pcdna3 yfp expression vector  (Addgene inc)

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 5. oxLDL induces uPAR association with TLR4. A. oxLDL-induced association of uPAR and TLR4 was assessed by co-immunoprecipitation. VSMC were treated with 4 μg/ml oxLDL for indicated time points, lysed and uPAR was immunoprecipitated using monoclonal anti-uPAR antibody. TLR4 in the immunoprecipitates was detected using polyclonal anti-TLR4 antibody. B. uPAR/TLR4 association was studied using immunocytochemistry. VSMC were treated with 4 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained for TLR4 (Alexa 488) and uPAR (Alexa 594). C. uPAR/TLR4 association was studied using Duolink PLA. VSMC were treated with 4.5 μg/ml oxLDL for 1 h, fixed with 4%PFA and stained as recommended by Duolink probes' provider. The number of Duolink signals per cell (right panel) was quantified using colony counting tool of ImageJ software. D. oxLDL-induced changes of SMA and myocardin expression were assessed by RT-PCR in VSMC in the presence of 5 μg/ml TLR4 antagonist Cli-095. E. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. Contractile proteins expression was analyzed by RT-PCR. F. SiCo- and TLR4si-transfected VSMC were treated with 7 μg/ml oxLDL for 48 h. G-CSF and GM-CSF expression was analyzed by RT-PCR. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Immunoprecipitation, Immunocytochemistry, Staining, Software, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 6. oxLDL-induced cytokine expression is mediated by NF-κB. A. VSMC were lentivirus-infected to express Gaussia luciferase under control of NF-κB responsive promoter as described in the Materials and methods section. 24 h after infection cells were transfected with SiCo or other siRNA as indicated. 24 h after transfection cells were stimulated with 5 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. B. HEK 293 cells were lentiviral infected to express Gaussia luciferase under control of NF-κB responsive promoter. Expression of uPAR was achieved by lentiviral infection. TLR4 and CD36 expression was achieved by cell transfection. Cells were stimulated with 8 μg/ml oxLDL for 2 h and luciferase activity in cell conditioned media was measured. C. Cells were stimulated with oxLDL in the presence of proteasome inhibitors. G-CSF and GM-CSF expression was assessed by RT-PCR. D. oxLDL- induced G-CSF and GM-CSF expression in the presence of NF-κB inhibitor BAY-11-7085 was assessed by RT-PCR. E. Primary human monocytes and macrophages were treated with conditioned medium of SiCo- and uPARsi-transfected VSMC stimulated with 8 μg/ml oxLDL. MCP-1 release was followed using Human CCL2/MCP-1 Quantikine ELISA Kit.* indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: Expressing, Infection, Luciferase, Control, Transfection, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of molecular and cellular cardiology

Article Title: oxLDL induces inflammatory responses in vascular smooth muscle cells via urokinase receptor association with CD36 and TLR4.

doi: 10.1016/j.yjmcc.2013.11.005

Figure Lengend Snippet: Fig. 7. In vivo evidence for uPAR-mediated events. A. Images captured from sub-fibrous cap area of the plaque stained for CD36 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. B. Images captured from sub-fibrous cap area of the plaque stained for TLR4 (Alexa 488), uPAR(Alexa 594) and DraQ5 as a nuclear stain. The right panel shows quantification of CD36/uPAR colocalization in plaque area and media. Scale bar 100 μm. * indicates significant difference (P b 0.05).

Article Snippet: After 18 h cells were transfected with luciferase constructs and incubated for 24 h. oxLDL stimulation was performed in serum for 6 h. TRL2 and TLR4 expression vectors reported by Dr. Golenbock were obtained from Addgene (plasmids 13016 and 13018, respectively); human CD36 expression vector was from InvivoGene; uPAR expression was achieved by lentiviral infection as described [22].

Techniques: In Vivo, Staining