mmp2  (Proteintech)

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Preparation and characteristics of self-assembling nanofiber hydrogel PA-TIMP-QK. (A) (1) The chemical structure of the PA-TIMP-QK created with ChemBioDraw Ultra 14.0. (2) The three-dimensional structure predicted by ChemBioDraw Ultra 14.0. (3) The PA-TIMP-QKs were assembled into cylindrical nanostructures by hydrophobic interaction. The image was created with Adobe Photoshop 2022. (4) The PA-TIMP-QK nanofibers formed a hydrogel after dissolving in water. The image was created with Adobe Photoshop 2022. (B) The PA-TIMP-QK gel in different concentrations. PA-TIMP-QK presented as a semitransparent gel. (C) Scanning electron microscopy of nanofiber gel networks. PA-TIMP-QK nanohydrogel had a porous structure with pore sizes ranging from 50 to 100 μm. Scale bars: 50 μm (left) and 20 μm (right). (D) Circular dichroism analysis of PA-TIMP-QK secondary structure. PA: Peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Electron Microscopy, Circular Dichroism

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: MMP-2 expression in cerebral ischemia/reperfusion rats and controlled release of QK from self-assembling nanofiber hydrogel PA-TIMP-QK. (A) Western blot of MMP-2 at 1, 3, 7, and 14 days after injection ( n = 3). (B) Collagen binding capacities of PA-TIMP-QK and PA-QK peptides ( n = 3). (C) Release capacities of PA-TIMP-QK and PA-QK at 12 and 24 hours ( n = 3). Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test (A) or Student’s t -test (B, C)). MMP-2: Matrix metalloproteinase-2; OD: optical density; PA: peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Expressing, Western Blot, Injection, Binding Assay

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Bioactivity evaluation of self-assembling nanofiber hydrogel PA-TIMP-QK in vitro. (A) MTT assay for detecting the proliferation activity of HUVECs. (B) The scratch assay for detecting the migration ability of HUVECs. Scale bars: 100 μm. (C) Statistical analysis of migration rate in the scratch assay ( n = 6). The migration area of the PA-TIMP-QK group was much larger than the control and VEGF165 groups. (D) Assessment of tube formation ability. The ability of the PA-TIMP-QK group to form tubules was stronger than the control group. The PA-QK and VEGF165 groups also had a stronger ability to form tubules than the control group. Scale bar: 50 μm. (E) Statistical analysis of tubes in the tube formation assay ( n = 6). (F) Statistical analysis of nodes in the tube formation assay ( n = 6). (G) MTT assay by OGD/R for detecting survival activity of PC12 cells. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). HUVECs: Human umbilical vein endothelial cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OGD/R: oxygen-glucose deprivation and reperfusion; PA: peptide amphiphilic molecule; PC12 cells: pheochromocytoma cells; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: In Vitro, MTT Assay, Activity Assay, Wound Healing Assay, Migration, Control, Tube Formation Assay

