Fig 1: CCL15 knockdown in HUVECs attenuates the migration of CFs towards HUVECs. (A) The mRNA expression levels in HUVECs each group were measured by reverse transcription-quantitative PCR. (B) Concentrations of CCL15 HUVEC culture supernatant in each group were measured by ELISA. (C) Migrated CFs were stained and imaged under a microscope (C-a) Control group, (C-b) NC group and (C-c) Si-CCL15 group. n=3. Scale bar, 100 µm. (D) The number of migrated CFs in each group. Untransfected HUVECs were used in Ctr groups and non-sense siRNA transfected HUVECs were used in the NC groups. ***P<0.001 and ****P<0.0001 vs. NC; ns vs. Ctr. HUVEC, human umbilical vein endothelial cell; CFs, circulating fibrocytes; Ctr, control; NC, negative control; si, small interfering; CCL, chemokine ligand; ns, non-significant.
Fig 2: CCL15 overexpression in HUVECs promotes the migration of CFs towards HUVECs. (A) The mRNA expression levels of CCL15 in HUVECs of each group were measured by reverse transcription-quantitative PCR. (B) Concentrations of CCL15 in HUVEC culture supernatants in each group were measured by ELISA. (C) Migrated CFs were stained and imaged under a microscope. (C-a) Control group, (C-b) Negative control group and (C-c) CCL15 group. n=3. Scale bar, 100 µm. (D) The number of migrated CFs in each group. Untransfected HUVECs were used in the Ctr groups and empty vector transfected HUVECs were used in the NC groups. ***P<0.001 and ****P<0.0001 vs. NC; ns vs. Ctr. HUVEC, human umbilical vein endothelial cell; CFs, circulating fibrocytes; Ctr, control; NC, negative control; CCL, chemokine ligand; ns, non-significant.
Fig 3: CCL15 is a vital chemokine that mediates the transmigration of CFs toward HUVECs. (A) Expression levels of the three chemokines in mono-cultured and co-cultured HUVECs were measured by gene chip microarray analysis and expressed as relative fluorescent signals. (B) Relative mRNA expression levels of CCL2, CCL15 and CXCL8 in the mono-cultured HUVECs and co-cultured HUVECs were measured by reverse transcription-quantitative PCR. (C) Concentrations of CCL2, CCL15 and CXCL8 in the medium of mono-cultured HUVECs and co-cultured HUVECs were measured by ELISA. (D) Migratory CFs were stained and imaged under a microscope. (D-a) Control group, (D-b) CXCL8 blocking group, (D-c) CCL2 blocking group and (D-d) CCL15 blocking group. n=3. Scale bar, 100 µm. (E) The number of migrated CFs in each group. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. HUVECs (parts A-C) or Ctr (part E). HUVEC, human umbilical vein endothelial cell; CFs, circulating fibrocytes; Ctr, control; CCL, chemokine ligand; CXCL, CXC chemokine ligand.
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