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. 2018 Sep 11;9(71):33515–33527. doi: 10.18632/oncotarget.26067

Figure 4. SSeCKS regulates the expression of E-Selectin and cancer cell adhesion on vascular endothelial cells.

Figure 4

(A) Protocol for the isolation of CD102+ lung endothelial cells (LEC) and CD102- lung fibroblasts (LF). (B) BLI of adhered (10 min) B16F10-luc or SM1WT1-LM3-luc on WT- or KO-LEC. Error bars, SEM for triplicate wells with LEC from 6 WT or KO lungs tested independently. *p < 0.01; **p < 0.001. (C) PCR array analysis of adhesion-regulating genes upregulated in KO- vs. WT-LEC or LF. All p values are < 0.05. (D) Relative mRNA expression of E-Selectin (Sele) in WT- or KO-LEC and -LF, normalized to Gapdh levels. Error bars, SEM for triplicate assays on samples from 6 lungs. *p < 0.01. (E) Immunoblot analysis of SSeCKS, Sele and Gapdh (protein loading control) from WT or KO-LEC. Band intensities were determined (for panels E, F and H) by Image J and normalized to Gapdh. (F) Immunoblot analysis of LEC treated (48 h) with control siRNA (siC) or siRNA for E-Selectin (siSele). (G) Adhesion of B16F10-luc to WT- or KO-LEC treated with siC or siSele. Error bars, SEM of triplicate wells performed independently on LEC from 6 lungs. (H) Expression of AKAP12, SELE or GAPDH (as a protein loading control) in HUVEC and HMVEC-L pre-treated (48 h) with control siRNA (C) or siRNA to AKAP12 (siA12) treatment. (I) Adhesion of B16F10-luc on HUVEC or HMVEC-L treated with siC or siAKAP12 (siAK12) as in panel H. Error bars, SEM of triplicate wells from two independent experiments. **p < 0.001.