Fig 1.
NF90/NF110 occupancy at promoters of immediate early genes in K562 cells.
(a) Average occupancy profile of NF90/NF110/NF110 at proximal promoters within 1000 bp upstream of transcription start site (TSS) of 49 immediate early genes (IEG), 58 delayed primary response genes (D-PRG), and 26 secondary response gene (SRG) defined by Tullai et al. x-axis: Relative position near TSS. y-axis: Fold-change over input of NF90/NF110/NF110 ChIP-Seq signal. (b-d) Signal tracks of NF90/NF110 chromatin occupancy determined by ChIP-seq at promoters of IEGs EGR1, FOS, and JUN; retrieved from UCSC genome browser [41]. NF90/NF110 ChIP-seq signal (fold change over input) is presented, aligned to peaks called by Irreproducible Discovery Rate (IDR) analysis that measures consistency between replicates. Relative position of the amplicons in the proximal promoter and gene body of each gene used for subsequent ChIP-PCR experiments are indicated.
Fig 2.
Dynamic associations of NF90/NF110 and NF45 with promoters of IEGs upon stimulation.
HEK293 cells were serum starved for 24 h, then stimulated by PMA (20 ng/ml) for indicated durations. Chromatin immunoprecipitation was performed with antibodies against NF90/NF110 (a) or NF45 (b). Abundance of IEG promoter fragments were assessed by quantitative polymerase chain reaction for input sheared chromatin, or specific immunoprecipitates. N = 3 biological replicates; One-way analysis of variance (ANOVA) test followed by post-hoc Student’s t-test corrected by Bonferroni; data reported as mean ±s.e.m of fold-change over input, * P <0.05, ** P < 0.01.
Fig 3.
Doxycycline-induced shRNAs specifically attenuated expression of NF90/NF110 or NF45 proteins.
HEK293 cells stably transfected with pINDUCER10-shNF90/NF110 (D2) or pINDUCER10-shNF45 (D5) were treated without or with doxycycline for 96 h, then serum starved for 12 h and treated with PMA (20 ng/ml) for 2 h. NF90/NF110 or NF45 protein expression was detected by immunoblotting.
Fig 4.
Reduced NF90/NF110 or NF45 expression attenuated inducible transcription of IEGs.
HEK293 D2 or D5 cells were treated without or with doxycycline, then serum-starved for 12 h and treated with PMA (20 ng/ml) for 0, 30, or 60 min. ChIP was performed with antibody against RNA Polymerase II, and real time transcription of EGR1, FOS, JUN and HBB gene bodies was measured by qPCR. N = 3 biological replicates; One-way analysis of variance (ANOVA) test followed by post-hoc Student’s t-test corrected by Bonferroni; data reported as mean ± s.e.m of fold-change over input, * P < 0.05, ** P< 0.01.
Fig 5.
Reduced NF90/NF110 or NF45 expression attenuated inducible expression of IEG RNAs.
HEK293 D2 or D5 cells were treated without or with doxycycline, then serum-starved for 12 h and treated with PMA (20 ng/ml) for 0, 15, or 30 min. Total RNA was prepared and used for reverse transcription qPCR to detect EGR1, FOS, and JUN mRNAs and pre-mRNAs. ACTB was used for normalization. N = 3 biological replicates; One-way analysis of variance (ANOVA) test followed by post-hoc Student’s t-test corrected by Bonferroni; data reported as mean ± s.e.m of fold-induction compared to 0 min, * P < 0.05, ** P < 0.01.
Fig 6.
Reduced NF90/NF110 or NF45 expression attenuated inducible expression of IEG proteins.
HEK293 D2 or D5 cells were treated without or with doxycycline, then serum starved for 12 h and treated with PMA (20 ng/ml) for 0, 2 h. Induction of IEGs EGR1, FOS and JUN were assessed by immunoblotting. GAPDH was used as a loading control.
Fig 7.
Reduced NF90/NF110 expression attenuated inducible expression IEGs by immunofluorescence analysis.
HEK293 cells stably transfected with pINDUCER10-shNF90/NF110 (D2) or pINDUCER10-shNF45 (D5) were treated without or with doxycycline for 96 h, then seeded on chamber slides, and serum starved for 24 h and stimulated by PMA (20 ng/ml) for 2 h for maximal protein expression of IEG. Slides were incubated with mouse monoclonal antibodies against NF90/NF110, and rabbit monoclonal antibodies against EGR1 (a-b), FOS (c-d), or JUN (e-f), then incubated with fluorescent conjugated secondary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594. Slides were counterstained with DAPI, then visualized by confocal fluorescent microscopy. Panels (b,d,f) show doxycycline-mediated shRNA knockdown compared to (a,c,e) respectively.
Fig 8.
Reduced NF45 expression attenuated inducible expression IEGs by immunofluorescence analysis.
HEK293 cells stably transfected with pINDUCER10-shNF90/NF110 (D2) or pINDUCER10-shNF45 (D5) were treated without or with doxycycline for 96 h, then seeded on chamber slides, and serum starved for 24 h and stimulated by PMA (20 ng/ml) for 2 h for maximal protein expression of IEG. Slides were incubated with mouse monoclonal antibodies against NF45, and rabbit monoclonal antibodies against EGR1 (a-b), FOS (c-d), or JUN (e-f), then incubated with fluorescent conjugated secondary antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594. Slides were counterstained with DAPI, then visualized by confocal fluorescent microscopy. Panels (b,d,f) show doxycycline-mediated shRNA knockdown compared to (a,c,e) respectively.