Journal: Redox Report : Communications in Free Radical Research

Article Title: Verbascoside targets endothelial HIF-1α/ Lysyl oxidase signaling to attenuate glomerular injury in diabetic nephropathy

doi: 10.1080/13510002.2025.2598110

Figure Lengend Snippet: LOX family upregulation in diabetic nephropathy is endothelial-specific. (a) Transcriptomic analysis of LOX family genes ( Lox, Loxl1 , Loxl2 , Loxl3 , and Loxl4 ) in db/db diabetic mouse kidneys vs. controls, showing upregulation of Lox , Loxl1, and Loxl2 . n = 4 (b) qPCR validation confirming increased renal mRNA levels of Lox , Loxl1, and Loxl2 in the DN model group vs. normal controls. n = 5. (c–f) Immunofluorescence co-localization analysis specifying upregulation of LOX and LOXL2 protein expression within kidney endothelial cells of db/db mice compared to controls. n = 4. White arrows indicate the expressions of LOX and LOXL2 outside the glomerular endothelial cells. (g) qPCR analysis demonstrates induction of Lox , Loxl1 , Loxl2 , and Loxl4 mRNA in mouse glomerular endothelial cells exposed to high glucose vs. normal glucose. n = 5. * p < 0.05, *** p < 0.001. Error bars indicate SD.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using the following: LOX antibody (abcam, ab-174316, 1:100), LOXL2 antibody (leading biology, LBIRA1098, 1:100), CD31 antibody (Santa Cruz, sc-18916, 1:250).

Techniques: Biomarker Discovery, Immunofluorescence, Expressing

Journal: Redox Report : Communications in Free Radical Research

Article Title: Verbascoside targets endothelial HIF-1α/ Lysyl oxidase signaling to attenuate glomerular injury in diabetic nephropathy

doi: 10.1080/13510002.2025.2598110

Figure Lengend Snippet: Proteomic profiling of the glomerular endothelial cell secretome under high glucose. (a) GO enrichment analysis of conditioned media from high glucose-treated GECs showing significant enrichment in pathways related to oxidative stress, lipid peroxidation, and protein oxidation. (b,c) GSEA confirming significant upregulation of oxidative stress and ECM-receptor interaction signaling pathways in HG-treated GECs. (d) Cross-referencing transcriptomic and proteomic datasets identifying commonly upregulated and commonly downregulated genes/proteins in diabetic kidneys and HG-EC CM. (e,f) Significant elevation of secreted LOX and LOXL2 proteins in HG-EC CM vs. control CM. n = 3; * p < 0.05, ** p < 0.01. Error bars indicate SD.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using the following: LOX antibody (abcam, ab-174316, 1:100), LOXL2 antibody (leading biology, LBIRA1098, 1:100), CD31 antibody (Santa Cruz, sc-18916, 1:250).

Techniques: Protein-Protein interactions, Control

Journal: Redox Report : Communications in Free Radical Research

Article Title: Verbascoside targets endothelial HIF-1α/ Lysyl oxidase signaling to attenuate glomerular injury in diabetic nephropathy

doi: 10.1080/13510002.2025.2598110

Figure Lengend Snippet: Inhibition of endothelial LOX/LOXL2 protects mesangial cells from high glucose-induced damage. (a–c) Suppression of mesangial cell proliferation and viability by conditioned media from HG-exposed GEnCs treated with BAPN (HG + BAPN-CM) or PAT-1251 (HG + PAT-1251-CM), compared to damaging HG-CM. n = 5. (d,e) Attenuation of HG-CM-induced lipid peroxidation and protein carbonylation in mesangial cells by endothelial LOX/LOXL2 inhibition. n = 5. (f,g) Suppression of fibrotic gene expression (FN1, Col4a1) in mesangial cells exposed to inhibitor-treated CM. n = 5. (h,i) Reduced collagen deposition in mesangial cells treated with BAPN-CM or PAT-1251-CM vs. HG-CM. n = 5. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars indicate SD.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using the following: LOX antibody (abcam, ab-174316, 1:100), LOXL2 antibody (leading biology, LBIRA1098, 1:100), CD31 antibody (Santa Cruz, sc-18916, 1:250).

Techniques: Inhibition, Gene Expression

Journal: Redox Report : Communications in Free Radical Research

Article Title: Verbascoside targets endothelial HIF-1α/ Lysyl oxidase signaling to attenuate glomerular injury in diabetic nephropathy

doi: 10.1080/13510002.2025.2598110

Figure Lengend Snippet: HIF-1α drives endothelial LOX/LOXL2-mediated mesangial injury. (a) Transcriptional upregulation of HIF-1α pathways in diabetic kidneys. n = 4. (b, c) Suppression of LOX and LOXL2 mRNA in HG-exposed GECs by HIF-1α inhibition. n = 5. (d-f) Suppression of mesangial proliferation/viability deficits by CM from HIF-inhibited GEnCs (HG + HIFi-CM). n = 5. (g, h) Reduction of oxidative stress markers in mesangial cells treated with HG + HIFi-CM. n = 5. (i-l) Attenuation of fibrotic gene expression (FN1, Col4a1) and collagen deposition in mesangial cells receiving HG + HIFi-CM. n = 5. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars indicate SD.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using the following: LOX antibody (abcam, ab-174316, 1:100), LOXL2 antibody (leading biology, LBIRA1098, 1:100), CD31 antibody (Santa Cruz, sc-18916, 1:250).

