Figure 5. EGF treatment induces HDAC4 and SUMO1 nuclear shuttling. (A) A431 cells were starved in serum-free medium for 6 h and then treated with EGF at indicated times. The cells were ﬁxed in formaldehyde and stained with anti-HDAC4 and anti-SUMO1 anti- bodies. The subcellular localization of HDAC4 (upper panel) and SUMO1 (lower panel) were determined by immunoﬂuorescence micro- scopy (green). DAPI was used to visualize nuclei distribution (blue). (B) A431 cells were transiently transfected with HA/LAP1 expression vector for 24 h and then the transfectants were starved in serum-free medium for 6 h. Cells were ﬁxed and subjected to indirect immunoﬂuor- escence staining. HA/LAP1 and endogenous HDAC4 (upper panel) or SUMO1 (lower panel) were stained with monoclonal anti-HA (1:100, red) and polyclonal anti-HDAC4 (1:100, green) or anti-SUMO1 (1:100, green) antibodies, respectively. Coexistence of HA/LAP1 with HDAC4 or SUMO1 were showed in ‘Merge’. in COX-2 transcription, we further addressed whether HDAC4 plays a functional role in the suLAP1-mediated repression of COX-2 transcription. Initially, a reporter assay was performed by cotransfection of the COX-2 reporter with HDAC4 or HDAC1 expression vectors. Interestingly, overexpression of HDAC4 attenuated COX-2 promoter activity regardless of EGF existence (Figure 6A); whereas, overexpression of HDAC1 showed no eﬀect in A431 cells although it has been implied to be a corepressor in the silencing of COX-2 transcription (44). To further verify whether HDAC4 plays a functional role through LAP1-binding with CEBP and CRE motifs, the COX-2 reporters bearing the wild-type or the double mutant of both CEBP and CRE motifs (mCEBP/CRE) Nucleic Acids Research, 2008, Vol. 36, No. 19 6073 were cotransfected with HDAC4 expression vectors. Com- pared to the HDAC4-mediated repressive eﬀect on the wild-type COX-2 promoter, HDAC4 lost around 40% of its repressive activity on the COX-2 reporter bearing the CEBP/CRE mutant (Figure 6B and Supplementary Figure S1). This suggests that the EGF-responsive ele- ments, CEBP and CRE motifs, play a functional role in response to the HDAC4-mediated repression of the COX- 2 promoter. Moreover, a knockdown approach was per- formed to further conﬁrm the HDAC4-exerted repressive eﬀect on COX-2 expression. Compared to the scrambled control, the western blot (Figure 6C, left panel) and RT–PCR assay (Figure 6C, right panel) show a consistent result that the silence of HDAC4 enhances COX-2 expres- sion regardless of EGF treatment. Furthermore, the spe- ciﬁc siRNAs of CEBPB or HDAC4 were cotransfected with the COX-2 reporter to detect whether this repressive eﬀect occurred through the promoter regulation. The silence of either CEBPB or HDAC4 can enhance both the basal and EGF-induced COX-2 promoter activities (Figure 6D). These results suggest that HDAC4 acts as a repressor on COX-2 transcription through the CEBPs- binding motifs, and that this repression not only functions during the re-inactivation of the COX-2 gene in long-term EGF treatment but also in the EGF-starved stage. suLAP1 cooperates with HDAC4 to repress COX-2 promoter activity HDAC4 plays a suppressive role in the regulation of the COX-2 promoter (Figure 6). suLAP1 is involved in the negative regulation of COX-2 transcription (Figure 3D and E). Further investigations were performed to deter- mine whether suLAP1 collaborated with HDAC4 to repress COX-2 promoter activity. To address this issue, a reporter assay was performed by cotransfecting the reporter of COX-2 promoter with various expression vec- tors as indicated in Figure 7A and Supplementary Figure S1. The cotransfected LAP1 and HDAC4 expres- sion vectors showed a cooperatively repressive ability against COX-2 promoter activity compared with transfec- tion with the LAP1 expression vectors only (Figure 7A, compare lane 2 with lane 4). This suggests that an increas- ing amount of LAP1 can enhance HDAC4-mediated repression of COX-2 transcription. In addition, the trans- fection of LAP1K174A not only attenuates the LAP1- mediated repression activity on the COX-2 reporter (Figure 7A, compare lanes 1 and 2 with lanes 5 and 6), but it also lost the HDAC4-mediated repressive activity on the COX-2 reporter from 42% to 11% (Figure 7A, com- pare lanes 2 and 4 with lanes 6 and 8). However, cotrans- fection with HDAC1 expression vectors shows a consistent COX-2 promoter activity (Figure 7A, compare lanes 2 and 3 with lanes 6 and 7). This suggests that suLAP1, at least in part, plays a functional role in HDAC4-mediated repression of the COX-2 promoter. Furthermore, whether LAP1 has a higher capability to interact with HDAC4 than LAP1K174A was addressed by a Co-IP assay with exogenously expressed HA/LAP1 or HA/LAP1K174A. Meanwhile, HA/LAP2, HA/ LAP2K151A and HA/LIP were recruited for comparison.