Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A) Role of endogenous BDNF and its receptor TrkB on CRC cell proliferation: effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell proliferation. The four cell lines were cultured for 24 h in FCS-free medium (FCS 10%, −) in the presence of exogenous BDNF (+), K252a (+) alone or in combination. Cell proliferation was determined by flow cytometry analysis using EdU Alexa Fluor 488. The data are presented as histograms of proliferating cells in relative units ± SEM of five independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free medium. (B, C) Effects of exogenous BDNF and suppressing endogenous TrkB receptor on cell survival. Apoptotic ratios of soluble nucleosomes were detected by ELISA Cell for WiDr, SW480, SW620, and COLO 205 induced by serum deprivation alone (FCS 10%, −) or in association either with exogenous BDNF (+), or with K252a (+), during 24–72 h of serum deprivation. Histograms, mean ratio of apoptotic cells ± SEM of at least three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone. (D, E) apoptotic ratio after 24 h serum deprivation alone (0% FCS) or with combination with a neutralizing anti-BDNF mAb (0% anti-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone.

Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Fine-Tuning Roles of Endogenous Brain-Derived Neurotrophic Factor, TrkB and Sortilin in Colorectal Cancer Cell Survival

doi: 10.1371/journal.pone.0025097

Figure Lengend Snippet: (A, B) Sortilin as a coreceptor of p75 NTR . Double staining (yellow) of sortilin (red) and p75 NTR (green) in SW480 cells (A) and SW620 (B) after 24 h of serum deprivation. (C) Colocalization of pro-BDNF and sortilin. Confocal microscopy study of a WiDr cells stained with an anti-pro-BDNF Ab (green) and an anti-sortilin Ab (red), and double staining (yellow) after 24 h of serum deprivation. (D, E) apoptotic ratios after 24 h serum deprivation alone (0% FCS) or combined with recombinant Pro-BDNF (0% Pro-BDNF). Histograms, mean ratio of apoptotic cells ± SEM of three independent experiments. *, p <0.05; **, p <0.01; ***, p <0.001, when compared with serum-free condition alone (0% FCS).

Article Snippet: Recombinant human BDNF (100 ng/ml), recombinant pro-BDNF (4 ng/ml), and K252a (200 nM) were purchased from Alomone labs.

Techniques: Double Staining, Confocal Microscopy, Staining, Recombinant

Journal: Frontiers in Immunology

Article Title: Role of LECT2 in exacerbating atopic dermatitis: insight from in vivo and in vitro models via NF-κB signaling pathway

doi: 10.3389/fimmu.2024.1439367

Figure Lengend Snippet: Effect of LECT2 on skin manifestations and pathological changes in DNCB-induced AD-like skin lesions. (A) Scheme of the animal experimental procedure. (B) Typical skin lesions on day 23 of the animal experiment and histological analysis with hematoxylin and eosin (H&E) staining showing epidermal and dermal structures and toluidine blue staining highlighting mast cell infiltration. (C) Determination of epidermal thickness. (D) Determination of dermal thickness. (E) Number of mast cells in slides under 200× magnification field of view, with toluidine blue-stained mast cells marked with red arrows, and the number of mast cells expressed as the mean total count in three fields of view. The data shown represent mean ± SEM and are representative of at least three independent experiments. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: Recombinant mouse LECT2 was purchased from CUSABIO Bioengineering Co., Ltd. (CSB-YP012855MO; Wuhan, China) and produced using a yeast culture system.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Role of LECT2 in exacerbating atopic dermatitis: insight from in vivo and in vitro models via NF-κB signaling pathway

doi: 10.3389/fimmu.2024.1439367

Figure Lengend Snippet: Effect of LECT2 on the expression of the barrier proteins (FLG, IVL, and LOR) and the inflammatory factors (IL-1β and IL-4) in DNCB-induced AD-like skin lesions. (A) The immunostaining for each protein was brown, with darker colors representing higher expression of the protein. (B) Western Blot analysis of the levels of barrier proteins (C) FLG, (D) IVL, and (E) LOR in DNCB-induced AD-like skin lesions. WT indicates wild-type mice, and KO indicates knockout mice. The data shown represent mean ± SEM and are representative of at least three independent experiments. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: Recombinant mouse LECT2 was purchased from CUSABIO Bioengineering Co., Ltd. (CSB-YP012855MO; Wuhan, China) and produced using a yeast culture system.