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Histological analysis of cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) H&E staining of ischemic brain at 14 days after injection. The PA-TIMP-QK group showed a more complete structure, fewer apoptotic cells, and more normal cell morphology compared with the other three groups. Scale bar: 100 μm. (B) Nissl staining of ischemic brain at 14 days after injection. The number of Nissl bodies in the PA-TIMP-QK group was higher and their distribution was more regular compared with the PBS and VEGF165 groups. The dashed box area shows typical morphological features. Scale bar: 100 μm. (C) Statistical analysis of Nissl bodies ( n = 6). (D) Immunofluorescence staining of neurons in ischemic brain (NeuN, red, Alexa Fluor® 594; nuclei, blue). There were more surviving neurons in the PA-TIMP-QK group than in the PBS, VEGF165 and PA-QK groups. Scale bar: 100 μm. (E) Statistical analysis of NeuN + cells ( n = 6). (F) Immunofluorescence staining of axons in ischemic brain (Tuj1, red; nuclei, blue). There were more axons in the PA-TIMP-QK group than in the PBS and VEGF165 groups. Scale bar: 100 μm. (G) Statistical analysis of Tuj1 + cells ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). H&E staining: Hematoxylin and eosin staining; NeuN: neuronal nuclei; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; Tuj1: beta III tubulin; VEGF165: vascular endothelial growth factor 165.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Injection, Staining, Immunofluorescence, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Immunofluorescence staining of capillaries and vessels in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of capillaries in ischemic brain (vWF, red, Alexa Fluor ® 594; nuclei, blue). The number of vWF-labeled capillaries in the PA-TIMP-QK group was greater than the PBS and VEGF165 groups at 14 days after treatment. Scale bar: 100 μm. (B) Statistical analysis of vWF + capillaries ( n = 6). (C) Immunofluorescence staining of vessels in ischemic brain (α-SMA, red, Alexa Fluor ® 488; nuclei, blue). PA-TIMP-QK promoted the regeneration of blood vessels compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (D) Statistical analysis of α-SMA + vessels ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165; vWF: von Willebrand factor; α-SMA: α-smooth muscle actin.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Immunofluorescence, Staining, Injection, Labeling, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Immunofluorescence staining of tight junction protein in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of endothelial cell-specific intercellular adhesion molecules with VE-cadherin in ischemic brain (red, Alexa Fluor ® 594; nuclei, blue). The fluorescence intensity of VE-cadherin was significantly increased in the PA-TIMP-QK group compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (B) Statistical analysis of mean fluorescence intensity of VE-cadherin ( n = 6). (C) Immunofluorescence staining of intercellular junction protein ZO-1 in ischemic brain (red, Alexa Fluor ® 594; nuclei, blue). The TJ formed with ZO-1 in the PA-TIMP-QK group was closer to that in the other three groups. Scale bars: 20 μm (upper) and 5 μm (lower). (D) Statistical analysis of mean fluorescence intensity of ZO-1 ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TJ: tight junction; VE-cadherin: vascular endothelial-cadherin; VEGF165: vascular endothelial growth factor 165; ZO-1: zonula occludens-1.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Immunofluorescence, Staining, Injection, Fluorescence, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Evaluation of cell inflammation and apoptosis and the activation of downstream signaling pathways in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of M1-type macrophages colabeled with CD68 and iNOS in the ischemic brain (CD68, red, Alexa Fluor ® 594; iNOS, green, Alexa Fluor ® 488; colocalization, yellow; nuclei, blue). CD68 and iNOS-colabeled M1 macrophages ratio in the PA-TIMP-QK group was much lower than that in the VEGF165 and PBS groups. Scale bar: 100 μm. (B) Statistical analysis of iNOS + /CD68 + macrophages ( n = 5). (C) Immunofluorescence staining of M2-type macrophages colabeled with CD68 and CD206 in the ischemic brain (CD68, red, Alexa Fluor ® 594; CD206, green, Alexa Fluor ® 488; colocalization, yellow; nuclei, blue). CD68 and CD206-colabeled M2 macrophages ratio in the PA-TIMP-QK group was much higher than that in the PBS and VEGF165 groups, but there was no statistical difference between the PA-TIMP-QK and PA-QK groups. Scale bar: 100 μm. (D) Statistical analysis of CD206 + /CD68 + macrophages ( n = 5). (E) TUNEL staining (TUNEL, green; nuclei, blue). The PA-TIMP-QK group had the fewest TUNEL-positive cells compared with the VEGF165 and PBS groups. Scale bar: 100 μm. (F) Statistical analysis of TUNEL + cells ( n = 6). (G) Western blot of p-AKT and AKT at 14 days after injection ( n = 4). (H) Western blot of p-ERK and ERK at 14 days after injection ( n = 4). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). AKT: The serine/threonine kinase; ERK: extracellular signal-regulated kinase; iNOS: inducible nitric oxide synthase; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling; VEGF165: vascular endothelial growth factor 165.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Activation Assay, Injection, Immunofluorescence, Staining, TUNEL Assay, Western Blot, Saline, End Labeling

