Fig. 6: The downregulation of COPS5 in MPI may result from the loss of a potential enhancer.

a Immunoblotting is performed to analyze the protein levels of HIF1A, COPS5, and RPS3 in MSF, MSF^HRAS\SV40LT, MPI-SF, and MPI-SF^HRAS\SV40LT. b Comparison of the protein levels of HIF1A, COPS5, and RPS3 in fibroblasts with and without expressing oncogenic HRAS(G12V) and SV40 LT. c A putative enhancer containing a predicted HIF1A binding motif at the upstream of COPS5 is specifically lost in MPI. The cyan box highlights the putative regulatory sequence of COPS5 (RSC) with ATAC-seq peaks in MSF but without ATAC-seq peaks in MPI-SF. d Luciferase reporter assays are conducted using HEK293T and NIH3T3 cell lines to assess the enhancer activity of RSC from mouse, MPI, and RSI. The negative control is the PGL3-promoter empty vector-transfected in HEK293T and NIH3T3. e A cell model of HEK293T that continuously expresses HIF1A is generated. The experiment was repeated independently three times with similar results. f The regulatory activity of mouse RSC significantly decreases when the 17 bp fragment containing the predicted binding motif is deleted. When the 17 bp fragments of mouse and RSI are respectively added to the RSC of MPI, the regulatory activity of the edited RSCs of MPI is significantly enhanced. g An antibody specific to HIF1A is used to enrich HIF1A along with its DNA targets in mouse and MPI fibroblasts, respectively. h ChIP-qPCR is performed using specific primers designed based on the RSC sequences. The results show that the concentration of mouse RSC is significantly higher than that of MPI RSC. The numbers of dots (n = 3) in (b), (d), (f), and (h) represent the number of independent experiments. All data are presented as mean ± SD. The P values are from two-tailed Student’s t tests. ns: not significant. Source data are provided as a Source Data file.