Techniques: Inhibition, Gene Expression

Journal: Redox Report : Communications in Free Radical Research

Article Title: Verbascoside targets endothelial HIF-1α/ Lysyl oxidase signaling to attenuate glomerular injury in diabetic nephropathy

doi: 10.1080/13510002.2025.2598110

Figure Lengend Snippet: Identification of Verbascoside as a dual LOX/LOXL2 inhibitor. (a-c) Virtual screening of a natural product library against LOX/LOXL2, prioritizing 11 candidates. (d) BLI kinetics revealing Verbascoside's high affinity for LOX (K D = 1.13 × 10 − 7 M) and LOXL2 (K D = 4.92 × 10 − 5 M). (e) Molecular docking interactions of Verbascoside and LOX at LOX binding site. (f) Analogous binding interactions at LOXL2 site. (g) Verbascoside inhibits LOX enzyme activity. n = 8. *** p < 0.001. Error bars indicate SD.

Article Snippet: Primary antibody incubation was performed overnight at 4°C using the following: LOX antibody (abcam, ab-174316, 1:100), LOXL2 antibody (leading biology, LBIRA1098, 1:100), CD31 antibody (Santa Cruz, sc-18916, 1:250).

Techniques: Binding Assay, Activity Assay

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Immunohistochemical analysis of LOXL2. LOXL2 expression was evaluated at high-power field (×400 magnification) by two experienced pathologists. a Negative for LOXL2. b One positive for LOXL2. c Two positive for LOXL2. d Three positive for LOXL2

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques: Immunohistochemical staining, Expressing

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Patient characteristics based on LOXL2 expression

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques:

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Pattern of recurrence based on LOXL2 expression

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques:

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Kaplan–Meier analysis of LOXL-2 expression. Survival was compared between the LOXL2-positive and -negative groups. The p-value was calculated using log-rank test. a Overall survival (p = 0.008). b Disease-free survival (p = 0.073). c Metastasis-free survival (p = 0.020)

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques: Expressing

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Multivariate analysis for overall survival and metastasis-free survival

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques:

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: LOXL2 expression analyzed in various breast cancer cell lines. In Western blot analysis, LOXL2 was only detected in the basal-like breast carcinoma cell lines (BT549, HBL100, HS578T, and MDA-MB-231)

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques: Expressing, Western Blot

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Analysis of LOXL2 expression in two basal-like breast cancer cell lines (MDA-MB-231 and BT549) with LOXL2 silencing. a Western analysis; Cells with LOXL2 silencing by siLOXL2 showed reduced LOXL2, Snail, Snai2, SPARC, p-FAK, and p-SRC at protein level. b RT-PCR analysis; LOXL2 silencing has an effect on reducing of LOXL2, Snail, Snail2, and SPARC at mRNA level

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Breast cancer research and treatment

Article Title: LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients

doi: 10.1007/s10549-013-2662-3

Figure Lengend Snippet: Silencing of LOXL2 suppresses the invasive potential of human breast cancer cells. a MDA-MB-231-siLOXL2 and BT549-siLOXL2 cells exhibited a more epithelial phenotype compared to si-control and WT. b Motility of MDA-MB-231 (WT, si-Control, and si-LOXL2) and BT549 (WT, si-Control, and si-LOXL2) cells was examined by wound-healing assay. Wound-healing assay was performed three times. Images were taken at 24 h. c Invasion capacity of MDA-MB-231 (WT, si-Control, and si-LOXL2) and BT549 (WT, si-Control, and si-LOXL2) cells were analyzed by a Matrigel invasion assay. Migrated tumor cells were taken at 5 h and stained with H&E. Microphotographic images were captured at ×100 magnification using an Olympus IX70 inverted microscope and the number of invading cells was counted in five random high-power fields (***p < 0.001)

Article Snippet: The membranes were blocked and probed with primary antibody against LOXL2 (Origene; Rockville, MD), Snail and Snai2 (Cell Signaling; Danvers, MA), EpCAM (Abcam, Cambridge, UK), SPARC (R&D System, Minneapolies, MN), pFAK and FAK (Santa Cruz, CA, USA), pSRC (Millipore, Billerica, MA, USA), SRC (Santa Cruz, CA, USA), CDH1 (BD Biosciences; Sparks, MD), and ß-actin (Sigma, St Louis, MO).

Techniques: Control, Wound Healing Assay, Invasion Assay, Staining, Inverted Microscopy

Journal: Oncotarget

Article Title: Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis.

doi: 10.18632/oncotarget.18170

Figure Lengend Snippet: Figure 1: Correlation between LOXL2 expression levels and migratory potential of different CRC cells in vitro. (A) LOXL2 expression in five CRC cell lines. Distilled water was used as a negative control (N.C.). (B) LOXL2 protein levels in CRC cells. MDA-MB-231 was used as a positive control (P.C.). (C) Motility of different CRC cells in vitro, determined in wound healing assay. Representative figures obtained in three independent experiment are shown.

