A549 clone sensitivity to methoxyamine (3 mM) before and after IDO induction.

Proliferation of each of 5 individual A549 cell clonal populations before (Panel A) and after (Panel B) IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Cultured medium was then replaced with fresh growth medium containing Methoxyamine (MX) (3 mM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. White bars: A549 clones transfected with scrambled, non-targeting control shRNA. Gray bars: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (n = 3 for determination of each value) ± SD. Results are normalized to control cells not treated with methoxyamine, without (panel A) or with (panel B) IFNγ treatment. Panel C: Induction of IDO in A549 clonal cell populations induces resistance to MX (3 mM). Results were obtained from 3 or 2 independent clonal cell populations with scrambled, non-targeting control shRNA or anti-IDO shRNA, respectively. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, *Significant difference, Student's t-test, p<0.05. Panel D: Relationship between IDO protein level (relative to actin) and resistance to methoxyamine (MX)(proliferation relative to untreated control cells). The R2 value of 0.83 represents a moderate positive relationship.

5株独立A549细胞克隆群体在经干扰素γ（Interferon γ, IFNγ）诱导吲哚胺2,3-双加氧酶（Indoleamine 2,3-dioxygenase, IDO）前（图版A）与诱导后（图版B）的增殖情况。将A549细胞克隆群体分别在含有或不含25 ng/ml IFNγ的培养基中培养48小时；随后更换培养基为含3 mM甲氧胺（Methoxyamine, MX）的新鲜生长培养基，令细胞继续增殖72小时。之后用胰酶消化细胞并计数活细胞数量。白色柱形代表转染了随机序列非靶向对照短发夹RNA（short hairpin RNA, shRNA）的A549克隆，灰色柱形代表转染了抗IDO shRNA的A549细胞。每根柱形均为3次重复实验的平均值（每个数值的测定均设3个重复，n=3），误差线表示标准差（standard deviation, SD）。实验结果以未接受甲氧胺处理的对照细胞为基准进行归一化，其中图版A未施加IFNγ处理，图版B则施加了IFNγ处理。

图版C：在A549细胞克隆群体中诱导IDO表达可使其对3 mM甲氧胺（MX）产生耐药性。该结果分别来自3株转染随机序列非靶向对照shRNA的独立克隆，以及2株转染抗IDO shRNA的独立克隆。每根柱形分别代表9次（白色柱形）或6次（黑色柱形）实验的平均值，误差线表示标准误（standard error of the mean, SEM）；* 表示差异具有统计学意义（Student's t检验，p<0.05）。

图版D：IDO蛋白水平（以肌动蛋白为内参）与甲氧胺（MX）耐药性（以未处理对照细胞的增殖率为参照）之间的相关性，其决定系数R²为0.83，表明二者存在中等程度的正相关关系。