Transcriptional response to c-MYC depletion in human carcinoma cells. (A) c-MYC downregulation in the cells studied does not have a consistent effect on MYC targets expression. Proteins were collected 3 days after control and c-MYC^KD (knockdown) short interfering RNA (siRNA) transfection and assayed for c-MYC downregulation. β-Tubulin analysis of the same extracts was used as a control for protein loading. Matching RNA samples were analyzed by quantitative radioactive reverse transcription-PCR for MYC family and MYC target (E-box- and Inr element-driven) genes expression: CUL1 (cullin 1), ID2 (inhibitor of DNA binding 2), CCND2 (cyclin D2), TERT (telomerase reverse transcriptase), P15^INK4b (cyclin-dependent kinase 4 inhibitor B), P21^CDKN1A (cyclin-dependent kinase inhibitor 1A). TBP (TATA box binding protein), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S rRNA were used for normalization. (B) Genome-wide patterns of transcriptional response to c-MYC depletion in human carcinoma cells. The cluster diagram depicts microarray data for genes that are regulated by siRNA-mediated c-MYC depletion. Mean fold-change values from independent replicates are given for 741 probe sets showing at least twofold regulation in one or more cell lines. White indicates unchanged expression, and red and blue shades indicate upregulation and downregulation, respectively, relative to control siRNA-transfected cells.

c-MYC represses expression of the CCL5 chemokine, which promotes the migration of MCF-7 cells. (A) Microarray expression profile (mean±s.e.m. from independent replicates) of CCL5 in control and c-MYC^KD (knockdown) cells. (B) CCL5 gene expression, normalized by 18S ribosomal RNA, was quantified by real-time reverse transcription–PCR analysis in control, c-MYC^KD, MIZ-1^KD or c-MYC/MIZ-1^DKD (double knockdown) MCF-7 cells. (C) Chromatin immunoprecipitations (ChIPs) were carried out with antibodies against c-MYC and MIZ-1, or a pre-immune serum (Ctrl). ChIP was evaluated using quantitative real-time PCR analysis, with primers in the CCL5 gene regulatory region. (D) Parental control and c-MYC^KD MCF-7 cells were treated with fetal calf serum (FCS) and recombinant CCL5 or an anti-CCL5-blocking antibody or left untreated and assayed for migration.

c-MYC depletion has versatile consequences on carcinoma cell proliferation and migration. (A) c-MYC western blot analysis, performed 3 days after transfection, in control and c-MYC^KD (knockdown) small interfering RNA (siRNA)-transfected human carcinoma cells. β-Tubulin analysis of the same extracts was used as a control for protein loading. (B) Matching RNA samples were analysed by quantitative radioactive reverse transcription–PCR for expression of the MYC genes. 18S rRNA was used for normalization. Control and c-MYC^KD cells were assayed for proliferation at given time points after siRNA transfection (C) and for their ability to migrate in response to fetal calf serum (FCS; D). Plotted values are the means±s.e.m. of three replicates. c-MYC downregulation by siRNA is shown in the insets. Ctrl, control. MYC, myelocytomatosis viral oncogene homologs.

Identification of c-MYC targets involved in carcinoma cell proliferation. (A–E) WNT5A gene repression contributes to the mitogenic function of c-MYC in MDA-MB-231 and HeLa cells. (A) WNT5A gene expression, normalized by 18S ribosomal RNA, was quantified by real-time reverse transcription–PCR analysis in control, c-MYC^KD (knockdown), MIZ-1^KD or c-MYC/MIZ-1^DKD (double knockdown) cells. (B) Chromatin immunoprecipitations (ChIPs) were carried out with antibodies against c-MYC and MIZ-1, or a pre-immune serum (Ctrl). ChIP was evaluated using quantitative real-time PCR analysis, with primers in the WNT5A gene regulatory region. (C–E) MDA-MB-231 and HeLa cells were treated (C) or transfected with the indicated expression vectors (D) or small interfering RNA (E) and counted 6 days later.