Article Snippet: Protein samples were separated using 8–10% SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked and probed with antibodies against LOXL2 (Origene, Rockville, MD, USA), SNAI1 (Cell Signaling, Danvers, MA, USA), phosphor-FAK;Tyr576, and FAK (Santa Cruz Biotechnology, CA, USA), phosphor-Src;Tyr418 (Millipore, Billerica, MA, USA), Src (Santa Cruz Biotechnology), CDH1 (BD Biosciences, Sparks, MD), VIM (Dako, Glostrup, Denmark), and β-actin (SigmaAldrich).

Techniques: Expressing, In Vitro, Negative Control, Positive Control, Wound Healing Assay

Journal: Oncotarget

Article Title: Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis.

doi: 10.18632/oncotarget.18170

Figure Lengend Snippet: Figure 2: Migratory potential alterations induced by the changes in LOXL2 expression. (A) Transwell migration of LOXL2- knockdown SW480 cells. Images were obtained 36 h after seeding. (B) Transwell migration of LOXL2-overexpressing SW620 cells. Images were obtained 72 h after seeding. Results were obtained from three independent experiments, and bar graphs in (A) and (B) represent cell number per image field (mean ± standard deviation).

Article Snippet: Protein samples were separated using 8–10% SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked and probed with antibodies against LOXL2 (Origene, Rockville, MD, USA), SNAI1 (Cell Signaling, Danvers, MA, USA), phosphor-FAK;Tyr576, and FAK (Santa Cruz Biotechnology, CA, USA), phosphor-Src;Tyr418 (Millipore, Billerica, MA, USA), Src (Santa Cruz Biotechnology), CDH1 (BD Biosciences, Sparks, MD), VIM (Dako, Glostrup, Denmark), and β-actin (SigmaAldrich).

Techniques: Expressing, Migration, Knockdown, Standard Deviation

Journal: Oncotarget

Article Title: Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis.

doi: 10.18632/oncotarget.18170

Figure Lengend Snippet: Figure 3: Effects of LOXL2 silencing/ectopic expression on the migration of CRC cells through the endothelial barrier. (A) SW480 cells transfected with siControl/siLOXL2 oligonucleotide were seeded on HUVECs, and images were obtained 36 h after seeding. (B) SW620 cells transfected with mock/LOXL2-expressing plasmids were seeded on HUVECs, and images were obtained 72 h after seeding. Results were obtained from three independent experiments and bar graphs in (A) and (B) represent cell number per image field (mean ± standard deviation).

Article Snippet: Protein samples were separated using 8–10% SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked and probed with antibodies against LOXL2 (Origene, Rockville, MD, USA), SNAI1 (Cell Signaling, Danvers, MA, USA), phosphor-FAK;Tyr576, and FAK (Santa Cruz Biotechnology, CA, USA), phosphor-Src;Tyr418 (Millipore, Billerica, MA, USA), Src (Santa Cruz Biotechnology), CDH1 (BD Biosciences, Sparks, MD), VIM (Dako, Glostrup, Denmark), and β-actin (SigmaAldrich).

Techniques: Expressing, Migration, Transfection, Standard Deviation

Journal: Oncotarget

Article Title: Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis.

doi: 10.18632/oncotarget.18170

Figure Lengend Snippet: Figure 4: Induction of EMT by LOXL2 through the activation of FAK/Src pathway and upregulation of Snail. (A) Expression of EMT-related molecules 24 and 48 h after the transfection of cells with LOXL2 siRNA/overexpressing plasmid. (B) CDH1 (E-cadherin), VIM (vimentin), and SNAI1 (Snail) expression in SW480 and SW620 cells 24 h after the transfection with siLOXL2 and LOXL2-overexpressing plasmid, respectively. GAPDH was used as an internal control. (C) Expression of EMT-related molecules in cells transfected with siLOXL2 and LOXL2-overexpressing plasmid.

Article Snippet: Protein samples were separated using 8–10% SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked and probed with antibodies against LOXL2 (Origene, Rockville, MD, USA), SNAI1 (Cell Signaling, Danvers, MA, USA), phosphor-FAK;Tyr576, and FAK (Santa Cruz Biotechnology, CA, USA), phosphor-Src;Tyr418 (Millipore, Billerica, MA, USA), Src (Santa Cruz Biotechnology), CDH1 (BD Biosciences, Sparks, MD), VIM (Dako, Glostrup, Denmark), and β-actin (SigmaAldrich).

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Role of LOXL2 in the epithelial-mesenchymal transition and colorectal cancer metastasis.

doi: 10.18632/oncotarget.18170

Figure Lengend Snippet: Figure 5: LOXL2 IHC analysis in CRC tissues and the correlation between this parameter and the survival of CRC patients. (A) IHC analysis of LOXL2 expression in CRC tissues. Representative images of normal colon tissue, CRC tissue with low LOXL2 expression, and CRC tissue with high LOXL2 expression are presented (magnification, 200 ×). (B) Correlation between the overall survival rates of CRC patients and LOXL2 expression. High LOXL2 expression was correlated with poor patient survival (log-rank Mantel- Cox test, p = 0.024).

Article Snippet: Protein samples were separated using 8–10% SDSpolyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes, which were blocked and probed with antibodies against LOXL2 (Origene, Rockville, MD, USA), SNAI1 (Cell Signaling, Danvers, MA, USA), phosphor-FAK;Tyr576, and FAK (Santa Cruz Biotechnology, CA, USA), phosphor-Src;Tyr418 (Millipore, Billerica, MA, USA), Src (Santa Cruz Biotechnology), CDH1 (BD Biosciences, Sparks, MD), VIM (Dako, Glostrup, Denmark), and β-actin (SigmaAldrich).