Techniques: Expressing, Immunostaining, Western Blot, Knock-Out

Journal: Frontiers in Immunology

Article Title: Role of LECT2 in exacerbating atopic dermatitis: insight from in vivo and in vitro models via NF-κB signaling pathway

doi: 10.3389/fimmu.2024.1439367

Figure Lengend Snippet: Effect of LECT2 on inflammatory factor levels in DNCB-induced AD-like skin lesions and DNCB-induced mice serum. The mRNA levels of (A) TNF-α, (B) IFN-γ, (C) IL-1β, (D) IL-4, (E) IL-6, (F) IL-13, (G) TSLP, and (H) RANTES were analyzed in DNCB-induced AD-like skin lesions by RT-qPCR. The protein levels of (I) TNF-α, (J) IgE, (K) histamine, (L) IL-4, and (M) IL-13 were analyzed in DNCB-induced mouse serum by ELISA. The data shown represent mean ± SEM and are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: Recombinant mouse LECT2 was purchased from CUSABIO Bioengineering Co., Ltd. (CSB-YP012855MO; Wuhan, China) and produced using a yeast culture system.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Role of LECT2 in exacerbating atopic dermatitis: insight from in vivo and in vitro models via NF-κB signaling pathway

doi: 10.3389/fimmu.2024.1439367

Figure Lengend Snippet: Effect of LECT2 on inflammatory factor levels in TNF-α/IFN-γ-induced HaCaT cells. The mRNA levels of (A) TNF-α, (B) IL-1β, (C) IL-4, (D) IL-6, (E) IL-13, (F) TSLP, and (G) RANTES were analyzed in TNF-α/IFN-γ-induced HaCaT cells by RT-qPCR. The protein levels of (H) IL-1β, (I) IL-4, (J) IL-6, and (K) IL-13 were analyzed in TNF-α/IFN-γ-induced HaCaT cells culture supernatant by ELISA. HaCaT cells were treated with LECT2 (400 ng/ml) for 1 hour to extract total RNA for RT-qPCR, and the cells were treated for 24 hours to collect cell culture supernatant for ELISA. The data shown represent mean ± SEM and represent at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: Recombinant mouse LECT2 was purchased from CUSABIO Bioengineering Co., Ltd. (CSB-YP012855MO; Wuhan, China) and produced using a yeast culture system.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Role of LECT2 in exacerbating atopic dermatitis: insight from in vivo and in vitro models via NF-κB signaling pathway

doi: 10.3389/fimmu.2024.1439367

Figure Lengend Snippet: Effect of LECT2 on barrier protein levels and NF-κB signaling pathway in TNF-α/IFN-γ-induced HaCaT cells. (A) Western Blot analysis of the levels of barrier proteins (B) FLG, (C) IVL, and (D) LOR in TNF-α/IFN-γ-induced HaCaT cells. (E) Western Blot analysis of the protein levels of (F) p-IKBα/IKBα, and (G) p-P65/P65 in total proteins of TNF-α/IFN-γ-induced HaCaT cells. (H) Western Blot analysis of the protein levels of (I) P65 in cytoplasmic proteins and (J) P65 in nuclear proteins of TNF-α/IFN-γ-induced HaCaT cells. (K) Immunofluorescence staining for P65 protein (labeled with Cy3, red). HaCaT cells were counterstained with DAPI (blue). HaCaT cells were treated with LECT2 (400 ng/ml) for 24 hours to extract total, cytoplasmic and nuclear proteins for Western Blot. The data shown represent mean ± SEM and represent at least three independent experiments. Scale bar: 20 μm * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: Recombinant mouse LECT2 was purchased from CUSABIO Bioengineering Co., Ltd. (CSB-YP012855MO; Wuhan, China) and produced using a yeast culture system.