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Animal behavior testing for rotarod test, grip strength test, and open field test at 7 and 14 days in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Statistical analysis of latency during the rotarod test ( n = 6). (B) Statistical analysis of speed during the rotarod test ( n = 6). (C) Statistical analysis of grip strength test ( n = 6). (D) Representative paths of rats in the open field test, tracked by the SMART 3.0 system. The total distances of the PA-TIMP-QK group at 7 days were longer than those in the PBS and VEGF165 groups. The total distances of the PA-TIMP-QK group at 14 days were longer than those in the PBS, VEGF165 and PA-QK groups. There was no significant difference in anxiety assessment after 7 days. On the 14 th day, the PA-TIMP-QK group spent significantly longer time in the central zone of the open field compared with the PBS, PA-QK groups. (E) Statistical analysis of total distance at 7 and 14 days ( n = 6). (F) Statistical analysis of time in the center zone at 7 and 14 days ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

Article Snippet: Next, 100 nM MMP-2 (50 μL/well, SinoBiological, Beijing, China, 10082-hnah) was added to the 96-well plate containing the biotin-labeled PA-TIMP-QK and biotin-labeled PA-QK peptides.

Techniques: Injection, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Preparation and characteristics of self-assembling nanofiber hydrogel PA-TIMP-QK. (A) (1) The chemical structure of the PA-TIMP-QK created with ChemBioDraw Ultra 14.0. (2) The three-dimensional structure predicted by ChemBioDraw Ultra 14.0. (3) The PA-TIMP-QKs were assembled into cylindrical nanostructures by hydrophobic interaction. The image was created with Adobe Photoshop 2022. (4) The PA-TIMP-QK nanofibers formed a hydrogel after dissolving in water. The image was created with Adobe Photoshop 2022. (B) The PA-TIMP-QK gel in different concentrations. PA-TIMP-QK presented as a semitransparent gel. (C) Scanning electron microscopy of nanofiber gel networks. PA-TIMP-QK nanohydrogel had a porous structure with pore sizes ranging from 50 to 100 μm. Scale bars: 50 μm (left) and 20 μm (right). (D) Circular dichroism analysis of PA-TIMP-QK secondary structure. PA: Peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Electron Microscopy, Circular Dichroism

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: MMP-2 expression in cerebral ischemia/reperfusion rats and controlled release of QK from self-assembling nanofiber hydrogel PA-TIMP-QK. (A) Western blot of MMP-2 at 1, 3, 7, and 14 days after injection ( n = 3). (B) Collagen binding capacities of PA-TIMP-QK and PA-QK peptides ( n = 3). (C) Release capacities of PA-TIMP-QK and PA-QK at 12 and 24 hours ( n = 3). Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test (A) or Student’s t -test (B, C)). MMP-2: Matrix metalloproteinase-2; OD: optical density; PA: peptide amphiphilic molecule; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Expressing, Western Blot, Injection, Binding Assay

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Bioactivity evaluation of self-assembling nanofiber hydrogel PA-TIMP-QK in vitro. (A) MTT assay for detecting the proliferation activity of HUVECs. (B) The scratch assay for detecting the migration ability of HUVECs. Scale bars: 100 μm. (C) Statistical analysis of migration rate in the scratch assay ( n = 6). The migration area of the PA-TIMP-QK group was much larger than the control and VEGF165 groups. (D) Assessment of tube formation ability. The ability of the PA-TIMP-QK group to form tubules was stronger than the control group. The PA-QK and VEGF165 groups also had a stronger ability to form tubules than the control group. Scale bar: 50 μm. (E) Statistical analysis of tubes in the tube formation assay ( n = 6). (F) Statistical analysis of nodes in the tube formation assay ( n = 6). (G) MTT assay by OGD/R for detecting survival activity of PC12 cells. Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). HUVECs: Human umbilical vein endothelial cells; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OGD/R: oxygen-glucose deprivation and reperfusion; PA: peptide amphiphilic molecule; PC12 cells: pheochromocytoma cells; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: In Vitro, MTT Assay, Activity Assay, Wound Healing Assay, Migration, Control, Tube Formation Assay