Techniques: Expressing

Journal: Bone Research

Article Title: Sorafenib inhibits ossification of the posterior longitudinal ligament by blocking LOXL2-mediated vascularization

doi: 10.1038/s41413-024-00327-7

Figure Lengend Snippet: LOXL2 and blood vessels were elevated in OPLL and ligament cell characterization. a , b Representative images for IF staining (left) of LOXL2 (red) and quantification (right). Scale bar = 100 μm. Values are means ± SD. n = 3 for the non-OPLL group and n = 5 for the OPLL group, *** P < 0.001 by Student’s t -test. c , d Representative images for IF staining (left) of EMCN (green) and CD31 (red) of non-OPLL or OPLL samples and corresponding quantitative analysis (right). Scale bar = 100 μm. Values are means ± SD. n = 3 for the non-OPLL group and n = 5 for the OPLL group, * P < 0.05 by Student’s t -test. e , f Representative images of LOXL2 protein of primary culture cells derived from non-OPLL or OPLL by western blotting (left) and quantification (right). Values are means ± SD. n = 5, * P < 0.05 by Student’s t -test. g Representative images of trilineage differentiation of ligament cells. The left panels represent Alcian blue staining for chondrogenic differentiation; the middle panels represent ALP staining for osteogenic differentiation, and the right panels represent oil red staining for adipogenic differentiation. The upper parts represent ligament cells in the growth medium (GM), and the lower parts represent ligament cells in the differentiation medium (DM). h Ligament cells derived from OPLL expressed typical MSC surface markers, positive for CD73 (99.9%), CD90 (99.5%), CD105 (99.3%), and negative for CD45 (0%). n = 5. i High-throughput sequencing was performed on ligament cells derived from OPLL and non-OPLL samples (GSE69787). The GO pathway enrichment analysis unveiled significant differences in angiogenesis, bone metabolism, and signaling pathways governing vasculogenesis while identifying co-regulatory network presence. The experiments were performed in three biological replicates

Article Snippet: Empty control plasmids (OE-Ctrl), wild-type LOXL2 overexpression plasmids (OE-LOXL2 WT), and point mutation LOXL2 plasmids (OE-LOXL2 Y689F) were procured from OriGENE Technologies, Inc.

Techniques: Staining, Derivative Assay, Western Blot, Next-Generation Sequencing, Protein-Protein interactions

Journal: Bone Research

Article Title: Sorafenib inhibits ossification of the posterior longitudinal ligament by blocking LOXL2-mediated vascularization

doi: 10.1038/s41413-024-00327-7

Figure Lengend Snippet: LOXL2 modulates ligament cell endothelial-like differentiation through HIF1A signaling pathway. a , b Increased expression of LOXL2 can enhance vasculogenesis, and BAPN did not inhibit the enhancement caused by overexpression of LOXL2. Ligament cells were transfected with LOXL2 plasmids (OE-LOXL2) or empty vector plasmids (OE-Ctrl), then treated with 2 mmol/L BAPN or PBS (Vehicle). Scale bars = 100 μm. Values are mean ± SD ( n = 3) ** P < 0.01, ns not significant by ANOVA. c Representative images of LOXL2 and HIF1A protein of ligament cells by western blotting. Ligament cells were treated with or without LOXL2 plasmids. d , e Representative immunofluorescence staining of HIF1A and quantification by fluorescence intensity. Ligament cells were transfected with or without LOXL2 plasmids. Scale bars = 50 μm. n = 3 and 100 cells were randomly selected. *** P < 0.001 by Student’s t -test. f , g Cell culture supernatant VEGFA and PDGF-BB production was measured via ELISA. After the knockdown or overexpression of LOXL2 for 24 h, medium supernatants of ligament cells were harvested to measure VEGFA and PDGF-BB concentrations. Values are mean ± SD ( n = 3). ** P < 0.01, *** P < 0.001 by Student’s t -test respectively. h , i Overexpression of wild-type LOXL2 (OE-LOXL2 WT) or a point-mutated LOXL2 variant (OE-LOXL2 Y689F), characterized by the absence of enzymatic activity, in ligament cells elicits enhancement in ligament cell vasculogenesis, as demonstrated through quantitative analysis. Scale bars = 100 μm. The presented data includes mean values ± SD ( n = 3). Statistical significance was determined by ANOVA, with asterisks indicating significance (** P < 0.001) and “ns” denoting non-significance. j Protein expression levels of LOXL2 and HIF1A within ligament cells were assessed through western blotting. GAPDH served as a loading control. Ligament cells were subjected to treatment with either wild-type or point-mutated LOXL2 variants. k Quantification of VEGFA and PDGF-BB production in the cell culture supernatant was achieved via ELISA. Following a 24-h overexpression of wild-type LOXL2 or the point-mutated LOXL2 variant, medium supernatants from ligament cells were collected to measure VEGFA and PDGF-BB concentrations. Data is presented as mean values ± SD ( n = 3). Statistical significance was determined by ANOVA, with triple asterisks indicating high significance (*** P < 0.001) and “not significant” denoting the absence of significance. All experiments were conducted with three independent biological replicates

Article Snippet: Empty control plasmids (OE-Ctrl), wild-type LOXL2 overexpression plasmids (OE-LOXL2 WT), and point mutation LOXL2 plasmids (OE-LOXL2 Y689F) were procured from OriGENE Technologies, Inc.

