Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Schematic depicting the protocol for hCECs, hCSSCs, and hiNSCs seeding onto silk scaffolds.

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques:

Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Evaluating the effects of the microenvironment (elevated glucose or dynamic perfusion) and different chemical stimulants (capsaicin and sodium lauryl sulfate) on cell viability and gene and protein expression using the corneal tissue model. ( Abbreviations : Interleukin-1 beta (IL-1β), Substance P (SP), Calcitonin gene-related peptide (CGRP); Gene names : CRCP -accessory protein for CGRP, SCN- sodium channel, KERA - keratocan (cornea-specific proteoglycan), LUM - lumican (proteoglycan abundant in the stroma)).

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques: Expressing, Concentration Assay, Permeability

Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Common secreted proteins related to nerve activation or tissue inflammation present in conditioned media isolated from corneal constructs. The relative concentration ranges of each protein detected in conditioned media in response to various chemical stimuli have been previously reported by our group ( Deardorff et al., 2018 ; Siran et al., 2018 ). ( Abbreviations : Substance P (SP), Calcitonin gene-related peptide (CGRP), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α), Matrix metalloproteinase-9 (MMP-9)).

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques: Activation Assay, Isolation, Construct, Concentration Assay

Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Phenotypic markers of each cell type present in the corneal construct. ( Abbreviations : Transient receptor potential cation channel subfamily V member 1 (TRPV1), Transient receptor potential cation channel subfamily A member 1 (TRPA1), Transient receptor potential cation channel subfamily M member 8 (TRPM8)).

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques: Construct, Marker

Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Troubleshooting guide for construct assembly and characterization.

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques: Construct, Cell Culture, Microscopy, Incubation, Sterility, Isolation, Lysis, Homogenization, Binding Assay

Journal: Current protocols in toxicology

Article Title: Assembly and Application of a 3D Human Corneal Tissue Model: A Step-by-Step Guide

doi: 10.1002/cptx.84

Figure Lengend Snippet: Relative timeframe for completion of each step in the assembly, stimulation, and characterization of the corneal tissue model. The amount of time may vary significantly depending on the user and the total number of constructs generated. These timeframes are based on 12 constructs per experiment.

Article Snippet: SUBCULTURING OF hCECs Materials Complete corneal epithelial media ( Refer to Reagents and Solutions Section ) Pipette T75 flask Primary human corneal epithelial cells (hCECs, ATCC, cat. no. ATCC® PCS-700-010TM) FNC Coating Mix® (Athena Environmental Sciences, cat. no. 0407) (Optional) 500 mL cell culture flask Seed and maintain hCECs 1.

Techniques: Construct, Generated, Incubation, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Imaging, Lysis, Protein Concentration, BIA-KA, Western Blot

Journal: Cell reports

Article Title: Microenvironmental stiffness induces metabolic reprogramming in glioblastoma

doi: 10.1016/j.celrep.2023.113175

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HYAL2 TaqMan ® Gene Expression Assays, Dye: FAM-MGB-small 250rxn cat#4331182 , Thermofisher , Hs00186841_m1.

Techniques: Virus, Plasmid Preparation, Recombinant, Sequencing, Gene Expression, Software

Journal: Molecular therapy. Nucleic acids

Article Title: Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.

doi: 10.1016/j.omtn.2023.08.001

Figure Lengend Snippet: Figure 6. dCasX-mediated p110d attenuation suppresses Akt activation and VEGF production in the mouse model of OIR (A and B) Retinal mRNA and proteins from P17 mice (n = 6) subjected to qPCR (A) and western blotting analyses with indicated antibodies (B), respectively. (C) The intensity of bands. The bar graphs are mean ± SD of three independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey honest significant difference (HSD) post hoc test. *p < 0.05, **p < 0.01. (D) Clarified vit- reous (5 mL) from the P17 mice with or without experiencing OIR was subjected to ELISA analysis by following the instructions of a Quantikine Mouse VEGF ELISA Kit.31 The bar graphs are mean ± SD of six mice. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. NS, not significant. *p < 0.05; **p < 0.01.

Article Snippet: This assay was conducted by following the instructions of a Quantikine Mouse VEGF ELISA Kit (Cat.MMV00; R&D Systems).

