Supplementary Information

ETV6/RUNX1 abrogates mitotic checkpoint function and targets its key player MAD2L1

Gerd Krapf, Ulrike Kaindl, Anna Kilbey, Gerhard Fuka, Andrea Inthal, Ruth Joas, Georg Mann, James C. Neil, Oskar A. Haas and E. Renate Panzer-Grümayer.

Methods

Patient samples and MAD2L1 mRNA quantification

MAD2L1 mRNA was amplified using published conditions (Pinto et al., 2007). Primary leukemic cells were obtained after informed consent of the patients’ parents and with approval of the Ethics Committee of St. Anna Children’s hospital.

Chromatin immunoprecipitation

ChIP was performed as described for the ChIP-IT kit (Active Motif, Carlsbad, CA). 1/10th of the immunoprecipitated DNA and 1/100th of input DNA were analyzed by PCR using oligonucleotides: MAD2L1#1 (f) 5′– cgaaattctgccagagggc -3′; MAD2L1#1 (r) 5′– ctttcatgtctgccggacg -3′; MAD2L1#2 (f) 5′– cgcgacatcacaggaagcc -3′; MAD2L1#2 (r) 5′– gctcccgggagagctgc-3′; p21WAF1 (f) 5’- ccattcccctaccccatgc -3’; p21WAF1 (r) 5’ cctaccatccccttcctcacc -3’; PHOX (GP91) (f) 5’- ccaatgattattagccaatttctg -3’; PHOX (GP91) (r) 5’- catggtggcagaggttgaatgt -3’. The hotstart PCR conditions were 5min at 95°C (1 cycle), 1min at 95°C, 1min at 57°C (MAD2L1 #1-test primer, PHOX – negative control) or 61°C (MAD2L1 #2 -test primer, p21WAF1 - positive control), 1min at 72°C (30 cycles), 10min at 72°C (1 cycle). The PCR products were resolved on a 1.5%TBE agarose gel.
The antibodies used were: anti c-Myc (Abcam-9E11) and anti N-cadherin negative control (610920 BD Transduction Labs, San Jose, CA, USA).

Figure 1

Mitotic checkpoint attenuation in E/R positive leukemic cell lines. (A) DNA content was analyzed by PI staining in the absence or presence (50ng/mL, 24 hrs) of nocodazole. Representative histograms of E/R+ (REH, AT-2) and E/R- (Nalm-6, K562) leukemic cell lines are shown. (B) Percentage of cells with 2N DNA content (G0/G1) of E/R+ or E/R- cell lines (as in A) upon activation of the mitotic checkpoint by nocodazole or paclitaxel. Boxes show the median and interquartile range (95 % of the values are within the range of the whiskers) of three independent experiments. (C) Mitotic indexes of E/R-positive leukemic cell lines (E/R+: REH, AT-2) and E/R-negative leukemic cell lines (E/R-: Nalm6, K562) were determined after nocodazole treatment (50ng/mL; for the indicated times). Box plots show the median and interquartile range from one representative experiment. Welch’s t-test, *, p≤0.05. All experiments were performed at least three times and at different occasions unless otherwise stated. (D/E) Apoptosis rates were analyzed by PI staining in E/R+ and E/R- leukemic cell lines (as in A). Percentages of PI positive cells are depicted under optimal culture conditions (D) and upon nocodazole treatment for 20 hrs. Welch’s t-test, p>0.8. (E). (F) Securin levels were determined by Western blot analysis using whole protein lysates from E/R+ and E/R- leukemic cell lines (as in A) upon nocodazole addition for 18 hrs. For the detection of securin a specific anti-PTTG-1 antibody (Zymed, SF, CA, USA) was used. The arrow indicates the full-length protein (28kDa; left part of the Figure). This band was used for protein quantification. Tubulin (DM1A, Calbiochem, Darmstadt, DE) was used as loading control. Right part of the figure, relative integrated intensity counts (I.I.) are blotted for E/R+ and E/R- cell lines. Welch’s t-test, *, p<0,05.

References

Pinto M, Soares MJ, Cerveira N, Henrique R, Ribeiro FR, Oliveira J et al (2007). Expression changes of the MAD mitotic checkpoint gene family in renal cell carcinomas characterized by numerical chromosome changes. Virchows Arch 450: 379-85.

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