IDO downregulation sensitized A549 xenografts to pemetrexed treatment.

<p><b>A</b>: Ten million A549 cells transfected with control shRNA (clone NC-3) or anti-IDO shRNA (clone 2–18) were injected into flanks of SCID mice. Once tumors reached ~300 mm³ animals were injected with 100,000 IU IFNγ i.p. (twice a week for four weeks) and 50 mg/kg pemetrexed i.p. (once per week for four weeks). Animals transplanted with A549 clonal cells transfected with control shRNA or anti-IDO shRNA received four weeks of IFNγ treatment alone <b>(B)</b> or IFNγ and pemetrexed <b>(C)</b>. Tumor growth was monitored with caliper measurement to estimate tumor volume. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to pemetrexed more than IDO knockdown alone.

<p>A549 cells were transfected with scrambled, non-targeting control siRNA or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, pemetrexed (30 nM) for 72, and then enumerated. Bars indicate mean relative cell numbers (n = 3 ± SD). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with pemetrexed, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing scrambled, non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with scrambled, non-targeting control siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with pemetrexed. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were each considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with pemetrexed, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

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

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</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. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 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. <b>Panel C</b>: 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 <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: 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.</p

Concurrent IDO and TS downregulation sensitizes A549 cells to 5FUdR more than TS knockdown alone.

<p>A549 cells were transfected with control or anti-thymidylate synthase (anti-TS) siRNA, treated with IFNγ (25 ng/ml) for 48 h, with 5FUdR (40 nM) for 72 h, and then enumerated. Bars indicate mean proliferation relative to appropriate controls ± SD (n = 3). <b>A</b>: Proliferation of clonal A549 cell populations induced with IFNγ and then treated with 5FUdR, but untransfected with siRNA of any kind. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. <b>B</b>: Proliferation of the same clonal A549 cell populations transfected with control non-targeting siRNA, TS siRNA #3, or TS siRNA #4, induced with IFNγ, and then treated with 5FUdR. Bars represent values normalized to values obtained from clones treated with IFNγ but untreated with pemetrexed or siRNA; those cells were considered to have a proliferation value of 100% after IFNγ treatment. <b>Gray bars</b>: clones containing anti-IDO shRNA. <b>White bars</b>: clones containing non-targeting control shRNA. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Data presented a representative experiment from two independent experiments. Results are normalized to control cells not treated with 5FUdR, but with treated with IFNγ. <b>Panel C</b>: shows the pooled results from panel A and B.</p

1. ON-Target Plus^® TS and BRCA2 siRNA target mRNA sequences.

<p>1. ON-Target Plus^® TS and BRCA2 siRNA target mRNA sequences.</p

A549 clone sensitivity to Gemcitabine and 5FUdR before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before <b>(Panel A and C)</b> and after <b>(Panel B and C)</b> IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h, treated with gemcitabine (10 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels A and B and SEM for panel C, (*<i>p</i><0.05). Results are normalized to control cells not treated with Gemcitabine, without (panel A) or with (panel B) IFNγ treatment. <b>Panels D-F</b>: Proliferation of each of 5 individual A549 cell clonal populations before and after IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h and, 5FUdR (200 nM) for 72 h, and then enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SD for Panels D and E and SEM for panel F ± SD. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. Results are normalized to control cells not treated with 5FUdR, without (panel A) or with (panel B) IFNγ treatment.</p

TS siRNA downregulation in A549 clonal populations.

<p>A549 clonal cell populations (NC-3, NC-10, and NC-30, each with stably-incorporated control, non-targeting scrambled shRNA; and 2–6 and 2–18, each with stably-incorporated anti-IDO shRNA) were seeded and grown overnight. Thymidylate synthase (TS) siRNA numbers 3 or 4, or scrambled, non-targeting control siRNA, were transiently transfected into each clonal population. Cells were lysed and protein was harvested 96 h later. TS and actin protein levels were determined by immunoblot. Results were quantified for each clone separately. <b>A</b>: TS protein quantification for each clonal population. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SEM. *Significant difference from the same cells transfected with scrambled control, non-targeting siRNA, Student's <i>t</i>-test, <i>p</i><0.05. <b>B</b>: Representative immunoblots of TS and actin protein in clone NC-3 after transfection of TS siRNA (siRNA numbers 3 and 4) or scrambled, non-targeting control siRNA (C2). <b>C</b>: Representative immunoblots of TS and actin protein in clone 2–18 after transfection of TS siRNA.</p

A549 clonal cell population sensitivity to FK866 (5 nM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before (<b>Panel A</b>) and after (<b>Panel B</b>) IDO induction with IFNγ. A549 clonal cell populations were cultured with or without IFNγ (25 ng/ml) for 48 h. Medium was then replaced with fresh growth medium containing FK866 (5 nM) and cells were allowed to proliferate for 72 h. Cells were then trypsinized and live cells were enumerated. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Results are normalized to control cells not treated with FK866, without (panel A) or with (panel B) IFNγ treatment. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SD, (*, p≤0.05). <b>Panel C</b>: A549 clonal resistance to FK866 before and after IFNγ-mediated IDO induction. Pooled results were obtained from 3 or 2 independent A549 clonal populations stably transfected with scrambled shRNA (white bars) or 2 anti-IDO shRNA (black bars) respectively, before and after induction of IDO. Each bar represents a mean of 9 (white bars) or 6 (black bars) values ± SEM, Significant difference, Student's <i>t</i>-test, <i>p</i><0.05. <b>Panel D</b>: Correlation analysis of the relationship between IDO protein content (relative to actin) and clonal population resistance to FK866 (proliferation relative to untreated control cells).</p

A549 clone sensitivity to pemetrexed (200 nM) before and after IDO induction.

<p>Proliferation of each of 5 individual A549 cell clonal populations before <b>(Panel A)</b> and after <b>(Panel B)</b> IDO induction with IFNγ. A549 clonal populations were cultured with or without IFNγ (25 ng/ml) for 48 h, then with pemetrexed (200 nM), and enumerated 72 h later. <b>White bars</b>: A549 clones transfected with scrambled, non-targeting control shRNA. <b>Gray bars</b>: A549 cells transfected with anti-IDO shRNA. Each bar represents the mean of 3 values (<i>n</i> = 3) ± SD. Results are normalized to control cells not treated with pemetrexed, without (panel A) or with (panel B) IFNγ treatment. <b>Panel C</b>: Induction of IDO in A549 clonal cell induces resistance to pemetrexed (200 nM). 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 <i>t</i>-test, <i>p</i><0.05.</p

Concurrent downregulation of IDO and BRCA2 did not sensitize A549 to 5FUdR.

<p>A549 clonal cells transfected with either scrambled shRNA (NC-3) or anti-IDO shRNA (2–18) were transiently transfected with breast cancer type-2 susceptibility protein (BRCA2) siRNA, induced with IFNγ (25 ng/ml) for 24 h, and then treated with 5FUdR (40 nM) for 72 h, at which time live cells were enumerated. Simultaneous downregulation of IDO and BRCA2 did not sensitize A549 to the TS-targeting drug 5FUdR to a greater degree than the knockdown of either gene alone. Bars represent the means of 3 independent measurements of cells (with or without downregulation of IDO) after BRCA2 siRNA transfection + 5FUdR treatment (<i>n</i> = 3 for each measurement) ± SD. Bars were normalized to values obtained from clones treated with IFNγ but untreated with 5FUdR or siRNA; those cells were considered to proliferate at a 100% level after IFNγ treatment. *Significant difference, Student's <i>t</i>-test, <i>p</i><0.05.</p