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay, Knockdown, Variant Assay, Activity Assay, Control

Journal: Bone Research

Article Title: Sorafenib inhibits ossification of the posterior longitudinal ligament by blocking LOXL2-mediated vascularization

doi: 10.1038/s41413-024-00327-7

Figure Lengend Snippet: Sorafenib attenuates the progression of the BMP-induced In vivo Ossification (BIO) model. a , b Representative pictures of capillary-like structures formed on Matrigel (left) of ligament cells overexpressing empty vector plasmids (OE-Ctrl) and LOXL2 plasmids (OE-LOXL2) with the treatment of sorafenib (90 nmol/L and 900 nmol/L) or DMSO (Vehicle); alongside quantification (right). Scale bars = 100 μm. Values are means ± SD ( n = 3). *** P < 0.001, * P < 0.005 by ANOVA. c , d Representative reconstructed 3D micro-CT transverse position images depict the ossification site following a 2-week treatment with 50 mg/kg Sorafenib, an LOXL2 enzyme inhibitor [(2-Chloropyridin-4-yl)methanamine hydrochloride] and a methylcellulose carrier alone (Vehicle). Colored bars within the images signify the density of ossified tissue, with higher density indicated at the top and lower density at the bottom. Quantitative analysis of structural parameters related to heterotopic bone formation, including Bone Volume (BV) and Trabecular Thickness (Tb.th), is presented. Scale bar = 2 mm. Data is reported as mean values ± SD ( n = 6). Statistical analysis was performed using ANOVA, with triple asterisks indicating high significance (*** P < 0.01), and “ns” denoting non-significance. e , f Safranin O-Fast Green (SOFG) staining of sections from the ossification site following a 2-week treatment period, accompanied by the quantification of the cartilage area ratio within the ossified tissue. The scale bar represents 100 μm. Data is presented as means ± SD ( n = 6). Statistical analysis was conducted using ANOVA, with triple asterisks indicating high significance (*** P < 0.001) and “ns” signifying non-significance. g – j The left panel displays representative images depicting the presence of SP7 and RUNX2 within the ossification site, while the right panel presents quantitative analysis. The scale bar corresponds to 50 μm. Error bars represent mean values ± SD ( n = 6). Statistical significance was determined by ANOVA, with *** P < 0.001 and “ns” indicating non-significance. k , l Representative images demonstrate immunofluorescence (IF) staining for EMCN (green) and CD31 (red) within the ossification site, followed by the corresponding quantitative analysis (right). The scale bar represents 50 μm. Data is reported as means ± SD ( n = 6). Statistical analysis was performed using ANOVA, with double asterisks indicating significance (** P < 0.01) and “ns” indicating non-significance. All experiments were conducted with three independent biological replicates

Article Snippet: Empty control plasmids (OE-Ctrl), wild-type LOXL2 overexpression plasmids (OE-LOXL2 WT), and point mutation LOXL2 plasmids (OE-LOXL2 Y689F) were procured from OriGENE Technologies, Inc.

Techniques: In Vivo, Plasmid Preparation, Micro-CT, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: LOXs oxidize lysine and hydroxylysine side chains on collagen and elastin to highly reactive semialdehydes called allysines, while producing hydrogen peroxide (H 2 O 2 ) and ammonia (NH 3 ) as byproducts. Allysines then spontaneously condense to form cross-linkages. In our approach to detect LOXL2 activity, adding biotin-hydrazide (BHZ) results in biotinylation of LOXL2-catalyzed peptidyl lysines.

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: Cells were incubated with 100 μM biotin-hydrazide (BHZ) for 24 h and then fixed and co-stained for LOXL2 (immunostaining; red), biotinylation (DTAF-streptavidin; green) corresponding to LOXs activity, and nuclei (DAPI; blue). A) Representative confocal microscopy images of i) control A7r5s cells, ii) wild type LOXL2 overexpression iii) catalytically inactive LOXL2-DM overexpression, iv) LOXL2 overexpression + LOXL2-specific inhibitor PAT-1251 (10 μM), v) LOXL2 overexpression + inhibitors BAPN (10 μM) + PAT-1251 (10 μM). (n = 12; Scale bar = 50 μm). Activity signal in each IF image was converted to mean gray value shown in bar graph (mean±SEM; *P<0.05 by student’s t-test). Representative western blotting images compare the expression of LOXs in the ECM of control A7r5 cells and A7r5 cells with wild type LOXL2 or LOXL2-DM overexpression. B) Representative confocal immunofluorescence microscopy images of A7r5 cells with LOXL2 overexpression. BHZ incorporation (green) was co-stained with collagen I or collagen IV (purple), and nuclei (blue). (n=5; Scale bar = 50 μm.) C) Representative confocal microscopy images of LOXs activity assay performed in wild-type human aortic smooth muscle cells (HASMC) and CRISPR-Cas9 mediated LOXL2 gene knockout HASMC T1 cells (Ci; Scale bar = 50 μm). Bar graph shows measured activity signal (n=24; mean±SEM; *P<0.05 by student’s t-test) (Cii). Representative western blotting images compare the expression of LOXs in the ECM of wild type HASMC cells and HASMC T1 cells (Ciii).

