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Blood, Vol. 113, Issue 22, 5558-5567, May 28, 2009
TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia
Blood Yu et al.
113: 5558
Supplemental materials for: Yu et al
Plasmid construction and in vitro DNA methylation
The −2.1-kb (−2100 to +10) sequence of the TSC-22 gene promoter was generated by PCR from mouse genomic DNA and the promoter sequences of −750-bp (−750 to +10), −320-bp (−320 to +10), and −43-bp (−43 to +10) were amplified from a plasmid containing the −2.1-kb fragment. The above amplified promoter fragments were cloned into a PCR2.1 vector (Invitrogen) and the sequences were confirmed by DNA sequencing. The sequenced fragments then were digested from the PCR2.1 vector by EcoRV and BamHI restriction enzymes and cloned into SmaI and BglII restriction sites of the pGL3 luciferase basic reporter vector (Promega, Madison, Wisconsin) to generate TSC-22-LUC reporter constructs. We amplified the murine TSC-22 coding sequence with a MYC tag at the N-terminus by PCR from cDNA, and the amplified fragment with a BglII restriction site before the start codon and a BamH1 restriction site after the stop codon. The amplified fragment was cloned into the same restriction sites of the retrovirus pMSCV-IRES-GFP-PURO vector (a kind gift from Dr. C. Baldus, The Ohio State University) and the sequences were confirmed by DNA sequencing. All PCR primer sequences are available on request. The −320-bp promoter insert was excised from the pGL3 vector with restriction enzymes XhoI and NcoI. The gel-purified insert was incubated overnight with 60 units SssI (CpG) methylase (methylated), or without (mock-methylated), as recommended by the manufacturer (New England BioLabs Inc., Beverly, MA). Following DNA purification, methylation status was confirmed using the restriction enzymes SmaI and NaeI, whose cleavage is blocked by methylation. Equal amounts of the methylated and mock-methylated DNA inserts were re-ligated separately into the pGL3 vector, and the re-ligated DNA was transfected into 293T cells for luciferase assays to compare the activities of the methylated or mock-methylated promoters. Genotyping of TSC-22 mice
Genotypes were determined by PCR analysis using primer 1 (5′-AGCCGAGTAGGACC
GAG-3′), primer 2 (5′-ATTGCAGTCTTGTTTCTGG-3′), and primer 3 (5′-CGCATAC
ATCAAATGGC-3′). Primer 1 is located upstream of the deleted TSC-22 region and primer 2 is within the region and primer 3 is downstream of the region. The combination of primers 1 and 2 resulted in a 2.5 kb band for wild-type, the 2.5 kb band and a 1.5 kb band for heterozygotes, and the 1.5 kb band for homozygotes. The set of primers 1 and 3 led to a 2 kb band for both wild-type and heterozygotes, and no band for homozygotes.
Files in this Data Supplement:
- Figure S1. RLGS analysis and transcript variants in the TSC-22D1 region (JPG, 243 KB)
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(A) A RLGS (Restriction Landmark Genomic Scanning) spot loss was identified in IL-15tg T or NK LGL leukemic (Leu) mice but not in IL-15tg mice with polyclonal T and NK cell expansion (Tg) or in wild-type (WT) mice. DNA sequencing indicates that the lost spot corresponds to the TSC-22D1 proximal promoter, which is located ~100 kb upstream of the TSC-22D1 transcription start site. (B) Schematic illustration of three transcript variants in the TSC-22D1 region analyzed by the Ensemble Genome Browser (http://www.ensembl.org/index.html).
- Figure S2. Ectopic expression of TSC-22 in the mouse leukemia L1210 cell line inhibits cell proliferation and decreases tumorigenicity in vivo (JPG, 226 KB)
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(A) L1210 cells transfected with a pMSCV vector containing TSC-22 in vitro grew significantly slower when compared to L1210 cells transfected with the empty vector (P < 0.05, n = 3). (B) At two weeks after injection, the average weight of tumors formed in vivo from L1210 cells expressing TSC-22 was significantly less than that of tumors formed from the cells transfected with the vector alone (P < 0.01, n = 6). (C) Photograph of the representative tumor pairs excised from sacrificed mice that had been injected with L1210 cells transfected with the pMSCV vector alone (top) or TSC-22 (bottom). The tumorigenicity assays in (B) and (C) were performed in NOD-SCID mice. Bulk transfected GFP(+) cells were purified by cell sorting. Error bars in (A)–(B) indicate standard deviations in one representative experiment of at least two with similar results.
- Figure S3. YAC-1 clones expressing TSC-22 form smaller tumors in mice than the clones expressing the pMSCV control vector (JPG, 132 KB)
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(A) The average weight of tumors collected from mice sacrificed at the end of in vivo experiments. The average weight of tumors formed in vivo from a representative YAC-1 clone expressing TSC-22 was significantly less than that of tumors formed from the clone transfected with the empty vector (P < 0.05, n = 6). (B) Photograph of the representative tumors excised from the sacrificed mice that had been injected with YAC-1 cells transfected with the pMSCV vector alone (top) or TSC-22 (bottom). Results of one representative clone of three with similar results are shown (A and B).
- Figure S4. Downregulation of TSC-22 expression in human NK cells by co-stimulation of IL-12 and IL-18 (JPG, 226 KB)
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(A) NK-92 human NK cell line (left panel) and primary human NK cells (right panel) were treated with or without IL-12 and IL-18 for 24 hr. (B) NK-92 cells were treated in the presence or absence of IL-12 and IL-18 co-stimulation for the indicated times. The TSC-22 transcript was measured by RT PCR and one of three representative experiments with similar results is shown (A and B).
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