Supplementalmaterials and methods

PCR genotyping

PCR conditions are 94oC for 45 sec, 60oC for 45 sec, followed by 72oC for 1 min, with a total of 30 cycles. Primers sequences are p1(TTCACACACAGTTTGCTCTCACC), p2(TGTGAAAAGATCTGTGATCTAAATGAGCC), and p3(CACTGCCAAAGTCACTATTAGGTTCC).

Generation of primary mouse embryonic fibroblasts and antibodies

MEFs were prepared from E12.5 embryos following standard procedures. Primary cells are cultured in DMEM supplemented with 10% FBS, L-Glutamine, Pen/Strep, and Non-essential amino acids. Anti-MLL C-terminal antibody has been described (Hsieh et al., 2003b). Anti-MLL2 C-terminal polyclonal antibodies were raised against purified MLL2 fragment encoding amino acids 2,277 to 2,422 (Covance).

FACS analyses

Thymocytes were purified and stained with anti-CD4 FITC conjugated and anti-CD8 PE conjugated antibodies (Pharmingen) before subjected to cytometric analysis with FACSCalibur (Becton-Dickinson). Data were analyzed with FlowJo software (Tree Star).

Cell death assays

Cells were labeled with anti-Annexin V FITC or APC conjugated antibody (Pharmingen) before subjected to flow cytometric analysis with FACSCalibur.

Quantitative RT PCR analyses

Primers sequences:

Taspase1

Forward CACAGCTGTGAGTACCTCAGGATG

Reverse CCACAAGCAGTGTCTGTTTATCTTGG

Cyclin D1

Forward CGTATCTTACTTCAAGTGCGTGCAGAAG

Reverse CAGGTTCCACTTGAGCTTGTTCAC

Cyclin D2

Forward GCAGAAGGACATCCAACCGTACATG

Reverse GGATGTGCTCAATGAAGTCGTGAGG

Cyclin E1

Forward GGATGAGAGCAGTTCTTCTGGATTGG

Reverse GACACAATGGTCAGAGGGCTTAGAC

Cyclin E2

Forward CATTCTGACCTGGAACCACAGATGAG

Reverse CAAGCACCATCAGTGACGTAAGC

Cyclin A2

Forward CACTGACACCTCTTGACTATCCAATGG

Reverse GCTGAAGCTTCCCTCTTAACACAGAC

Cyclin B1

Forward GAGCCTGAACCTGAACTTGAACATG

Reverse CCTGTATTAGCCAGTCAATGAGGATAGC

Cyclin B2

Forward CCTGAGGATGTCTCCATGAAGGAAG

Reverse GTTTCCTGCAGAAGCCGAAACTTG

p16Ink4A

Forward GAACTCGAGGAGAGCCATCTGGAG

Reverse CGTAGTTGAGCAGAAGAGCTGCTAC

p21

Forward GAGAACGGTGGAACTTTGACTTCGTC

Reverse CGCTTGGAGTGATAGAAATCTGTCAGG

p27

Forward GGTCTCAGGCAAACTCTGAGGACC

Reverse AGCTGTTTACGTCTGGCGTCGAAG

ARF

Forward GTTCTTGGTCACTGTGAGGATTCAGC

Reverse CCTCTTCTCAAGATCCTCTCTAGCCTC

Western blot analyses

Western blots using commercially available antibodies against p16Ink4a (Santa Cruz, sc-1207), p21 (Santa Cruz, sc-397), p27 (Santa Cruz, sc-528), ARF (Novus, 200-174), Cyclin D1 (Santa Cruz, sc-450), Cyclin E (Cell Signaling), Cyclin A (Sigma), anti-phosphoRb (S807/811) (Cell Signaling),Actin (Sigma), Flag (Sigma), Myc (Santa Cruz), and Protein C (Roche) were performed according to the suppliers’ recommendations.

Chromatin immunoprecipitation assays

Oligo sequences

Cyclin E1-1p

Forward TTG TTG TCC TCC CAG GTC TGA

Reverse CAG CAG GGA GGC CAC TCA

Taqman probe FAM-TTC CAA GCC CAA GTC CTG AGC CAT G-IBFQ

Cyclin E1-2p

Forward CGG ACC ACA GCA ACA TGA AA

Reverse GGC CAC ATT TGC CTT CCT T

Taqman probe FAM-CTC CGA CCT TTC AGT CCG CTC CAG AA -IBFQ

Cyclin E2-1p

Forward GCC CGG CCT ATA TAT TGA GTT G

Reverse GCC CCT GTC CCC AAT ACC T

Taqman probe FAM-CTG AGC CGA GCT GTG GAG GGT CTG-IBFQ

Cyclin E2-2p

Forward GGG ATC ACT TCA CGG AGA GAT T

Reverse TCC AGA AGA GCA GAA GGA AAG G

Taqman probe FAM-AAG CAC GGT TTT CTC CTT TAA TTC CCC CC-IBFQ

p16p

Forward ACTGAATCTCCGCGAGGAAA

Reverse CTGTCTGCAGCGGACTCCAT

Taqman probe FAM-CGAACTCGAGGAGAGCCATCTGGAGC- IBFQ

Soft agar assays

Primary MEFs were infected with retrovirus expressing E1a-IRES-Ras, Myc-IRES-Ras, or DNp53-IRES-Ras, and selected against Puromycin (1ug/ml) for four days. Soft agar assays were performed as described with minor modifications (Maeda et al., 2005). One hundred thousand cells were plated on a 6 cm dish and positive colonies (diameter > 200 um) were scored 2-3 weeks after plating.

Supplemental Figures

Supplemental Figure S1 Tissue expression of Taspase1 in mice. Total RNA was obtained from mice at 6 weeks of age for quantitative RT-PCR analyses. RNA input was normalized with 18S rRNA (ABI). Representativedata from three mice are presented. The expression of Taspase1 in heart is arbitrarily set at 1.

Supplemental Figure S2 Western blot analyses of CdkIs, ARF, and Cyclins onasynchronized passage 2wild type and Taspase1-/- MEFs.

Supplemental Figure S3 Taspase1-/- MEFs have fewer cycling cells. Asynchronized MEFs were cultured in the presence of BrdU for 24 hours before subjected to FACS analysis using anti-BrdU FITC conjugated antibody. Representative data from three independent experiments are presented.

Supplemental Figure S4 RNAi-mediated knockdown of p16 in Taspase1-/- MEFs. Western blot analyses of MEFs infected with lentivirus expressing indicated shRNA. ShRNA against firefly luciferase was used as a negative control. ShRNA against p16 reduced the expression of p16 in Taspase1-/-cells to nearwild type levels.

References

Maeda, T., Hobbs, R. M., Merghoub, T., Guernah, I., Zelent, A., Cordon-Cardo, C., Teruya-Feldstein, J., and Pandolfi, P. P. (2005). Role of the proto-oncogene Pokemon in cellular transformation and ARF repression. Nature 433, 278-285.

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