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International immunopharmacology

Article Title: GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17.

doi: 10.1016/j.intimp.2024.111603

Figure Lengend Snippet: Fig. 2. GTS-21 regulated the ACE/ACE2 ratio and glycocalyx shedding in LPS-ALI mice A-D ELISA kits were used to quantify the concentrations of major renin- angiotensin system (RAS) regulating enzymes (ACE, Ang II, ACE2, and Ang 1-7) in the lung tissue (n = 6 per group). E Protein levels of important RAS- regulating enzymes (ACE, AT1R, ACE2, and MasR) (n = 3 per group). The relative protein levels are displayed. F Levels of NF-κB phosphoproteins (p-P65 and p- IκB) (n = 3 per group). The relative protein levels are displayed. The immunofluorescence of HS, SDC-1, and surfactant protein D in the lungs is shown in G in representative images and densitometry (scar bar, 20 mm, n = 3 per group). H Evans Blue staining tested lung permeability (n = 3 for each group). Using ELISA, we assessed the BALF concentrations of HS I and HPA J; SP-D in serum K and BALF L (n = 5 for each group). M Relative protein levels of SDC-1, HPA, EXT-1, and SP-D in The lung tissues (n = 3 for each group). All data were shown as mean ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: AT1R (cat. no. 25343–1-ap) and MasR (cat. no. 20080–1-ap) were bought from Proteintech.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Permeability

Journal: International immunopharmacology

Article Title: GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17.

doi: 10.1016/j.intimp.2024.111603

Figure Lengend Snippet: Fig. 5. Effect of macrophage polarization on ACE/ACE2 ratio and glycocalyx shedding in epithelial cells. A The experimental procedure for RAW264.7 cells su pernatant with different polarization states were added to the culture medium to test their effect on alveolar epithelial cells. B The expressions of key renin- angiotensin system regulatory enzymes (ACE, AT1R, ACE2, and MasR) and main component of NF-κB pathway (p-P65 and p-IκB) were detected. C Representa tive image and densitometry of immunofluorescence of the main component of glycocalyx (HS and SDC-1) and surfactant protein-D in MLE-12 cells (Scar bar, 20 μm). D Expression of the main components of the glycocalyx (SDC-1), key regulatory enzymes (HPA and EXT1), and SP-D was quantified in MLE-12 cells. Based on three independent experiments, all data were shown as mean ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: AT1R (cat. no. 25343–1-ap) and MasR (cat. no. 20080–1-ap) were bought from Proteintech.

Techniques: Immunofluorescence, Expressing

Journal: International immunopharmacology

Article Title: GTS-21 attenuates ACE/ACE2 ratio and glycocalyx shedding in lipopolysaccharide-induced acute lung injury by targeting macrophage polarization derived ADAM-17.

doi: 10.1016/j.intimp.2024.111603

Figure Lengend Snippet: Fig. 7. Effect of ADAM-17 on ACE/ACE2 ratio and glycocalyx shedding in epithelial cells Experimental procedure of RAW264.7 cells with recombinant ADAM17 (rADAM-17) or siRNA ADAM-17 to elevate and inhibit the activity of ADAM-17. Macrophage supernatant was used to test its effect on epithelial cells. A The ex pressions of key renin-angiotensin system regulatory enzymes (ACE, AT1R, ACE2, and MasR) and main component of NF-κB pathway (p-P65 and p-IκB) were determined. B Representative image and densitometry of immunofluorescence of main component of glycocalyx (HS and SDC-1) and surfactant protein-D in the MLE- 12 cells (Scar bar, 20 μm). C The expression levels of the main components of the glycocalyx (SDC-1), key regulatory enzymes (HPA and EXT1), and SP-D were quantified in MLE-12 cells. Based on three independent experiments, all data were shown as mean ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: AT1R (cat. no. 25343–1-ap) and MasR (cat. no. 20080–1-ap) were bought from Proteintech.

Techniques: Recombinant, Activity Assay, Immunofluorescence, Expressing

Journal: Cells

Article Title: S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis

doi: 10.3390/cells10082168

Figure Lengend Snippet: c-Src-dependent FAK activation regulates S1P-induced effects on VEGF production and EPC angiogenesis. ( A , B ) Osteoblasts were pretreated with c-Src (PP2) (10 μM) or FAK inhibitors (10 μM), or transfected with siRNAs (c-Src or FAK), then stimulated with S1P. VEGF levels were quantified by qPCR and ELISA. ( C , D ) Collected CM was administered to EPCs, and EPC angiogenesis was determined. ( E ) Osteoblasts were transfected with c-Src or FAK siRNAs and Western blot determined c-Src or FAK expression. ( F ) After incubating osteoblasts with S1P, Western blot determined c-Src and FAK phosphorylation. ( G , H ) Osteoblasts were pretreated with c-Src (PP2) (10 μM) or FAK inhibitors (10 μM), then stimulated with S1P. Western blot quantified c-Src and FAK phosphorylation and total protein. Results are expressed as the mean ± S.D. ( n = 3). * p < 0.05 versus the control; # p < 0.05 versus S1P alone.