Techniques: Western Blot, Immunofluorescence, Staining, Labeling

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF improves native collateral blood flow during AMI . A , Representative myocardial perfusion images of the control, vector, and PEDF group during AMI by positron emission tomography scan (arrows indicate perfusion in ischemic areas). B , Quantification of ischemic myocardial volume, * P <0.05 vs the indicated group. C , Quantification of SUV ‐mean, * P <0.05 vs the indicated group. D , Quantification of ischemic myocardial volume in AMI 4 hours, * P <0.05, not significant ( NS ), P >0.05 vs the control group. E , Quantification of SUV ‐mean in AMI 4 hours. *** P <0.001, NS , P >0.05 vs the control group. F , Quantification of the expansion of ischemic myocardial volume (delta ischemic myocardial volume), NS , P >0.05, * P <0.05 vs the control group. G , Quantification of the decline of SUV ‐mean (delta SUV ‐mean), NS >0.05, * P <0.05 vs the control group, n (control)=8; n (vector and PEDF )=5, each bar represents the mean± SEM . AMI indicates acute myocardial infarction; NS , not significant; PEDF , pigment epithelium–derived factor; PET , positron emission tomography; SUV , standardized uptake volume.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: Control, Plasmid Preparation, Positron Emission Tomography, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF promotes the process of ventricular remodeling and protects the ischemic myocardium and cardiac function. A , Representative figures of the Evans blue/2,3,5‐triphenyltetrazolium–stained myocardial tissues in each indicated experimental condition, with quantification of the infarct size, *** P <0.001 vs the sham group, ### P <0.001 vs the control group, NS , P >0.05 vs the indicated group, n=6. B , Cardiac function was measured by transthoracic M‐mode echocardiography. C , Left ventricular ejection fraction ( EF %), ** P <0.01, vs the sham group, ## P <0.01, vs the control group, NS , P >0.05 vs the indicated group. D , Left ventricular fractional shortening ( FS %), *** P <0.05, vs the sham group, ## P <0.01, vs the control group, NS , P >0.05 vs the indicated group, n=6. E , Representative Masson staining of the heart sections 1 day‐4 weeks post‐ AMI ; the black arrows indicate the infarct areas. The results of AMI 3 days are shown in the blue box; the results of AMI 4 weeks are shown in the red box, bar=50 μm. F , Quantitative analysis of mean‐left ventricular wall thickness (mean‐ LVWT ), ** P <0.01, NS , P >0.05 vs the control group. G , Quantitative analysis of the fibrotic area, ** P <0.01, *** P <0.001 vs the indicated group, n=6. AMI indicates acute myocardial infarction; NS , not significant; PEDF , pigment epithelium–derived factor.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: Staining, Control, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF induces the CCMR remodeling in vivo. A , Representative figures of CCMR in the infarct area in each indicated experimental condition (bar=50 μm). B , Quantification of the density of CCMR , * P <0.05, *** P <0.001 vs the respective result of 5 minutes, *** P <0.001 vs the indicated group. C , Total cross‐sectional area of CCMR , NS , P >0.05, *** P <0.001 vs the control group. D , Quantification of the mean diameter of CCMR , NS , P >0.05, *** P <0.001 vs the control group, n=6. E , Representative image of the lanthanum nitrate perfusion experiment (bar=1 μm). F , Confocal immunofluorescence analysis of the expression and distribution of VE ‐cadherin in CCMR vessels (bar=10 μm). CCMR indicates coronary collateral microcirculation reserve; DAPI, 40,6‐diamidino‐2‐phenylindole; NS, not significant; PEDF, pigment epithelium‐derived factor; VE‐cadherin, vascular endothelial‐cadherin.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: In Vivo, Control, Immunofluorescence, Expressing, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF remodels nascent blood vessels in vitro. A and B , Experimental protocol of HCAEC tube formation assay and cardiac explant Matrigel assay. C , HCAEC tube formation in Matrigel in response to pre‐ PEDF or to post‐ PEDF . D , Percentage of area covered with tubes, * P <0.05, ** P <0.01, *** P <0.001 vs the respective result of 12 hours, ### P <0.001 vs the indicated group, n=4 to 6 (bar=50 μm). E , Cardiac explant Matrigel assay. F , Quantification of density of area covered with nascent vessels, * P <0.05, *** P <0.001 vs the respective result of 3 days. ### P <0.001 vs the indicated group. G , Quantification of the average diameter of nascent vessels, * P <0.05, *** P <0.001, NS , P >0.05 vs the respective result of 3 days, ### P <0.001 vs the indicated group, n=6 (bar=50 μm). HCAEC indicates human coronary artery endothelial cells; NS , not significant; PEDF , pigment epithelium–derived factor.