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Histological analysis of cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) H&E staining of ischemic brain at 14 days after injection. The PA-TIMP-QK group showed a more complete structure, fewer apoptotic cells, and more normal cell morphology compared with the other three groups. Scale bar: 100 μm. (B) Nissl staining of ischemic brain at 14 days after injection. The number of Nissl bodies in the PA-TIMP-QK group was higher and their distribution was more regular compared with the PBS and VEGF165 groups. The dashed box area shows typical morphological features. Scale bar: 100 μm. (C) Statistical analysis of Nissl bodies ( n = 6). (D) Immunofluorescence staining of neurons in ischemic brain (NeuN, red, Alexa Fluor® 594; nuclei, blue). There were more surviving neurons in the PA-TIMP-QK group than in the PBS, VEGF165 and PA-QK groups. Scale bar: 100 μm. (E) Statistical analysis of NeuN + cells ( n = 6). (F) Immunofluorescence staining of axons in ischemic brain (Tuj1, red; nuclei, blue). There were more axons in the PA-TIMP-QK group than in the PBS and VEGF165 groups. Scale bar: 100 μm. (G) Statistical analysis of Tuj1 + cells ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). H&E staining: Hematoxylin and eosin staining; NeuN: neuronal nuclei; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; Tuj1: beta III tubulin; VEGF165: vascular endothelial growth factor 165.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Injection, Staining, Immunofluorescence, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Immunofluorescence staining of capillaries and vessels in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of capillaries in ischemic brain (vWF, red, Alexa Fluor ® 594; nuclei, blue). The number of vWF-labeled capillaries in the PA-TIMP-QK group was greater than the PBS and VEGF165 groups at 14 days after treatment. Scale bar: 100 μm. (B) Statistical analysis of vWF + capillaries ( n = 6). (C) Immunofluorescence staining of vessels in ischemic brain (α-SMA, red, Alexa Fluor ® 488; nuclei, blue). PA-TIMP-QK promoted the regeneration of blood vessels compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (D) Statistical analysis of α-SMA + vessels ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165; vWF: von Willebrand factor; α-SMA: α-smooth muscle actin.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Immunofluorescence, Staining, Injection, Labeling, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Immunofluorescence staining of tight junction protein in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of endothelial cell-specific intercellular adhesion molecules with VE-cadherin in ischemic brain (red, Alexa Fluor ® 594; nuclei, blue). The fluorescence intensity of VE-cadherin was significantly increased in the PA-TIMP-QK group compared with the PBS and VEGF165 groups. Scale bar: 100 μm. (B) Statistical analysis of mean fluorescence intensity of VE-cadherin ( n = 6). (C) Immunofluorescence staining of intercellular junction protein ZO-1 in ischemic brain (red, Alexa Fluor ® 594; nuclei, blue). The TJ formed with ZO-1 in the PA-TIMP-QK group was closer to that in the other three groups. Scale bars: 20 μm (upper) and 5 μm (lower). (D) Statistical analysis of mean fluorescence intensity of ZO-1 ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TJ: tight junction; VE-cadherin: vascular endothelial-cadherin; VEGF165: vascular endothelial growth factor 165; ZO-1: zonula occludens-1.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Immunofluorescence, Staining, Injection, Fluorescence, Saline

