FIG 9.

Inhibition of canonical NF-κB signaling pathway by cystatin E/M protein. (A) Basal expression of nonphosphorylated IκBα was seen in HeLa cells and was not altered after doxycycline treatment. However, IκBα expression increased in CST6 cells after doxycycline treatment, correlating to decreased phosphor lation of IκBα. Treatment with TNF-α enhanced phospho-IκBα expression in both the HeLa and CST6 cell lines, and the expression was not altered in HeLa cells after doxycycline treatment. On the other hand, treatment with doxycycline led to reduced expression of phospho-IκBα in CST6 cells. (B) The basal IKKβ expression level was high in the control CST6 cells, phospho-IKKβ expression was visualized only with the addition of TNF-α, and the expression was reduced in doxycycline-treated cells. (C) Induced cystatin E/M expression did not inhibit endogenous activation of p100 (NF-κB2) to produce p52 in CST6 cells. In HEK 293T cells, exogenous NIK expression led to activation of myc-tag p100 to produce myc-p52. While this activation was inhibited by cIAP1/2 expression, inhibition was not observed with the expression of cystatin E/M. (D) Mechanistic representation showing inhibition of the IKKβ-mediated canonical and not the NIK-mediated noncanonical NF-κB signaling pathway by cystatin E/M. TNFR, tumor necrosis factor receptor; TNFSFR, tumor necrosis factor superfamily receptor.