microscope (original magnification 200x) and ( b ) quantitated using customized macros generated in Image-Pro Plus 5.1. Data are presented as means ± SD n = 3. *p ≤ 0.05 vs vector control **p ≤ 0.01 vs vector control. ( c ) RF6A monkey choroid endothelial cells were subjected to tube formation assay in the presence of conditioned medium (CM) of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5) in the presence of anti-bFGF or SB-3CT (lower panel) and compared to the absence of inhibitors (upper panel). ( d ) Migration of RF6A choroidal endothelial cells towards conditioned medium of RPE cells expressing empty vector (V), wild-type TIMP3 (W1) or S179C-TIMP3 (M1, M5). Migrating cell number is expressed as mean +/− SD of quadruplicate samples.

Article Snippet: Antibodies: Anti-bFGF antibody (neutralizing) (EMD Millipore/Calbiochem, Darmstadt Germany); Monoclonal anti-TIMP3 antibody (EMD Millipore/Chemicon, Temecula CA); FGFR1 rabbit polyclonal antibody and phosphor FGFR1 rabbit monoclonal antibody (Epitomics, Inc. Burlingame, CA); Anti-MAP kinase and anti-MAP kinase, phospho-specific rabbit polyclonal antibodies (EMD Millipore/Calbiochem, Darmstadt Germany).

Techniques: Expressing, Incubation, Plasmid Preparation, Inverted Microscopy, Generated, Tube Formation Assay, Migration