Compared to the vector control H6c7-pBp cells [4]. Three separate shRNA sequences were used to downregulate LCN2 expression in H6c7KrT cells, resulting in reduction of LCN2 mRNA levels by 76 , 92 
, and 65 , as compared to the non-silencing (NS) shRNA (Fig. 2A). MNS web Protein lysates and conditioned media also showed concordant reduction in LCN2 protein expression (Fig. 2B). The LCN2KD2 shRNA sequence was selected for subsequent experiments as it yielded maximal knockdown of LCN2 expression. Stable transduction 
of LCN2KD2 and NS shRNA into the BxPC3 and HPAF-II cell lines decreased LCN2 mRNA expression by 78 and 87 , respectively (Fig. 2C). We stably transduced the low LCN2 expressing PANC1 cells with a LCN2 expression construct or a control empty vector (EV). Q-PCR and Nafarelin immunoblot analyses confirmed the overexpression (Fig. 2D). Secreted production of LCN2 was consistent with intracellular protein levels. Modifying LCN2 expression inLCN2 in Pancreatic CancerFigure 6. LCN2 promotes resistance to gemcitabine and angiogenesis. Effect of gemcitabine on the growth of tumors formed by (A) BxPC3NS and CN2KD2 cell lines and (B) with PANC1-EV and CN2 cell lines [*denotes significance between vehicle treated cell lines (n = 10 per group, p,0.0001, mixed model multiple regression) {denotes significance between vehicle and gemcitabine treated mice injected with BxPC3 LCN2KD2 (n = 10 per group, p = 0.0003, mixed model multiple regression)]. (C) Protein lysates isolated from BxPC3 and PANC1 xenografts were assayed for caspase-3 cleavage after gemcitabine treatment (n = 10). (D) Representative histological images of xenografts formed by BxPC3 NS and CN2KD2, and PANC1-EV and CN2 cells after H E, and immunostaining for LCN2, Ki67, and murine CD31. (E) Vascular density in five hot spots at high magnification in the BxPC3 NS and CN2KD2, and PANC1-EV and CN2 xenografts. The mRNA expression of (F) HIF1A and (G) VEGF in the BxPC3 15755315 and PANC1 xenografts. Gene expression was compared between KD and NS, or LCN2 and EV. [*denotes significance between KD and NS, or LCN2 and EV (n = 20; p,0.05, student t-test)]. doi:10.1371/journal.pone.0046677.gLCN2 in Pancreatic Cancercontributes to the invasiveness of PDAC cells by promoting MMP-9 activity.LCN2 Promotes Tumorigenicity in the Xenograft ModelThe expression of LCN2 has been demonstrated to enhance breast tumour formation and progression [13]. To determine how LCN2 affects tumorigenicity in PDAC, BxPC3, HPAF-II, and PANC1 cells were implanted subcutaneously into SCID mice. Knocking-down LCN2 in both BxPC3 and HPAF-II cells significantly reduced tumor growth when compared to the NS xenografts (p,0.01; Fig. 5A ). Consistently, PANC1 LCN2 cells had markedly increased tumor growth compared to the EV xenografts (p,0.01; 1326631 Fig. 5C). Gelatin zymography was employed to examine the effects of suppressing or overexpressing LCN2 on MMP-9 activity in PDAC xenografts. Diminishing LCN2 expression reduced MMP-9 activity by 35 and 79 in BxPC3 and HPAF-II xenografts, respectively (Fig. 5D , S2A). Whereas, in PANC1 xenografts there was little MMP-9 activity and LCN2 expression promoted MMP-9 activity in half of xenografts assessed (Fig. 5F). Together, LCN2 expression influences tumourigenicity in vivo and promotes MMP-9 activity in PDAC cell lines that highly express LCN2.LCN2 Enhances Gemcitabine Resistance in PDAC Cells in vitroGemcitabine is used as a first-line chemotherapeutic agent in PDAC [23]. To determine if LCN2 