processed according to the same ChIP assay protocol using the indicated antibodies. The primers were as fol- lows: F-186, CTGGGTTTCCGATTTTCTCA and R-49, GAGTTCCTGGACGTGCTCCT. The ampliﬁed DNA products were resolved by agarose gel electrophoresis and conﬁrmed by sequencing. Immunofluoresce assay A431 cells were seeded onto glass coverslips. Twenty-four hours after reseeding, cells were starved in serum-free medium for 8 h prior to EGF treatment. Fixation was performed in 4% formaldehyde in PBS at room tempera- ture for 20 min. After three times of washing by PBS, cells were permeabilized with 1% Triton X-100 in PBS for 5 min. Next, cells were preincubated with 0.5% Tween 20 in 0.5% BSA in PBS for 15 min, blocked with 1% BSA in PBS for 1 h at room temperature and then incu- bated for 1 h at room temperature with following primary antibodies diluted in 1% BSA in PBS: anti-HDAC4 (1:100). After washing with PBS for three times, cells were incubated with the relative secondary antibodies (Alexa Fluor 488 anti-rabbit 1:150) in 1% BSA/PBS for 1 h at room temperature followed by washing in PBS. Coversilps were then mounted with ProLong Gold anti- fade reagent with 40-6-diamidino-2-phenylindole (DAPI) (Invitrogen). RESULTS Expression of CEBPBs is induced after long-term EGF treatment in A431 cells We previously reported that CEBPD can act as a bifunc- tional regulator and is involved in both basal and EGF- induced COX-2 expression (14). Since CEBPBs can form a heterodimer with CEBPD and are implied to be involved in the COX-2 gene regulation, we decided to investigate whether the CEBPBs also participate in the EGF-regu- lated COX-2 transcription. To elucidate the relationships between CEBPD and three isoforms of CEBPB in EGF- regulated COX-2 transcription, their expression patterns were ﬁrst detected by RT–PCR assay and western blot. Upon EGF treatment, the COX-2 expression was imme- diately induced ( 1 h), which is consistent with the increase of CEBPD expression (Figure 1A and B, lane 1–6). This expression started to decrease after 3 h of EGF treatment. Interestingly, the induction of three iso- forms of the CEBPB expression occurred in the long-term EGF treatment, along with the decrease of COX-2 expres- sion (Figure 1A and B, lane 4–8), although several studies have suggested that LAP1 and LAP2 act as positive reg- ulators, upregulating the COX-2 expression (12,39). However, the lagging phase induction of LAP1 and LAP2 conﬂicts with the positive regulation model of COX-2 transcription. According to this observation, we hypothesized that the slowly induced CEBPBs may play diﬀerent roles in COX-2 gene regulation compared with the immediately induced CEBPD. Nucleic Acids Research, 2008, Vol. 36, No. 19 6069 Figure 1. Expression of COX-2 and CEBPs upon long-term EGF treat- ment in A431 cells. (A) A431 cells were starved in serum-free medium for 6 h then treated with 25 ng/ml of EGF. Total RNA was harvested at the indicated times and an RT–PCR assay performed for the following genes: COX-2, CEBPD, CEBPB and GAPDH. (B) The conditions were the same as those described in (A); the cell lysates were harvested for western blot and protein expressions of COX-2, CEBPD, CEBPB and GAPDH were detected by speciﬁc antibodies. CEBPB participation in repression of EGF-induced COX-2 expression To verify the repressive eﬀect of CEBPBs on COX-2 expression, a loss-of-function assay using the knockdown approach was performed. Silencing of the three isoforms of CEBPB reverses the COX-2 transcripts and protein expression (Figure 2A). This suggests that the CEBPBs indeed do play negative roles on COX-2 expression. As mentioned above, LAP1 and LAP2 consist of transactiva- tion and DNA binding/dimerization domains, whereas LIP lacks the N-terminal transactivation domain. The implication arising from this is that LIP can act as a dominant-negative form protein, repressing transcrip- tional activation through the formation of a heterodimer with the rest of the C/EBP family members. The three CEBPB variants are translated from one transcript, and the 50-untranslated region (50-UTR) of CEBPB mRNA plays a critical role in regulating the selected-translation initiation of isoforms (20,40). To further dissect the eﬀect of individual CEBPB isoforms on the regulation of the COX-2 transcription, expression vectors bearing coding cDNA of CEBPB isoforms, HA/LAP1, HA/LAP2 or HA/LIP, without the 50-UTR were constructed for their speciﬁc translation (Figure 2B). Compared with the con- trol vectors upon EGF treatment, the well-characterized negative regulator HA/LIP shows an inhibitory eﬀect on the COX-2 promoter activity. Interestingly, both transfec- tants of HA/LAP1 and HA/LAP2 show a consistent repressive eﬀect in response to EGF treatment (Figure 2C, right panel). In spite of this, the COX-2