Figure 3.
Figure 3. Effects of K-11706 and K13144 on Epo promoter/enhancer. (A) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the wild-type (Pwt) Epo promoter/enhancer with a Luc reporter construct in Hep3B cells. The wild-type (Pwt) Epo promoter/enhancer with a Luc reporter construct was transfected into 8 × 105 Hep3B cells and incubated with 15 U/mL rhIL-1β, 220 U/mL rhTNF-α, or 100 nM K-11706 under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hours. Hypoxic induction of Luc gene expression is represented here as a hypoxia/normoxia ratio shown as fold induction. Six separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with control, P < .005; **, significance compared with 15 U/mL IL-1β, P < .005; ***, significance compared with 220 U/mL TNF-α, P < .005; ****, significance compared with control, P < .025. (B) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the mutant type (Pm6: TACG-TAAA) in the Epo enhancer with a Luc reporter construct in Hep3B cells. A mutant form (Pm6: TACG-TAAA) of the Epo enhancer with a Luc reporter construct was transfected into 8 × 105 Hep3B cells. Experimental conditions were the same as those described in the legend for panel A. Eight separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with control, P < .005; **, significance compared with 15 U/mL IL-1β, P < .005; ***, significance compared with 220 U/mL TNF-α, P < .005; ****, significance compared with control, P < .05. (C) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the mutant-type (Pm7: AGATAAC-ATATAAA) in the Epo promoter with a Luc reporter construct in Hep3B cells. A mutant form (Pm7: AGATAAC-ATATAAA) of the Epo promoter with Luc reporter construct was transfected into 8 × 105 Hep3B cells. Experimental conditions were the same as those described in the legend for panel A. Five separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with 15 U/mL IL-1β, P < .001; **, significance compared with 220 U/mL TNF-α, P < .005; ***, significance compared with control, P < .001.

Effects of K-11706 and K13144 on Epo promoter/enhancer. (A) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the wild-type (Pwt) Epo promoter/enhancer with a Luc reporter construct in Hep3B cells. The wild-type (Pwt) Epo promoter/enhancer with a Luc reporter construct was transfected into 8 × 105 Hep3B cells and incubated with 15 U/mL rhIL-1β, 220 U/mL rhTNF-α, or 100 nM K-11706 under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 hours. Hypoxic induction of Luc gene expression is represented here as a hypoxia/normoxia ratio shown as fold induction. Six separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with control, P < .005; **, significance compared with 15 U/mL IL-1β, P < .005; ***, significance compared with 220 U/mL TNF-α, P < .005; ****, significance compared with control, P < .025. (B) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the mutant type (Pm6: TACG-TAAA) in the Epo enhancer with a Luc reporter construct in Hep3B cells. A mutant form (Pm6: TACG-TAAA) of the Epo enhancer with a Luc reporter construct was transfected into 8 × 105 Hep3B cells. Experimental conditions were the same as those described in the legend for panel A. Eight separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with control, P < .005; **, significance compared with 15 U/mL IL-1β, P < .005; ***, significance compared with 220 U/mL TNF-α, P < .005; ****, significance compared with control, P < .05. (C) Effects of K-11706 and K-13144 on the inhibition of the induction by IL-1β and TNF-α of the mutant-type (Pm7: AGATAAC-ATATAAA) in the Epo promoter with a Luc reporter construct in Hep3B cells. A mutant form (Pm7: AGATAAC-ATATAAA) of the Epo promoter with Luc reporter construct was transfected into 8 × 105 Hep3B cells. Experimental conditions were the same as those described in the legend for panel A. Five separate experiments (quadruple samples) were performed. Error bars represent 1 SD. * indicates significance compared with 15 U/mL IL-1β, P < .001; **, significance compared with 220 U/mL TNF-α, P < .005; ***, significance compared with control, P < .001.

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