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Evaluation of cell inflammation and apoptosis and the activation of downstream signaling pathways in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Immunofluorescence staining of M1-type macrophages colabeled with CD68 and iNOS in the ischemic brain (CD68, red, Alexa Fluor ® 594; iNOS, green, Alexa Fluor ® 488; colocalization, yellow; nuclei, blue). CD68 and iNOS-colabeled M1 macrophages ratio in the PA-TIMP-QK group was much lower than that in the VEGF165 and PBS groups. Scale bar: 100 μm. (B) Statistical analysis of iNOS + /CD68 + macrophages ( n = 5). (C) Immunofluorescence staining of M2-type macrophages colabeled with CD68 and CD206 in the ischemic brain (CD68, red, Alexa Fluor ® 594; CD206, green, Alexa Fluor ® 488; colocalization, yellow; nuclei, blue). CD68 and CD206-colabeled M2 macrophages ratio in the PA-TIMP-QK group was much higher than that in the PBS and VEGF165 groups, but there was no statistical difference between the PA-TIMP-QK and PA-QK groups. Scale bar: 100 μm. (D) Statistical analysis of CD206 + /CD68 + macrophages ( n = 5). (E) TUNEL staining (TUNEL, green; nuclei, blue). The PA-TIMP-QK group had the fewest TUNEL-positive cells compared with the VEGF165 and PBS groups. Scale bar: 100 μm. (F) Statistical analysis of TUNEL + cells ( n = 6). (G) Western blot of p-AKT and AKT at 14 days after injection ( n = 4). (H) Western blot of p-ERK and ERK at 14 days after injection ( n = 4). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). AKT: The serine/threonine kinase; ERK: extracellular signal-regulated kinase; iNOS: inducible nitric oxide synthase; PA: peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling; VEGF165: vascular endothelial growth factor 165.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Activation Assay, Injection, Immunofluorescence, Staining, TUNEL Assay, Western Blot, Saline, End Labeling

Journal: Neural Regeneration Research

Article Title: A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

doi: 10.4103/NRR.NRR-D-23-01322

Figure Lengend Snippet: Animal behavior testing for rotarod test, grip strength test, and open field test at 7 and 14 days in cerebral ischemia/reperfusion after self-assembling nanofiber hydrogel PA-TIMP-QK injection. (A) Statistical analysis of latency during the rotarod test ( n = 6). (B) Statistical analysis of speed during the rotarod test ( n = 6). (C) Statistical analysis of grip strength test ( n = 6). (D) Representative paths of rats in the open field test, tracked by the SMART 3.0 system. The total distances of the PA-TIMP-QK group at 7 days were longer than those in the PBS and VEGF165 groups. The total distances of the PA-TIMP-QK group at 14 days were longer than those in the PBS, VEGF165 and PA-QK groups. There was no significant difference in anxiety assessment after 7 days. On the 14 th day, the PA-TIMP-QK group spent significantly longer time in the central zone of the open field compared with the PBS, PA-QK groups. (E) Statistical analysis of total distance at 7 and 14 days ( n = 6). (F) Statistical analysis of time in the center zone at 7 and 14 days ( n = 6). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). PA: Peptide amphiphilic molecule; PBS: phosphate buffered saline; QK: vascular endothelial growth factor mimic peptide-KLTWQELYQLKYKGI; TIMP: matrix metalloproteinase-2 cleaved peptide-PLGLAG; VEGF165: vascular endothelial growth factor 165.

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies: anti-MMP-2 (rabbit, 1:1000, Abcam, Cat# ab181286, RRID: AB_3073889), anti-AKT (rabbit, 1:1000, ABclonal, Cat# A17909, RRID: AB_2861754), anti-phosphorylated serine/threonine kinase Akt (p-AKT; rabbit, 1:1000, ABclonal, Cat# AP0140, RRID: AB_2770900), anti-extracellular regulated protein kinases 1/2 (ERK1/2; rabbit, 1:800, ZENBIO, Chengdu, Sichuan, China, Cat# 343830, AB_3073887), p-ERK1/2 (rabbit, 1:800, ZENBIO, Cat# R22917, RRID: AB_3073888), and β-actin rabbit monoclonal antibody (1:5000, ABclonal, Cat# AC038, RRID: AB_2863784).

