Figure 5. MCAM is required for the induction of VCAM1 by HERV-K dUTPase.

(A–D) PAECs were transfected with siRNA targeting MCAM (MC) or with nontargeting siRNA (Con) for 48 hours, then treated as described for Figure 2. (A) SNAIL, IL-6, and VCAM1 gene expression, assessed in whole cell lysates by qPCR. (B) VCAM1 protein expression was assessed in whole cell lysates by immunoblot and densitometric quantification 72 hours after HERV-K dUTPase treatment in whole cell lysates using GAPDH as a loading control. (C) SNAIL protein in nuclear extracts was assessed by immunoblot and densitometric quantification using Lamin-B as the loading control. (D) Secreted IL-6 was measured by ELISA in the EC media 24 hours following the addition of HERV-K dUTPase. (E–H) PAECs were transfected with siRNAs targeting MCAM and TLR4 (M+T) or with Con before HERV-K dUTPase treatment as described in A–D. (E) MCAM, TLR4, SNAIL, IL-6, and VCAM1 mRNA were assessed as in A. (F) SNAIL protein expression was assessed by immunoblot and densitometric quantification, as in B. (G) Secreted IL-6 was measured by ELISA as in C. (H) VCAM1 protein expression was assessed by immunoblot and densitometric quantification as in D. For all panels, data are expressed as fold change compared with Veh/Con, and show n = 3, mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Veh/Con and ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 versus H.dUTP/Con by a 1-way ANOVA followed by Tukey multiple comparison test.