Figure 1
Figure 1. PKK regulates NF-κB activity in DLBCL cells. (A) Overexpression of PKK activates NF-κB in DLBCL cells. SUDHL-6 cells stably carrying pMIG vector or expressing Flag-tagged PKK-N were cultured in serum-free medium for 20 hours. The expression of the indicated proteins involved in NF-κB signaling was analyzed by Western blotting. p-IκBα, p-IKKα, and p-IKKβ represent phosphorylated forms of IκBα, IKKα, and IKKβ, respectively. Analysis of γ-tubulin was used as the equal loading control. (B) Suppression of PKK expression inhibits NF-κB activity in ABC DLBCL cells. OCI-Ly10 cells were infected with lentiviruses expressing either a control shRNA (L-shControl) or a PKK-specific shRNA (L-shPKK-1). Three days after infection, RNA levels of PKK in the indicated cells were analyzed by RT-PCR (left), and expression of the indicated proteins involved in NF-κB signaling was analyzed by Western blotting (right). Analysis of GAPDH and γ-tubulin was used as loading controls. (C) PKK knockdown leads to inhibition of NF-κB DNA-binding activity in ABC DLBCL cells. OCI-Ly10 cells were infected with lentiviruses expressing a control shRNA (L-shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2). Sixty-four hours after infection, nuclear extracts were prepared and the DNA-binding activity of the indicated NF-κB subunit was analyzed as described in “Methods.” The assays were performed in triplicates. shControl-expressing cells contain significantly higher NF-κB DNA-binding activity than cells expressing an shPKK (P < 0.01 for all NF-κB subunits, except for RelB subunit). (D) Suppression of PKK expression results in decreased NF-κB activity in GCB DLBCL cell line SUDHL-6 cells. SUDHL-6 cells stably expressing either the control shRNA (shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2) were cultured in serum-free medium overnight. The levels of PKK mRNA in the indicated cells were analyzed by RT-PCR (right), and the expression of the indicated proteins was analyzed on Western blots (right). (E) Suppression of PKK expression inhibits NF-κB activity in GCB DLBCL cell line OCI-Ly7 cells. OCI-Ly7 cells stably expressing either shControl or shPKK-1 were cultured in serum-free medium overnight. Expression of PKK mRNA (left) and the indicated proteins involved in NF-κB signaling (right) were analyzed as described in panel D. (F) PKK knockdown reduces NF-κB DNA-binding activity in GCB DLBCL cells. SUDHL-6 cells stably expressing the indicated shRNA were cultured in serum-free medium overnight. Nuclear extracts were prepared, and the DNA-binding activity of p65 and p50 were assayed as described in panel C. The difference between control cells and cells expressing an shPKK in NF-κB DNA-binding activity is statistically significant (P < .01).

PKK regulates NF-κB activity in DLBCL cells. (A) Overexpression of PKK activates NF-κB in DLBCL cells. SUDHL-6 cells stably carrying pMIG vector or expressing Flag-tagged PKK-N were cultured in serum-free medium for 20 hours. The expression of the indicated proteins involved in NF-κB signaling was analyzed by Western blotting. p-IκBα, p-IKKα, and p-IKKβ represent phosphorylated forms of IκBα, IKKα, and IKKβ, respectively. Analysis of γ-tubulin was used as the equal loading control. (B) Suppression of PKK expression inhibits NF-κB activity in ABC DLBCL cells. OCI-Ly10 cells were infected with lentiviruses expressing either a control shRNA (L-shControl) or a PKK-specific shRNA (L-shPKK-1). Three days after infection, RNA levels of PKK in the indicated cells were analyzed by RT-PCR (left), and expression of the indicated proteins involved in NF-κB signaling was analyzed by Western blotting (right). Analysis of GAPDH and γ-tubulin was used as loading controls. (C) PKK knockdown leads to inhibition of NF-κB DNA-binding activity in ABC DLBCL cells. OCI-Ly10 cells were infected with lentiviruses expressing a control shRNA (L-shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2). Sixty-four hours after infection, nuclear extracts were prepared and the DNA-binding activity of the indicated NF-κB subunit was analyzed as described in “Methods.” The assays were performed in triplicates. shControl-expressing cells contain significantly higher NF-κB DNA-binding activity than cells expressing an shPKK (P < 0.01 for all NF-κB subunits, except for RelB subunit). (D) Suppression of PKK expression results in decreased NF-κB activity in GCB DLBCL cell line SUDHL-6 cells. SUDHL-6 cells stably expressing either the control shRNA (shControl) or a PKK-specific shRNA (shPKK-1 or shPKK-2) were cultured in serum-free medium overnight. The levels of PKK mRNA in the indicated cells were analyzed by RT-PCR (right), and the expression of the indicated proteins was analyzed on Western blots (right). (E) Suppression of PKK expression inhibits NF-κB activity in GCB DLBCL cell line OCI-Ly7 cells. OCI-Ly7 cells stably expressing either shControl or shPKK-1 were cultured in serum-free medium overnight. Expression of PKK mRNA (left) and the indicated proteins involved in NF-κB signaling (right) were analyzed as described in panel D. (F) PKK knockdown reduces NF-κB DNA-binding activity in GCB DLBCL cells. SUDHL-6 cells stably expressing the indicated shRNA were cultured in serum-free medium overnight. Nuclear extracts were prepared, and the DNA-binding activity of p65 and p50 were assayed as described in panel C. The difference between control cells and cells expressing an shPKK in NF-κB DNA-binding activity is statistically significant (P < .01).

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