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. Author manuscript; available in PMC: 2023 Mar 17.
Published in final edited form as: Cell Chem Biol. 2021 Nov 29;29(3):517–529.e5. doi: 10.1016/j.chembiol.2021.11.005

Fig. 4. p97 inhibition impairs the transcriptional activity of E2F1 by downregulating the CCND1/CDK complex.

Fig. 4

(a) The putative transcription factor binding sites (TFBSs) of the 33 proteins which were specifically downregulated by p97 inhibition. Data was analyzed using the g:Profiler. Blue indicates the gene is a potential target of transcription factor c-Myb or E2F1. Blank indicates it is not a target gene. (b) Depiction of the known regulatory network of the CCND1/CDK/RB1/E2F1 pathway. (c) Log2 fold change of the proteins regulating E2F1 function which were significantly affected by MG, CB and NMS in our TMT results. (d) The dysregulation of E2F1 related proteins as confirmed by western blot. HCT116 cells were treated with 1 μM MG132, 2 μM CB-5083 or 4 μM of NMS-874 for 6h. (e) The transcriptional activity of E2F1 was measured using E2F1 reporter assay. HCT116 cells were transfected with pGL2-AN plasmid for 24h. Then cells were plated in 384 well plate and incubated for 16h. The luminescence were detected after 8h treatment with MG132, CB-5083 or NMS-873. Data are shown as mean ± SD, n=3. (f) qRT-PCR analysis of ATF3, DHFR and CCND1 RNAs following treatment with MG132, CB-5083 or NMS-874. HCT116 cells were treated for 2, 6 and 24h using the same concentration as proteomic and western blot assay (n=4). (See Fig. S4 and Table S4).