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. 2013 May 28;110(24):9920–9925. doi: 10.1073/pnas.1305172110

Fig. 1.

Fig. 1.

Induction of TET1 and HOX gene expression upon depletion of HMGA2 in 1833 cells, a bone-tropic derivative of human breast cancer cell line MDA-MB-231, or in MMTV–Wnt1 transgenic mouse breast tumors. (A, B, and D–H) We stably transduced 1833 cells with HMGA2 shRNA (shHMGA2) or control SCR sh. (A) Gene expression array analysis showing up-regulation of TET1 and 20 of 39 HOX genes in HMGA2-depleted cells. (B) The expression levels of HOXA genes are shown. *Fold change (fc) < 2; **fc > 2 based on the signal intensity of gene expression arrays. (C) Genomic transcription units of human HOXA genes on chromosome 7 viewed using the UCSC Genome Browser (40). HOXA genes are transcribed from right to left with the following order: 5′UTR (thin blue bar), Coding Sequence (thick blue bar), and 3′UTR (thin blue bar). Bar length is proportional to length of DNA sequence. (D–H) qRT-PCR and immunoblotting analyses validated induction of TET1 and HOXA gene expression in HMGA2-depleted cells. (D–F) HMGA2 (D), TET1 (E), or HOXA4/5/6/7/9/11 (F) mRNA analyzed by qRT-PCR (GAPDH as normalization control). (G) HMGA2, TET1, and HOXA9/7 protein analyzed by immunoblotting (GAPDH as control). (H) Genome-wide 5hmC levels analyzed by dot blot assay. (I and J) Loss of Hmga2 in MMTV–Wnt1 transgenic mouse breast tumors induced Tet1 and Hoxa9/7 expression. Wnt1 transgenic mice were crossed with Hmga2-specific knockout mice. Mouse primary breast tumors were obtained from Hmga2 wild-type (Hmga2+/+), heterozygous (Hmga2+/−), or null (Hmga2−/−) mice. (I) Murine Hmga2, Tet1, and Hoxa9/7 mRNA analyzed by qRT-PCR (with mouse Gapdh as normalization control). (J) Murine Tet1 and Hoxa9 protein and 5hmC levels analyzed by immunostaining. (D–F, H, and I) Data are means ± SEM; n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.