Journal: Nature communications

Article Title: Activation of E-prostanoid 3 receptor in macrophages facilitates cardiac healing after myocardial infarction.

doi: 10.1038/ncomms14656

Figure Lengend Snippet: Figure 5 | Ep3 regulates CX3CR1 and VEGF expression through TGFb1 signalling. (a,b)Western blot analysis of CCR2, CX3CR1, phospho-Smad2, phospho-Smad3 and VEGF in Mos/Mps from Ep3F/F and Ep3F/F;LysMCre mice at 16 h (a) and 96 h (b)after a zymosan challenge. (c) Western blot analysis of CCR2, CX3CR1, phospho-Smad2, phospho-Smad3 and VEGF in cultured Mps with or without PGE2 stimulation. (d) Effect of Ep3 deletion on Mp migration in response to PGE2. Data represent mean±s.e.m. *Po0.05 versus Ep3F/F, #Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 5. (e) Effect of TGFb1 blocker SB525334 on Ep3-mediated VEGF and CX3CR1 mRNA expression in cultured Mps. Mp-Ep3Tg, Mp-specific Ep3a transgenic mice. Data represent mean±s.e.m. *Po0.05 versus wild-type (WT), **Po0.01 versus indicated (unpaired two-tailed t-test); n ¼ 5. (f) Representative immunostaining of CD68 (red) and VEGF (green) in peri-infarct zones of hearts from Mp-Ep3Tg mice at day 14 post MI. The solid box outlines the region enlarged below. Yellow arrow, CD68 þ/VEGF þ cells. Scale bar 20 mm. IZ, infarct zone. (g) Quantitation of VEGF signalling in CD68 þ cells in injured hearts as shown in f. Data represent mean±s.e.m. *Po0.05 versus WT, #Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 5. (h) Effect of SB525334 on Ep3-mediated VEGF and CX3CR1 protein expression in cultured Mps. (i) Effect of SB525334 on PGE2/Ep3-mediated Mp migration in vitro. Data represent mean±s.e.m. *Po0.05 versus WT, #Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 4–6.

Article Snippet: Equal quantities of proteins from total cell lysates were separated by 10% SDS–PAGE gel and probed with antibodies against CX3CR1 (1:500, Abcam), CCR2 (1:500, Abcam), p-Smad2 (1:1,000, CST), p-Smad3 NATURE COMMUNICATIONS | 8:14656 | DOI: 10.1038/ncomms14656 | www.nature.com/naturecommunications 11 (1:1,000, CST), T-Smad3 (1:1,000, CST), T-Smad2/3 (1:1,000, CST), VEGF (1:1,000, Proteinteh), and GAPDH (1:2,000, CST), and then conjugated with an HRP-labelled secondary antibody in blocking buffer for 1–2 h at room temperature.

Techniques: Expressing, Western Blot, Cell Culture, Migration, Two Tailed Test, Transgenic Assay, Immunostaining, Quantitation Assay, In Vitro

Journal: Nature communications

Article Title: Activation of E-prostanoid 3 receptor in macrophages facilitates cardiac healing after myocardial infarction.

doi: 10.1038/ncomms14656

Figure Lengend Snippet: Figure 7 | CX3CR1 and VEGF are TGFb1-targeting genes in mice and humans. (a,b) Schematic illustration of predicted Smad binding elements (SBEs) in the promoter of murine Cx3cr1 (a) and Vegf (b) genes. S ¼ SBE. (c) Gel electrophoresis of PCR-amplified SBE-containing fragments in the promoter of the murine Cx3cr1 gene using anti-Smad2/3 immunoprecipitation. (d) Effect of the TGFb1 blocker SB525334 on S3 þ S4 fragment-mediated luciferase activity. *Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 4. (e) Effect of mutations of S3 (mS3) and S4 (mS4) on S3 þ S4 fragment-mediated transcription activity. *Po0.05 versus the S3 þ S4 group, n ¼ 4. (f) Effect of L-798,106 on S3 þ S4 fragment-mediated luciferase activity in RAW264.7 cells. *Po0.05 versus no treatment (unpaired two-tailed t-test);n ¼ 4. (g) Gel electrophoresis of PCR-amplified SBE-containing fragments in the promoter of the murine Vegf gene using anti-Smad2/3 immunoprecipitation. (h) Effect of SB525334 on the functional SBEs (S10 þ S20) in murine Vegf promoter. *Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 4. (i) Effect of L798106 on S10 þ S20 fragment-mediated luciferase activity in RAW264.7 cells. *Po0.05 versus no treatment (unpaired two-tailed t-test); n ¼ 5. (j,k) Schematic illustration of the predicted SBE(s) on the human CX3CR1 gene (hS, (j)) and the human VEGF gene (hS0, (k)). (l) Gel electrophoresis of PCR-amplified SBE-containing fragments in the promoter of the human CX3CR1 gene. (m) Effect of SB525334 on hS-mediated transcription activity in THP-1 cells. *Po0.05 as indicated (unpaired two-tailed t-test); n ¼ 5. (n) Effect of L-798,106 on hS- mediated transcription activity in THP-1 cells. *Po0.05 versus no treatment (unpaired two-tailed t-test); n ¼ 5. (o) Gel electrophoresis of PCR-amplified SBE-containing fragments in the promoter of the human VEGF gene. (p) Effect of SB525334 on hS30–hS50 fragment-mediated transcription activity in THP-1 cells. *Po0.05 as indicated, n ¼ 5. (q) Effect of L-798,106 on hS30–hS50 fragment-mediated transcription activity in THP-1 cells. *Po0.05 versus no treatment (unpaired two-tailed t-test); n ¼ 5. (r) Schematic diagram of EP3-mediated regulation of CX3CR1 and VEGF genes in Mos/Mps through TGFb1.

Article Snippet: Equal quantities of proteins from total cell lysates were separated by 10% SDS–PAGE gel and probed with antibodies against CX3CR1 (1:500, Abcam), CCR2 (1:500, Abcam), p-Smad2 (1:1,000, CST), p-Smad3 NATURE COMMUNICATIONS | 8:14656 | DOI: 10.1038/ncomms14656 | www.nature.com/naturecommunications 11 (1:1,000, CST), T-Smad3 (1:1,000, CST), T-Smad2/3 (1:1,000, CST), VEGF (1:1,000, Proteinteh), and GAPDH (1:2,000, CST), and then conjugated with an HRP-labelled secondary antibody in blocking buffer for 1–2 h at room temperature.

Techniques: Binding Assay, Nucleic Acid Electrophoresis, Immunoprecipitation, Luciferase, Activity Assay, Two Tailed Test, Functional Assay