Techniques: Injection, Saline

Journal: Molecular Medicine Reports

Article Title: PLOD2 exacerbates cervical squamous cell carcinoma by suppressing p53 by binding to YAP1

doi: 10.3892/mmr.2024.13388

Figure Lengend Snippet: PLOD2 silencing suppresses the migration, and promoted apoptosis and senescence in SiHa cells. (A) Cell migration was measured by wound healing assay at 0 h and the ending timepoint and (B) Transwell assay at the ending timepoint. (C) Western blotting was performed to assess the levels of invasion-associated proteins (MMP2 and MMP9). (D) Cell apoptosis was examined by flow cytometric analysis. (E) Western blotting was used to assess apoptotic-related protein (Bcl-2, Bax, cleaved caspase 3 and caspase 3) levels in SiHa cells. (F) The expression level of SA-β-Gal was detected by SA-β-Gal staining. (G) The levels of senescence-associated secretory phenotype genes (IL-6, IL-1β, IL-8 and CCL20) were identified by RT-qPCR. Results are the mean ± SD. ***P<0.001. PLOD2, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2; NC, negative control; SA-β-Gal, β-galactosidase.

Article Snippet: After blocking with 5% BSA (Biofroxx; neoFroxx) for 1 h at room temperature, the membranes were cultured with primary antibodies PLOD2 (1:1,000; Abcam; cat. no. ab313765), MMP2 (1:3,000; Proteintech Group, Inc.; cat. no. 66366-1-Ig), MMP9 (1:500; Proteintech Group, Inc.; cat. no. 27306-1-AP), Bcl-2 (1:1,000; Affinity Biosciences; cat. no. AF6139), Bax (1:1,000; Affinity Biosciences; cat. no. AF0120), caspase 3 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 9662), cleaved caspase 3 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 9661), YAP1 (1:5,000; Abcam; cat. no. ab52771) and GAPDH (Proteintech Group, Inc.; cat. no. 60004-1-Ig; 1:5,000) overnight at 4°C and with secondary antibodies HRP-conjugated Goat Anti-Mouse IgG (H+L; 1:2,000; Proteintech Group, Inc.; cat. no. SA00001-1) or HRP-conjugated Goat Anti-Rabbit IgG (H+L; 1:2,000; Proteintech Group, Inc.; cat. no. SA00001-2) at 37°C for 2 h. The ELC A solution was mixed with the B solution in equal proportions and visualization of the protein bands was achieved using the ECL detection system (Thermo Fisher Scientific, Inc.) in accordance with standard protocols , and analysis of protein density was assessed by Image J software v1.50 (National Institutes of Health).

Techniques: Migration, Wound Healing Assay, Transwell Assay, Western Blot, Expressing, Staining, Quantitative RT-PCR, Negative Control

Journal: Molecular Medicine Reports

Article Title: PLOD2 exacerbates cervical squamous cell carcinoma by suppressing p53 by binding to YAP1

doi: 10.3892/mmr.2024.13388

Figure Lengend Snippet: PLOD2 regulates YAP1 to promote proliferation, migration and invasion of SiHa cells. (A) mRNA and (B) protein levels of YAP1 in SiHa cells after YAP1 overexpression were detected by RT-qPCR and western blotting. (C) CCK-8 assay was used to measure the cell proliferation of SiHa cells. (D) Wound healing assay (at 0 h and the ending timepoint) and (E) Transwell assay (at the ending timepoint) were used to detect cell migration. (F) Western blotting was performed to assess the levels of invasion-associated proteins (MMP2 and MMP9). Results are the mean ± SD. ***P<0.001. PLOD2, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2; YAP1, Yes-associated protein 1; Ov-NC, overexpression vector-negative control; Ov-YAP1, overexpression vector-YAP1; siRNA-NC, small interfering RNA-negative control; siRNA-PLOD2, small interfering RNA-PLOD2.