promotes su.Compared to the vector control H6c7-pBp cells [4]. Three separate shRNA sequences were used to downregulate LCN2 expression in H6c7KrT cells, resulting in reduction of LCN2 mRNA levels by 76 , 92 , and 65 , as compared to the non-silencing (NS) shRNA (Fig. 2A). Protein lysates and conditioned media also showed concordant reduction in LCN2 protein expression (Fig. 2B). The LCN2KD2 shRNA sequence was selected for subsequent experiments as it yielded maximal knockdown of LCN2 expression. Stable transduction of LCN2KD2 and NS shRNA into the BxPC3 and HPAF-II cell lines decreased LCN2 mRNA expression by 78 and 87 , respectively (Fig. 2C). We stably transduced the low LCN2 expressing PANC1 cells with a LCN2 expression construct or a control empty vector (EV). Q-PCR and immunoblot analyses confirmed the overexpression (Fig. 2D). Secreted production of LCN2 was consistent with intracellular protein levels. Modifying LCN2 expression inLCN2 in Pancreatic CancerFigure 6. LCN2 promotes resistance to gemcitabine and angiogenesis. Effect of gemcitabine on the growth of tumors formed by (A) BxPC3NS and CN2KD2 cell lines and (B) with PANC1-EV and CN2 cell lines [*denotes significance between vehicle treated cell lines (n = 10 per group, p,0.0001, mixed model multiple regression) {denotes significance between vehicle and gemcitabine treated mice injected with BxPC3 LCN2KD2 (n = 10 per group, p = 0.0003, mixed model multiple regression)]. (C) Protein lysates isolated from BxPC3 and PANC1 xenografts were assayed for caspase-3 cleavage after gemcitabine treatment (n = 10). (D) Representative histological images of xenografts formed by BxPC3 NS and CN2KD2, and PANC1-EV and CN2 cells after H E, and immunostaining for LCN2, Ki67, and murine CD31. (E) Vascular density in five hot spots at high magnification in the BxPC3 NS and CN2KD2, and PANC1-EV and CN2 xenografts. The mRNA expression of (F) HIF1A and (G) VEGF in the BxPC3 15755315 and PANC1 xenografts. Gene expression was compared between KD and NS, or LCN2 and EV. [*denotes significance between KD and NS, or LCN2 and EV (n = 20; p,0.05, student t-test)]. doi:10.1371/journal.pone.0046677.gLCN2 in Pancreatic Cancercontributes to the invasiveness of PDAC cells by promoting MMP-9 activity.LCN2 Promotes Tumorigenicity in the Xenograft ModelThe expression of LCN2 has been demonstrated to enhance breast tumour formation and progression [13]. To determine how LCN2 affects tumorigenicity in PDAC, BxPC3, HPAF-II, and PANC1 cells were implanted subcutaneously into SCID mice. Knocking-down LCN2 in both BxPC3 and HPAF-II cells significantly reduced tumor growth when compared to the NS xenografts (p,0.01; Fig. 5A ). Consistently, PANC1 LCN2 cells had markedly increased tumor growth compared to the EV xenografts (p,0.01; 1326631 Fig. 5C). Gelatin zymography was employed to examine the effects of suppressing or overexpressing LCN2 on MMP-9 activity in PDAC xenografts. Diminishing LCN2 expression reduced MMP-9 activity by 35 and 79 in BxPC3 and HPAF-II xenografts, respectively (Fig. 5D , S2A). Whereas, in PANC1 xenografts there was little MMP-9 activity and LCN2 expression promoted MMP-9 activity in half of xenografts assessed (Fig. 5F). Together, LCN2 expression influences tumourigenicity in vivo and promotes MMP-9 activity in PDAC cell lines that highly express LCN2.LCN2 Enhances Gemcitabine Resistance in PDAC Cells in vitroGemcitabine is used as a first-line chemotherapeutic agent in PDAC [23]. To determine if LCN2 promotes su.
