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. 2015 Sep 2;89(22):11396–11405. doi: 10.1128/JVI.02151-15

FIG 3.

FIG 3

ΔNp73α binds to TLR9 promoter on NF-κB site C. (A) ChIP was performed in HPV38 E6/E7 HFK using anti-ΔNp73α antibody. Simultaneously, part of the total chromatin fraction (1/10) was used as input. qPCR was performed using specific primers flanking the NF-κB RE within the TLR9 promoter. The histogram shows the relative amount of the promoter bound by ΔNp73α after subtraction of the background of nonspecific IgG control expressed as a percentage of the input. Data are the means from three independent experiments. **, P < 0.01. (B) HPV38 E6/E7 HFK transiently expressing HA-ΔNp73α was processed for the oligonucleotide pulldown assay. Cell lysate was incubated with biotinylated (btn) probes containing the NF-κB RE of the TLR9 promoter, either wild type (pTLR9wt) or mutated (pTLR9mut). DNA-associated proteins were recovered by precipitation with streptavidin beads and analyzed by IB (left side). The intensity of the protein bands in three independent experiments was quantified (right side). *, P < 0.05; **, P < 0.01. (C) Scheme of the TLR9 promoter luciferase constructs: the full-length (−3227/−1) construct containing the 4 NF-κB RE (sites A, B, C, and D) and the deletion (−1017/−1 and −290/−1) constructs. (D) RPMI 8226 cells were cotransfected with TLR9 promoter constructs (−3227/−1, −1017/−1, and −290/−1) cloned in front of the luciferase reporter gene with pcDNA3 empty vector or expressing ΔNp73α. After 48 h, cells were harvested and luciferase activity was measured. Data are the means from three independent experiments performed in triplicate. **, P < 0.01. (E) RPMI 8226 cells were cotransfected with TLR9 promoter constructs wild-type (−3227/−1) or NF-κB point mutated in site C (mutC) cloned in front of the luciferase reporter gene with pcDNA3 empty vector or expressing ΔNp73α. After 48 h, cells were harvested and luciferase activity was measured. Data are the means from three independent experiments performed in triplicate. **, P < 0.01. (F) RPMI 8226 cells were cotransfected with TLR9 promoter constructs (−3227/−1 or −1017/−1) cloned in front of the luciferase reporter gene with pLXSN empty vector or pLXSN HPV38E7 construct and ΔNp73α sense (S) or AS oligonucleotides. After 48 h, cells were harvested and luciferase activity was measured. Data are the means from three experiments. **, P < 0.01.