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, pPanel 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.

本数据集展示了5株独立A549细胞克隆群体在经干扰素γ（IFNγ）诱导吲哚胺2,3-双加氧酶（IDO）前后的增殖情况，其中图A为诱导前，图B为诱导后。将A549细胞克隆群体置于添加或不添加25 ng/ml干扰素γ的培养基中培养48小时，随后弃去原培养基，更换为含3 mM甲氧胺（Methoxyamine, MX）的新鲜完全培养基，继续让细胞增殖72小时。之后对细胞进行胰酶消化并计数活细胞数量。白色柱形代表转染了乱序非靶向对照短发卡RNA（shRNA）的A549细胞克隆，灰色柱形代表转染了抗IDO短发卡RNA的A549细胞。每根柱形代表3次独立实验的平均值±标准差（SD），每个数值均通过3次重复测定得到。所有结果均以未接受甲氧胺处理的对照组细胞为基准进行归一化，其中对照组分别为未添加干扰素γ（图A）与添加干扰素γ（图B）的细胞。
图C：A549细胞克隆群体经IDO诱导后，可产生对3 mM甲氧胺的耐药性。该结果分别来自3株转染乱序非靶向对照shRNA的独立克隆群体，以及2株转染抗IDO shRNA的独立克隆群体。每根柱形分别代表9次（白色柱形）或6次（黑色柱形）测定的平均值±标准误（SEM），*代表经t检验（Student's t-test）存在显著性差异。
图D：IDO蛋白水平（以肌动蛋白为参照）与甲氧胺（MX）耐药性（以未经处理的对照组细胞增殖率为参照）之间的相关性。决定系数（R²）为0.83，表明二者存在中等程度的正相关关系。