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Incubation, Staining, Immunostaining, Activity Assay, Confocal Microscopy, Control, Over Expression, Western Blot, Expressing, Immunofluorescence, Microscopy, CRISPR, Gene Knockout

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: Cells overexpressing LOXL2 or LOXL2DM were stained for LOX, LOXL1, LOXL3 and AOC3 separately, and co-stained with DAPI (blue). No significant increase in expression was seen for these proteins in AdLOXL2 and AdLOXL2-DM cells.

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: Cells were incubated with 100 μM biotin-hydrazide (BHZ), with or without vitamin E (50 μM α-Tocopherol) for 24 h and then fixed and co-stained for LOXL2 (purple; immunofluorescent staining), biotinylation corresponding to LOXs activity (green; DTAF-streptavidin), and nuclei (blue; DAPI). Activity signal in each IF image was converted to mean gray value shown in bar graph (n=12). BHZ incorporation was unchanged in cells with the application of vitamin E. (n = 6; Scale bar = 50 μm. *P<0.05 by student’s t-test.)

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Incubation, Staining, Activity Assay

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: A) H 2 O 2 -resorufin coupled LOX activity assay in cell culture medium from A7r5 cells with and without adenoviral-induced overexpression of LOXL2 or LOXL2-DM Activity was calculated as the initial rate of reaction calculated as the slope of fluorescence (relative fluorescence unit (RFU)) per unit time (1 h) normalized to the average signal of AdLOXL2 samples. (n=15; *P<0.05 by student’s t-test). B) Representative Western blot image comparing total and biotinylated LOXL2 and LOXL2DM after incubation with increasing concentrations of BHZ for 2h or 24h followed by enrichment with Streptavidin agarose beads. Western blots showed no meaningful levels of biotinylated-LOXL2.

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Activity Assay, Cell Culture, Over Expression, Fluorescence, Western Blot, Incubation

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: A) A7r5 cells overexpressing LOXL2 were seeded on coverslips and incubated with different concentrations of biotin-hydrazide (BHZ; 0, 50, 100, or 150 μM) for 24, 48, or 72 h. After being fixed and stained for immunofluorescence with DTAF-streptavidin, cells were imaged with confocal microscopy in five independent coverslip locations per condition. (Scale bar = 250 μm). B) LOXs activity signal from each image was converted to grayscale and analyzed for mean gray value. (n = 10)

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Incubation, Staining, Immunofluorescence, Confocal Microscopy, Activity Assay

Journal: bioRxiv

Article Title: A Novel In Situ Activity Assay for Lysyl Oxidases

doi: 10.1101/2021.04.30.442175

Figure Lengend Snippet: A) Representative Western blot images of LOXL2, LOX, LOXL1 and LOXL3 expression in aortic matrix of young (Y, < 3 months old) and old (O, > 18 months old) wild-type (WT) and LOXL2+/− littermate mice (n = 8). AOC3 is detected in the cytosolic fraction of aortic tissue homogenates but not in ECM and AOC3 expression level is similar across four groups. GAPDH was used as loading control. Bar graph shows the densitometry analysis of Western blots (mean ± SEM; n=6; *P<0.05 by 2-way ANOVA). B) Representative confocal Z-stack maximum projection immunofluorescence images show LOXL2 activity in aortic rings from young and old, WT and LOXL2+/− mice. Freshly isolated aortic rings were incubated with 200 μM biotin-hydrazide (BHZ) in DMEM + ITS for 24 h, then fixed and stained for LOXL2 (red), LOXs activity (green), and nuclei (blue). (n=6; Scale bar = 100 μm). C) Representative confocal images of control aortic rings incubated without BHZ. Samples were stained for LOXL2 (red), endogenous biotinylation (green), and nuclei (blue). (n = 6; Scale bar = 100 μm). D) Bar graph of LOXs activity calculated as mean gray value of the IF images (mean ± SEM; n=12, *P<0.05 by 2-way ANOVA). The overall LOXs activity in each group corresponds positively to the expression levels of LOXs in aortic ECM shown in A).

Article Snippet: LOXL2+/− heterozygous mice were produced by using cryopreserved sperm in which the LOXL2 gene was disrupted by insertion of a long terminal repeat-trapping cassette into exon 1 (Taconic Inc.).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Activity Assay, Isolation, Incubation, Staining

Journal: Journal of cellular physiology

Article Title: Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

doi: 10.1002/jcp.27695

Figure Lengend Snippet: FIGURE 1 LOXL2 regulates migration in EC. (a) LOXL2 mRNA expression is decreased in shLOXL2 expressing EC compared to shCtrl expressing EC (n = 4 + SD, Student’s t test). (b) Doxycycline‐induced overexpression of LOXL2 mRNA is confirmed by stimulating EC expressing pInducer20‐LOXL2 (Ind LOXL2) with PBS (Vehicle) or doxycycline (Dox) for 24 hr. (c) Western blot analysis demonstrating reduced LOXL2 protein expression in shLOXL2 expressing EC, and increased LOXL2 protein expression when stimulating pInducer20‐LOXL2 infected EC (Ind LOXL2), but not wildtype EC (WT), with doxycycline, using GAPDH as a loading control. (d) Representative pictures of a migration scratch assay using shCtrl and shLOXL2 expressing EC at t = 0 and t = 6 hr. (e) Quantification of shCtrl and shLOXL2 expressing endothelial cell migration over 6 hr (n = 5 + SD, Student’s t test). (f) Representative pictures of a migration scratch assay when overexpressing LOXL2 in EC at t = 0 and t = 6 hr. (g) Quantification of LOXL2 overexpressing endothelial cell migration over 6 hr (n = 3 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐ buffered saline; shCtrl: shRNA control; shLOXL2: short hairpin‐mediated LOXL2 knockdown

