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. 2016 Oct 10;126(11):4187–4204. doi: 10.1172/JCI78260

Figure 6. SPARCL1 is a marker of EC quiescence.

Figure 6

(A) MVECs were plated on chamber slides and grown to subconfluent and confluent states. Cells were costained by IHC for SPARCL1/Ki-67 or SPARCL1/IL-33 (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 75 μm. Graph depicts the relative numbers (percentages) of Ki-67– and nuclear IL-33–positive cells per optical field. Error bars indicate SD. (B) HUVECs were transiently transfected with a human SPARCL1 expression plasmid. Cells were incubated 24 hours after transfection for 2 hours with 10 μM EdU. Afterward, the Click-iT reaction was performed according to the manufacturer’s protocol, and SPARCL1 was stained (EdU, pink; SPARCL1, green; DAPI, blue). Scale bar: 50 μm. The relative amount of EdU-positive cells was determined in both SPARCL1-positive and SPARCL1-negative cells (n = 1,883). Error bars indicate SD. (C) HUVECs were plated on chamber slides and grown until confluence was reached in some areas. The cells were immunocytochemically stained for SPARCL1/Ki-67 or SPARCL1/IL-33, and confocal images of areas with inhomogenous confluence were acquired (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 100 μm. (D) Confluent cell layers of MVECs were scratched and immunocytochemically stained for SPARCL1 after 0, 14, and 48 hours (SPARCL1, green, DAPI, blue). The migration front is marked by a dashed white line, and cells migrating into the scratch that had lost SPARCL1 expression are labeled with arrowheads. Scale bar: 250 μm. (E) HUVECs were grown in parallel dishes for 1, 15, or 22 days. On day 15, cells from 1 dish were reseeded at a low density (subcultivated - at day 15) and grown for an additional 8 days (subcultivated - at day 22). The slides were immunofluorescently costained for SPARCL1/Ki-67 and SPARCL1/IL-33 expression (SPARCL1, green; Ki-67/nuclear IL-33, pink, arrowheads; DAPI, blue). Scale bar: 100 μm. (A and B) ***P < 0.001, by Student’s t test . (A, B, and D) One representative experiment of two independent experiments is depicted.