Article Snippet: After blocking with 5% BSA (Biofroxx; neoFroxx) for 1 h at room temperature, the membranes were cultured with primary antibodies PLOD2 (1:1,000; Abcam; cat. no. ab313765), MMP2 (1:3,000; Proteintech Group, Inc.; cat. no. 66366-1-Ig), MMP9 (1:500; Proteintech Group, Inc.; cat. no. 27306-1-AP), Bcl-2 (1:1,000; Affinity Biosciences; cat. no. AF6139), Bax (1:1,000; Affinity Biosciences; cat. no. AF0120), caspase 3 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 9662), cleaved caspase 3 (1:1,000; CST Biological Reagents Co., Ltd.; cat. no. 9661), YAP1 (1:5,000; Abcam; cat. no. ab52771) and GAPDH (Proteintech Group, Inc.; cat. no. 60004-1-Ig; 1:5,000) overnight at 4°C and with secondary antibodies HRP-conjugated Goat Anti-Mouse IgG (H+L; 1:2,000; Proteintech Group, Inc.; cat. no. SA00001-1) or HRP-conjugated Goat Anti-Rabbit IgG (H+L; 1:2,000; Proteintech Group, Inc.; cat. no. SA00001-2) at 37°C for 2 h. The ELC A solution was mixed with the B solution in equal proportions and visualization of the protein bands was achieved using the ECL detection system (Thermo Fisher Scientific, Inc.) in accordance with standard protocols , and analysis of protein density was assessed by Image J software v1.50 (National Institutes of Health).

Techniques: Migration, Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Plasmid Preparation, Negative Control, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer

doi: 10.3892/ijo.2024.5704

Figure Lengend Snippet: Antibodies used in the present study.

Article Snippet: MMP2 , Western blotting , Cell Signaling Technology, Inc. , 40994.

Techniques: Western Blot, Immunofluorescence

Journal: International Journal of Oncology

Article Title: Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer

doi: 10.3892/ijo.2024.5704

Figure Lengend Snippet: HT inhibits the migration and invasion of human BC cells. (A and B) Transwell migration and invasion assays were conducted to assess the migratory and invasive abilities of BC cells. Magnification, ×200. (C and D) Western blotting was performed to measure the level of MMP2 protein expression in BC cells. Data are presented in terms of mean ± SD values. Two-groups comparisons were performed using Student's t-test, whereas one-way ANOVA followed by Bonferroni's post hoc test was used to compare mean values among multiple groups. * P<0.05 and **** P<0.0001, vs. NT group. HT, hyperthermia; BC, bladder cancer; NT, normal temperature; MMP, matrix metalloproteinase.

Article Snippet: MMP2 , Western blotting , Cell Signaling Technology, Inc. , 40994.

Techniques: Migration, Western Blot, Expressing

Journal: Experimental and Therapeutic Medicine

Article Title: Formononetin suppresses the malignant progression of papillary thyroid carcinoma depending on downregulation of CBX4

doi: 10.3892/etm.2024.12746

Figure Lengend Snippet: FMNT treatment represses the migration, invasion, EMT and angiogenesis of papillary thyroid carcinoma cells. TPC-1 cells were treated with 0, 10, 30 and 100 µM FMNT for 24 h. (A) The migratory ability of TPC-1 cells was evaluated by wound healing assay (magnification, x100). (B) The invasive ability of TPC-1 cells was evaluated by Transwell assay (magnification, x100). (C) Expression levels of MMP2 and MMP9 in TPC-1 cells were determined by western blot analysis. (D) Expression levels of E-cadherin, N-cadherin and Vimentin in TPC-1 cells were determined by western blotting. (E) HUVECs were incubated with the conditioned media of TPC-1 cells at 37˚C for 24 h. In vitro angiogenesis of HUVECs was evaluated using a tube formation assay (magnification, x200). (F) VEGF and (G) VEGFR2 levels in TPC-1 cells were determined by reverse transcription-quantitative PCR. * P<0.05, ** P<0.01 and *** P<0.001 vs. the Control group. FMNT, formononetin; HUVECs, human umbilical vein endothelial cells.

