FIGURE 5.

NFκB pathway controls PML expression through regulation of IL6 level. A, shown is efficiency of NEMO knockdown by specific siRNA detected on mRNA level 2 days after transfection in BJ. B, down-regulation of PML, NEMO, and the activated and total form of STAT3 in BJ detected on immunoblot 2 and 4 days after transfection of NEMO siRNA; GAPDH was used as loading control. Down-regulation of PML (D) and IL6 mRNA (D) levels quantified by qRT-PCR in BJ cells 2 days after NEMO knockdown is shown. The values represent the average of three independent experiments and are given as the -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E. β-Actin was used as a reference gene. E, shown is immunoblot detection of the STAT3-activated form after NEMO knockdown and NEMO knockdown with the addition of recombinant human IL6 protein (5000 pg/ml) into medium of BJ cells. Shown is down-regulation of PML (F), NEMO (G), and IRF1 (H) mRNA levels 2 days after NEMO knockdown and NEMO knockdown with the addition of recombinant human IL6 protein (5000 pg/ml) quantified by real time qRT-PCR in BJ cells. The values represent the average of two independent experiments and are shown as -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E.; β-actin was used as a reference gene. Shown is down-regulation of PML mRNA (I) and protein levels detected as PML NBs (J) 2 days after NEMO, STAT3, or combined NEMO and STAT3 knockdown by specific siRNAs in BJ cells. The mRNA values represent the average of two independent experiments and are shown as -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E. β-Actin was used as a reference gene. Bar, 15 μm. Asterisks (*, **, and ***) represent p value <0.05, <0.01, and <0.005, respectively.