Article Snippet: The primary antibodies used were LOXL2 (cat. no. AF2639; R&D Systems, Minneapolis, MN), GAPDH (cat. no. 2118; Cell Signaling Technology, Danvers, MA), α‐SMA (cat. no. TA310169; Origene), PECAM‐1 (cat. no. sc‐1505; Santa Cruz Biotechnology), VE‐cadherin (cat. no. TA804746; Origene), Snail (cat. no. TA500366; Origene), Fibronectin/HRP (cat. no. P0246; Dako, Glostrup, Denmark), FAK (cat. no. 13009; Cell Signaling Technology), p‐FAK Y397 (cat. no. 8556; Cell Signaling Technology), PKB/Akt (cat. no. 9272; Cell Signaling Technology), and p‐PKB/Akt S473 (cat. no. 4060; Cell Signaling Technology).

Techniques: Migration, Expressing, Over Expression, Western Blot, Infection, Control, Wound Healing Assay, Saline, shRNA, Knockdown

Journal: Journal of cellular physiology

Article Title: Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

doi: 10.1002/jcp.27695

Figure Lengend Snippet: FIGURE 2 LOXL2 regulates angiogenic sprouting in EC. (a) Representative pictures of an angiogenic sprouting assay using shCtrl and shLOXL2 expressing EC at t = 72 hr. (b) Quantification of sprout length in a 72 hr angiogenic sprouting assay using shCtrl and shLOXL2 expressing EC (n = 4 + SD, Student’s t test). (c) Representative pictures of an angiogenic sprouting assay in pInducer20‐LOXL2 (Ind LOXL2) expressing EC stimulated with PBS (Vehicle) or doxycycline (Dox) at t = 72 hr. (d) LOXL2 overexpression increases sprouting length in an angiogenic sprouting assay (n = 4 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐buffered saline; shCtrl: shRNA control; shLOXL2: short hairpin‐mediated LOXL2 knockdown

Article Snippet: The primary antibodies used were LOXL2 (cat. no. AF2639; R&D Systems, Minneapolis, MN), GAPDH (cat. no. 2118; Cell Signaling Technology, Danvers, MA), α‐SMA (cat. no. TA310169; Origene), PECAM‐1 (cat. no. sc‐1505; Santa Cruz Biotechnology), VE‐cadherin (cat. no. TA804746; Origene), Snail (cat. no. TA500366; Origene), Fibronectin/HRP (cat. no. P0246; Dako, Glostrup, Denmark), FAK (cat. no. 13009; Cell Signaling Technology), p‐FAK Y397 (cat. no. 8556; Cell Signaling Technology), PKB/Akt (cat. no. 9272; Cell Signaling Technology), and p‐PKB/Akt S473 (cat. no. 4060; Cell Signaling Technology).

Techniques: Expressing, Over Expression, Saline, shRNA, Control, Knockdown

Journal: Journal of cellular physiology

Article Title: Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

doi: 10.1002/jcp.27695

Figure Lengend Snippet: FIGURE 3 LOXL2‐induced increase in migration and sprouting is independent of enzymatic activity. (a) A schematic representation of LOXL2 (upper panel), and the catalytically inactive H26Q‐H628Q‐LOXL2 mutant (LOXL2 H626/628Q). (b) Western blot analysis demonstrating the inducible overexpression of an H26Q‐H628Q‐LOXL2 mutant in EC by stimulation with doxycycline (Dox) for 24 hr compared to PBS (Vehicle). (c) Representative pictures of a migration scratch assay when overexpressing wildtype LOXL2 (Ind LOXL2) and the H26Q‐H628Q‐ LOXL2 mutant (Ind dLOXL2) in EC at t = 0 and t = 6 hr. (d) Overexpression of either wildtype LOXL2 or LOXL2 H626/628Q in EC results in comparable increased migration in scratch migration assays (n = 4 + SD, Student’s t test). (e) Representative pictures of an angiogenic sprouting assay in inducible wildtype LOXL2 or LOXL2 H626/628Q expressing EC stimulated with PBS (Vehicle) or doxycycline (Dox) at t = 72 hr. (f) Overexpression of either wildtype LOXL2 or LOXL2 H626/628Q in EC results in a comparable increase in sprouting length in angiogenic sprouting assays (n = 4 + SD, Student’s t test); *p < 0.05 and **p < 0.01. EC: endothelial cells; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; PBS: phosphate‐buffered saline [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The primary antibodies used were LOXL2 (cat. no. AF2639; R&D Systems, Minneapolis, MN), GAPDH (cat. no. 2118; Cell Signaling Technology, Danvers, MA), α‐SMA (cat. no. TA310169; Origene), PECAM‐1 (cat. no. sc‐1505; Santa Cruz Biotechnology), VE‐cadherin (cat. no. TA804746; Origene), Snail (cat. no. TA500366; Origene), Fibronectin/HRP (cat. no. P0246; Dako, Glostrup, Denmark), FAK (cat. no. 13009; Cell Signaling Technology), p‐FAK Y397 (cat. no. 8556; Cell Signaling Technology), PKB/Akt (cat. no. 9272; Cell Signaling Technology), and p‐PKB/Akt S473 (cat. no. 4060; Cell Signaling Technology).

