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. 2017 Apr 3;198(10):4074–4085. doi: 10.4049/jimmunol.1600823

FIGURE 3.

FIGURE 3.

Role of MAPK in ICAM-1–mediated gene expression. (A) Total RNA was isolated from untreated (NT) or 4 h ICAM-1 cross-linked (XL) GPNTs and analyzed by semiquantitative RT-PCR for message levels of VCAM-1, TNF-α, COX-2, CCL2, ICAM-1, and GAPDH. (B and C) Confluent hCMEC/D3 (B) or human dermal MVEC (hDMEC) (C) were either left untreated or subjected to ICAM-1 cross-linking. At the indicated times TNF-α concentration in the culture supernatant was determined by ELISA. Shown are mean levels ± SEM of TNF-α above those in control cells from three independent experiments. (D and E) hCMEC/D3 (D) or human dermal MVEC (hDMEC) (E) were left untreated (NT) or subjected to ICAM-1 cross-linking (XL) for 24 h. The concentration of CXCL8, CXCL10, CCL2, CCL3, and CCL4 in the supernatant was measured by multianalyte flow assay. Where indicated anti–TNF-α (1 μg/ml) was included during the stimulation period to determine if altered chemokine secretion was a consequence of TNF-α induction. Shown are mean concentrations ± SEM of chemokines in the culture supernatant as determined from three independent experiments. (F) Confluent GPNT ECs were either left untreated (NT) or treated with 200 U/ml TNF-α for 12 h or subjected to ICAM-1 cross-linking (XL) for 12 h prior to immunoblot analysis of VCAM-1 and tubulin. Shown is a representative blot and densitometric quantification of three independent experiments. Where indicated cross-linking was performed in the presence of 50 μM U0126, SP600125, or SB202190. White separation lines indicate where lanes from the same blots were joined. (G) GPNT ECs were transiently nucleofected with pVCAM-1-luc, reseeded and allowed to grow to confluence (48–72 h posttransfection), and then subjected to ICAM-1 cross-linking (XL) for 6 h or stimulation with IL-1β (IL1β). Where indicated cross-linking was performed in the presence of 50 μM U0126, SP600125, or SB202190. Mean ± SEM luciferase activity (relative to untreated cells) was determined from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.