Article Snippet: After blocking in 5% non-fat milk for 1 h at 37˚C, membranes were probed with primary antibodies [anti-CBX4 (1:1,000; cat. no. ab4189; Abcam), anti-NANOG (1:1,000; cat. no. ab109250; Abcam), anti-OCT4 (1:1,000; cat. no. ab19857; Abcam), anti-CD133 (1:2,000; cat. no. ab222782; Abcam), anti-MMP2 (1:1,000; cat. no. ab92536; Abcam), anti-MMP9 (1:1,000; cat. no. ab76003; Abcam), anti-E-cadherin (1:1,000; cat. no. ab40772; Abcam), anti-N-cadherin (1:5,000; cat. no. ab76011; Abcam) and anti-Vimentin (1:1,000; cat. no. ab92547; Abcam)] overnight at 4˚C and subsequently incubated with HRP-conjugated secondary antibodies (1:20,000, cat. no. ab6721; Abcam) for 1 h at 37˚C.

Techniques: Migration, Wound Healing Assay, Transwell Assay, Expressing, Western Blot, Incubation, In Vitro, Tube Formation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Formononetin suppresses the malignant progression of papillary thyroid carcinoma depending on downregulation of CBX4

doi: 10.3892/etm.2024.12746

Figure Lengend Snippet: FMNT treatment represses the migration, invasion, EMT and angiogenesis of papillary thyroid carcinoma cells by downregulating CBX4 expression. FMNT-treated TPC-1 cells were transfected with oe-CBX4 or oe-NC. (A) The migratory ability of TPC-1 cells was evaluated by wound healing assay (magnification, x100). (B) The invasive ability of TPC-1 cells was evaluated using a Transwell assay (magnification, x100). (C) Expression levels of MMP2 and MMP9 in TPC-1 cells were determined by western blotting. (D) Expression levels of E-cadherin, N-cadherin and Vimentin in TPC-1 cells were determined by western blot analysis. (E) HUVECs were incubated with the CM of TPC-1 cells at 37˚C for 24 h. In vitro angiogenesis of HUVECs was evaluated by tube formation assay (magnification, x200). (F and G) VEGF and VEGFR2 levels in TPC-1 cells were determined by reverse transcription-quantitative PCR. *** P<0.001 vs. the Control group; ## P<0.01 and ### P<0.001 vs. FMNT + oe-NC. FMNT, formononetin; CBX4, chromobox homolog 4; oe, overexpression; NC, negative control; HUVECs, human umbilical vein endothelial cells.

Article Snippet: After blocking in 5% non-fat milk for 1 h at 37˚C, membranes were probed with primary antibodies [anti-CBX4 (1:1,000; cat. no. ab4189; Abcam), anti-NANOG (1:1,000; cat. no. ab109250; Abcam), anti-OCT4 (1:1,000; cat. no. ab19857; Abcam), anti-CD133 (1:2,000; cat. no. ab222782; Abcam), anti-MMP2 (1:1,000; cat. no. ab92536; Abcam), anti-MMP9 (1:1,000; cat. no. ab76003; Abcam), anti-E-cadherin (1:1,000; cat. no. ab40772; Abcam), anti-N-cadherin (1:5,000; cat. no. ab76011; Abcam) and anti-Vimentin (1:1,000; cat. no. ab92547; Abcam)] overnight at 4˚C and subsequently incubated with HRP-conjugated secondary antibodies (1:20,000, cat. no. ab6721; Abcam) for 1 h at 37˚C.

Techniques: Migration, Expressing, Transfection, Wound Healing Assay, Transwell Assay, Western Blot, Incubation, In Vitro, Tube Formation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Over Expression, Negative Control