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: In Vitro, Tube Formation Assay, Matrigel Assay, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF induces AJ s that are better organized on membranes and enhanced their tolerance to hypoxia in HCAEC tubes. A , Representative immunofluorescence staining images of HCAEC tubes (bar=50 μm [left], bar=10 μm [right]). B , Western blot determination of VEGFR 2 and VEGFR 3 protein expression, ** P <0.01, *** P <0.001 vs the indicated group. C , Immunofluorescence images of VE ‐cadherin of HCAEC tubes (bar=10 μm). D , Western blot determination of VE ‐cadherin in membrane and cytoplasm under normoxia or OGD , * P <0.05, ** P <0.01 vs the indicated group, n=3. E , Verification of HIF ‐1α expression, NS , P >0.05, * P <0.05 vs the indicated group, ## P <0.01 vs the normal group, n=5. AJs indicates adherens junctions; HCAEC , human coronary artery endothelial cells; HIF ‐1α, hypoxia‐inducing factor‐1α; NS , not significant; OGD , glucose and oxygen deprivation; PEDF , pigment epithelium–derived factor; VE ‐cadherin, vascular endothelial‐cadherin; VEGFR 2, vascular endothelial growth factor receptor 2.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Membrane, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: PEDF induces nascent blood vessel remodeling that may be associated with the NO and Notch‐1 pathway. A , Western blotting showing the levels of protein related to FSS in myocardial tissue and HCAEC s. B , Quantitative analysis of protein levels in myocardial tissue, * P <0.05, ** P <0.01, vs the control group, respectively. C , Quantitative analysis of protein levels in HCAEC s, * P <0.05, ** P <0.01, vs the control group, respectively. D , HCAEC tube formation in Matrigel in response to IMR ‐1 or l ‐ NMMA , *** P <0.001 vs the control group, ## P <0.01, NS , P >0.05 vs the PEDF group, n=6. E , Quantification of the nascent vessel area, *** P <0.001 vs the control group, # P <0.05, ## P <0.01, NS , P >0.05 vs the PEDF group, n=6. F , Quantification of the average diameter, *** P <0.001 vs the control group, # P <0.05, NS , P >0.05 vs the PEDF group, n=6. G , Western blot determination of VEGFR 2 and VEGFR 3 protein. H , Quantification of VEGFR 2 and VEGFR 3 protein levels, NS , P >0.05 vs the indicated group, n=5. I , Western blot determination of VE ‐cadherin in membrane and cytoplasm in response to IMR ‐1 or l ‐ NMMA , * P <0.05, NS , P >0.05 vs the indicated group, n=5. FSS indicates fluid shear force; HCAEC , human coronary artery endothelial cells; IMR ‐1, inhibitor of mastermind recruitment‐1; l ‐ NMMA , l ‐nitromonomethylarginine; NS , not significant; PEDF , pigment epithelium–derived factor; VEGFR 2/3, vascular endothelial growth factor receptor 2/3.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: Western Blot, Control, Membrane, Shear, Derivative Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pigment Epithelium–Derived Factor Increases Native Collateral Blood Flow to Improve Cardiac Function and Induce Ventricular Remodeling After Acute Myocardial Infarction

doi: 10.1161/JAHA.119.013323

Figure Lengend Snippet: Oxidative stress does not participate in the collateral remodeling induced by PEDF . A , mt ROS production was monitored by Mito SOX ™ Red in HCAEC s, with ( B ) quantification. ROS production was observed by red fluorescence of Mito SOX ™ by fluorescence microscopy and analyzed by Image‐Pro Plus software (scale bar=50 μm; n=5). C , NOX activity was assessed in all experimental groups using the NOX activity assay kit (n=5). *** P <0.001, NS, P >0.05 vs the normal group. HCAEC indicates human coronary artery endothelial cells; NOX , NADPH oxidase; NS , not significant; OGD , oxygen‐glucose deprivation; PEDF , pigment epithelium–derived factor; ROS , reactive oxygen species.

Article Snippet: Recombinant rat PEDF (GenBank accession number: NM_177927 ) was synthesized by Cusabio Biotech, Co, Ltd (Wuhan, China).

Techniques: Fluorescence, Microscopy, Software, Activity Assay, Derivative Assay