Select Cancer Testes Antigens of the MAGE-A, -B, and -C Families Are Expressed in Mast Cell Lines and Promote Cell Viability In Vitro and In Vivo  Bing Yang, Sean O'Herrin, Jianqiang Wu, Shannon Reagan-Shaw, Yongsheng Ma, Minakshi Nihal, B. Jack Longley  Journal of Investigative Dermatology  Volume 127, Issue 2, Pages 267-275 (February 2007) DOI: 10.1038/sj.jid.5700548 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 MAGE siRNAs inhibit MAGE gene expression and mast cell viability in vitro. (a) Select MAGE siRNA Smart-Pools inhibit the growth of human HMC1.1 and murine P815 mast cell lines, but not the HaCat transformed human keratinocyte cell line. The final concentration of each siRNA is 100nM. Bars are shown as % of growth compared to non-transfected cells. Control bar (clear) is growth of nontransfected cells and is by definition 100%. Nonspecific bar indicates “non-sense” siRNA. Cells were counted 3 days after transfection with 100nM siRNA in Lipofectamine 2000. MAGE-A complex reagent targets all MAGE-A genes expressed in HMC1.1 except MAGE-A1, which differs significantly in sequence from the other MAGE-A genes. The HMC1.1 cell line does not express significant protein levels of MAGE-B and the P815 line does not express significant levels of mMage-a family members. There is no murine homolog of the human MAGE-C family. The effective MAGE siRNAs showed a dose–response relationship between 50 and 150nM (data not shown). * indicates statistical significant difference from nonspecific siRNA treated group, P<0.05. (b) Representative results with siRNAs targeting individual MAGE-A family members. Except A1, the rest of tested MAGE-A siRNAs showed growth inhibition in HMC-1.1 cells. (c) RT-PCR validates gene targeting by showing loss of amplifiable MAGE mRNAs after treatment with specific siRNAs. Twenty-four hours after transfection with 100nM of siRNA, total RNA was extracted from cells, reverse transcribed, and amplified with primers bracketing the siRNA target sequences. Gel electrophoresis and staining with ethidium bromide show loss of amplimers with MAGE specific siRNA but not with control siRNA. Specific targeting in HMC1 cells was further validated by immunoblotting showing knockdown of MAGE-A total, MAGE-A1 or MAGE-C2 protein by MAGE-A complex, MAGE-A1, or MAGE-C2 siRNAs, respectively. HMC1.1 human mast cells were transfected with 100nM MAGE siRNA in Lipofectamine 2000 and MAGE protein was identified by Western blotting 48hours after transfection. Antibodies against effective individual MAGE proteins (A2, A3, A5, A6, and A12) were not available. Journal of Investigative Dermatology 2007 127, 267-275DOI: (10.1038/sj.jid.5700548) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 MAGE siRNAs induce apoptosis in mast cell lines. Fluorescence microscopy of acridine orange-ethidium bromide stained HMC1.1 cells ((a) 12hour) and P815 cells ((d) 3hour) after transfection with nonspecific siRNA shows that essentially all fluoresence green due to uptake of the vital dye acridine orange. After transfection with MAGE-A siRNA ((b) HMC1.1) or mMage-b siRNA ((e) P815), dead apoptotic cells show condensed and fragmented nuclei that stain red with ethidium bromide. (a and b) Bar=50μm and (d and e) bar=25μm. Statistically significant differences of 25.3% ((c) HMC1.1) and 27.3% ((f) P815) were seen in the number of dead apoptotic cells in MAGE siRNA treated cells vs nonspecific siRNA treated cells (P<0.05 HMC1.1, P<0.05 P815). (g and h) TUNEL analysis by flow cytometry at same times after transfection shows MAGE siRNA induced apoptosis is not inhibited by the general caspase inhibitor zVAD-FMK. The percentage of apoptotic cells is given in the upper right corner of each panel. Journal of Investigative Dermatology 2007 127, 267-275DOI: (10.1038/sj.jid.5700548) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 MAGE siRNAs inhibit mast cell proliferation. Flow cytometry showed an increase in the percentage of cells in S phase after treatment with MAGE siRNAs. (a) HMC 1.1 cells were stained with PI showed 46% of cells in S phase 24hour after transfection with MAGE-A complex siRNA, compared to 31% in S phase for cells treated with control siRNA. (b) Similar studies with P815 cells which had 60% in S-phase after mMage-b siRNA treatment compared with 51% after treatment with nonspecific siRNA. *Indicates statistical significant difference from nonspecific siRNA treated groups, P<0.05. Journal of Investigative Dermatology 2007 127, 267-275DOI: (10.1038/sj.jid.5700548) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 MAGE siRNAs inhibit mast cell tumor growth in vivo. (a) Kaplan–Meier plot. P815 cells were transfected with 100nM mMage-b siRNA (si-STABLE-PLUS, Dharmacon) or l00nM control siSTABLE-PLUS siRNA, both administered in Lipafectamine™ 2000. After 6hours, equal numbers of viable cells were injected subcutaneously into the flanks of syngenic DBA/2 mice and tumor size measured every other day by two independent and blinded investigators. (b) Linear Regression analysis shows that tumor diameters grew an average of 0.62mm per day postinoculation in the nonspecific siRNA treated group and 0.50mm per day for the mMage-b siRNA treated group, a statistically significant difference with P<0.01. The observed difference for mice with tumors treated with MAGE siRNA compared to mice with tumors treated with nonspecific siRNA was statistically significant with P<0.01 according to a log-rank analysis of both (c) mean and (d) median data. NS=non-specific siRNA. Journal of Investigative Dermatology 2007 127, 267-275DOI: (10.1038/sj.jid.5700548) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions