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. Author manuscript; available in PMC: 2019 May 1.
Published in final edited form as: Am J Transplant. 2017 Nov 23;18(5):1096–1109. doi: 10.1111/ajt.14544

Figure 1. Monocyte adherence to HLA class I Ab-stimulated endothelium is suppressed when the mTOR pathway is inhibited in ECs.

Figure 1

Primary endothelial cells (ECs) were stimulated with Ab against Integrin β3 (anti-ITGB3), HLA class I (anti-HLA I; clone W6/32) or thrombin for 5 min. CFSE-labeled MonoMac6 (MM6 cells) or human monocytes enriched from third-party peripheral blood (PBMC-derived monocytes) were pre-treated with polyclonal human IgG to block FcγR interactions, then allowed to adhere to ECs for 20 min, then un-adhered monocytes were washed off and adherent monocytes in 8–10 fields were imaged and quantified. (A) Shown are mean numbers of MM6 cells (left panel) or PBMC-derived monocytes (right panel) adherent to treated ECs over untreated ECs ± SEM from five independent experiments. ****P<0.0001, ns=not significant when comparing treated ECs to untreated ECs by two-way ANOVA with Tukey’s multiple comparisons test (B) ECs were pre-treated with Rapa, RAD or no inhibitor for 24 h, or BAPTA-AM for 30 min before monocytes were allowed to adhere. Shown are fold changes in mean number of MM6 cells (left panel) or PBMC-derived monocytes (right panel) adherent to treated ECs over untreated control ECs (dotted line) ± SEM from five independent experiments. *P<0.05, **P<0.01, ***P<0.001 for ECs given the indicated inhibitor to ECs given no inhibitor by two-way ANOVA with Tukey’s multiple comparisons test (C) Representative images showing CFSE-labeled MM6 cells adherent to the endothelial monolayer following indicated treatment. scale bars = 100 µm (D) ECs were pre-treated with no inhibitor, Rapa or RAD for 2 h (targets mTORC1 or 2 respectively), or BAPTA-AM for 30 min before MM6 cells were allowed to adhere. Shown are fold changes in mean numbers of MM6 cells adherent to treated ECs over untreated control ECs (dotted line) ± SEM from five independent experiments. *P<0.05 for ECs given the indicated inhibitor to ECs given no inhibitor by two-way ANOVA with Tukey’s multiple comparisons test (E) ECs were pre-treated with control siRNA or siRNA directed against mTOR, Raptor (mTORC1), or Rictor (mTORC2) before MM6 cells were allowed to adhere. Shown are fold changes in numbers of MM6 cells adherent to treated ECs over untreated control ECs (dotted line) ± SEM from five independent experiments. **P<0.01, ***P<0.001 for ECs given the indicated siRNA to ECs given no siRNA by two-way ANOVA with Tukey’s multiple comparisons test