Article Snippet: Antibodies against the phosph- FAK (catalog number: sc-374668) and c-Src (catalog number: SC-12928-R), total protein of c-Src (catalog number: SC-5266) and FAK (catalog number: SC-1688), VEGF (catalog number: SC-7269), SphK1 (catalog number: sc-365401) and β-actin (catalog number: sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Phospho-proteomics, Control

Journal: Cells

Article Title: S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis

doi: 10.3390/cells10082168

Figure Lengend Snippet: S1P facilitates VEGF synthesis and EPC angiogenesis via the inhibition of miR-16-5p. ( A ) The open-source software enabled identification of miRNAs that possibly disturb the transcription of VEGF. ( B ) Osteoblasts were transfected with S1P. MiR-16-5p expression was determined by the qPCR assay. ( C , D ) The osteoblasts were transfected with miR-16-5p mimic, then stimulated by S1P. Levels of VEGF were determined by qPCR and ELISA. ( E , F ) Collected CM was administered to the EPCs, and angiogenesis was quantified. ( G ) A schematic representation of human VEGF containing the miR-16-5p binding site. ( H ) The luciferase plasmids with, or without miR-16-5p mimic, were transfected into osteoblasts, before stimulating them with S1P. Measurement of luciferase activity revealed VEGF promoter activity. ( I ) Osteoblasts were transfected with S1P 1 or S1P 3 siRNA for 24 h, or pretreated with c-Src or FAK inhibitors for30 min, then stimulated with S1P for 24 h. MiR-16-5p expression was quantified by qPCR. Results are expressed as the mean ± S.D. ( n = 3). * p < 0.05 versus the control; # p < 0.05 versus S1P alone.

Article Snippet: Antibodies against the phosph- FAK (catalog number: sc-374668) and c-Src (catalog number: SC-12928-R), total protein of c-Src (catalog number: SC-5266) and FAK (catalog number: SC-1688), VEGF (catalog number: SC-7269), SphK1 (catalog number: sc-365401) and β-actin (catalog number: sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Inhibition, Software, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Activity Assay, Control

Journal: Cells

Article Title: S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis

doi: 10.3390/cells10082168

Figure Lengend Snippet: S1P knockdown reduces the in vivo severity of RA. ( A ) After infecting osteoblasts with control or SphK1 shRNA, Western blot determined SphK1 and VEGF expression. CIA mice received intra-articular injections of ~7.1 × 10 6 PFU SphK1 shRNA or control shRNA on day 14 and were sacrificed on day 49. ( B ) Representative µ-CT images of the hind paws taken on day 49. ( C ) A digital plethysmometer quantified the amounts of hind paw swelling. ( D – F ) µ-CT SkyScan Software quantified BMD, trabecular numbers and bone volume. ( G ) Histological sections of ankle joints were stained with H and E or Safranin O/fast green, and immunostained with VEGF, CD31, CD34 and CD133. Results are expressed as the mean ± S.D. ( n = 3). * p < 0.05 versus the control group; # p < 0.05 versus S1P alone.

Article Snippet: Antibodies against the phosph- FAK (catalog number: sc-374668) and c-Src (catalog number: SC-12928-R), total protein of c-Src (catalog number: SC-5266) and FAK (catalog number: SC-1688), VEGF (catalog number: SC-7269), SphK1 (catalog number: sc-365401) and β-actin (catalog number: sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, In Vivo, Control, shRNA, Western Blot, Expressing, Software, Staining

Journal: Cells

Article Title: S1P Increases VEGF Production in Osteoblasts and Facilitates Endothelial Progenitor Cell Angiogenesis by Inhibiting miR-16-5p Expression via the c-Src/FAK Signaling Pathway in Rheumatoid Arthritis

doi: 10.3390/cells10082168

Figure Lengend Snippet: Schematic diagram summarizes the mechanisms of S1P-induced EPC angiogenesis during RA pathogenesis. S1P induces VEGF expression in osteoblasts by suppressing miR-16-5p expression via the S1P 1 and S1P3 receptors, and the c-Src and FAK signaling pathways, and promotes EPC angiogenesis in RA.

Article Snippet: Antibodies against the phosph- FAK (catalog number: sc-374668) and c-Src (catalog number: SC-12928-R), total protein of c-Src (catalog number: SC-5266) and FAK (catalog number: SC-1688), VEGF (catalog number: SC-7269), SphK1 (catalog number: sc-365401) and β-actin (catalog number: sc-47778) were bought from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Protein-Protein interactions