Techniques: Migration, Activity Assay, Mutagenesis, Western Blot, Over Expression, Wound Healing Assay, Expressing, Saline

Journal: Journal of cellular physiology

Article Title: Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

doi: 10.1002/jcp.27695

Figure Lengend Snippet: FIGURE 4 LOXL2 knockdown in EC reduces EndMT, but LOXL2 overexpression does not induce EndMT. (a) LOXL2 knockdown in EC reduces mRNA expression of mesenchymal markers α‐SMA, calponin 1, and fibronectin, and increases mRNA expression of endothelial markers PECAM‐1 and VE‐cadherin (n = 3, + SD, Student’s t test). (b) LOXL2 knockdown in EC increases protein expression of endothelial markers PECAM‐1 and VE‐cadherin, and reduces protein expression of mesenchymal markers α‐SMA and fibronectin. (c) The LOXL2 expression is increased in EC after stimulation with TGF‐β for 24 hr. (d) LOXL2 knockdown results in delayed TGF‐β‐mediated upregulation of α‐SMA in EC. (e) LOXL2 overexpression does not affect mRNA expression of mesenchymal markers α‐SMA, calponin 1, and fibronectin, or expression of endothelial markers PECAM‐1 and VE‐cadherin (n = 3, + SD, Student’s t test). (f) LOXL2 overexpression does not affect protein expression of endothelial markers PECAM‐1 and VE‐cadherin, or expression of mesenchymal markers α‐SMA and fibronectin; *p < 0.05, **p < 0.01, and ***p < 0.001. EC: endothelial cells; EndMT: endothelial‐to‐mesenchymal transition; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; LOXL2: lysyl oxidase‐like 2; mRNA: messenger RNA; PBS: phosphate‐buffered saline; PECAM‐1: platelet endothelial cell adhesion molecule 1; shCtrl: shRNA control; α‐SMA: α‐smooth muscle actin; shLOXL2: short hairpin‐mediated LOXL2 knockdown; TGF‐β: transforming growth factor‐β; VE‐cadherin: vascular endothelial cadherin

Article Snippet: The primary antibodies used were LOXL2 (cat. no. AF2639; R&D Systems, Minneapolis, MN), GAPDH (cat. no. 2118; Cell Signaling Technology, Danvers, MA), α‐SMA (cat. no. TA310169; Origene), PECAM‐1 (cat. no. sc‐1505; Santa Cruz Biotechnology), VE‐cadherin (cat. no. TA804746; Origene), Snail (cat. no. TA500366; Origene), Fibronectin/HRP (cat. no. P0246; Dako, Glostrup, Denmark), FAK (cat. no. 13009; Cell Signaling Technology), p‐FAK Y397 (cat. no. 8556; Cell Signaling Technology), PKB/Akt (cat. no. 9272; Cell Signaling Technology), and p‐PKB/Akt S473 (cat. no. 4060; Cell Signaling Technology).

Techniques: Knockdown, Over Expression, Expressing, Saline, shRNA, Control

Journal: Journal of cellular physiology

Article Title: Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition.

doi: 10.1002/jcp.27695

Figure Lengend Snippet: FIGURE 5 LOXL2 regulates EMT–EndMT‐associated signaling pathways PKB/Akt and FAK. (a) Knockdown of LOXL2 in EC reduces FAK Y397 phosphorylation, and reduces PKB/Akt expression and phosphorylation. (b) Overexpression of LOXL2 in EC increases FAK Y397 phosphorylation and PKB/Akt S473. (c) Overexpression of the catalytically inactive H26Q‐H628Q‐LOXL2 mutant increases FAK Y397 phosphorylation and PKB/Akt S473. EC: endothelial cells; EndMT: endothelial‐to‐mesenchymal transition; EMT: epithelial‐to‐mesenchymal transition; GAPDH: glyceraldehyde 3‐phosphate dehydrogenase; FAK: focal adhesion kinase; LOXL2: lysyl oxidase‐like 2; PKB: protein kinase B

Article Snippet: The primary antibodies used were LOXL2 (cat. no. AF2639; R&D Systems, Minneapolis, MN), GAPDH (cat. no. 2118; Cell Signaling Technology, Danvers, MA), α‐SMA (cat. no. TA310169; Origene), PECAM‐1 (cat. no. sc‐1505; Santa Cruz Biotechnology), VE‐cadherin (cat. no. TA804746; Origene), Snail (cat. no. TA500366; Origene), Fibronectin/HRP (cat. no. P0246; Dako, Glostrup, Denmark), FAK (cat. no. 13009; Cell Signaling Technology), p‐FAK Y397 (cat. no. 8556; Cell Signaling Technology), PKB/Akt (cat. no. 9272; Cell Signaling Technology), and p‐PKB/Akt S473 (cat. no. 4060; Cell Signaling Technology).

Techniques: Protein-Protein interactions, Knockdown, Phospho-proteomics, Expressing, Over Expression, Mutagenesis