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. 2019 Jul 11;10:3055. doi: 10.1038/s41467-019-11044-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Expression of LIF is required for in vitro and in vivo malignancies of human pancreatic cancer cells. a The ability to form spheres in 3D culture, reduced by the depletion of KRAS, could be rescued by addition of LIF at 100 ng/mL. Sphere size is shown in the left panel, and sphere-forming efficiency (numbers of formed spheres/numbers of seeded cells) in the right panel (N = 6, *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001). a The ability of Panc1.0 to form spheres in 3D culture, reduced by knocking down KRAS, could be rescued by the addition of LIF, but not IL-6 at the same concentration (100 ng/mL) (N = 6, ***P < 0.001). b Representative western blots (left panel) showing the depletion of LIF protein expression by CRISPR/Cas9 and reduced phosphorylated STAT3 at Y705 in mouse pancreatic tumor cells. The ability to form spheres in 3D culture (middle panel), reduced by depletion of LIF, can be rescued by addition of mouse LIF at 100 ng/mL (N = 6, *P < 0.05, **P < 0.01). (Right panel) Overall survival rate of syngeneic mice which were orthotopically injected with mouse pancreatic cancer cells (n = 5). c Representative ELISA (left panel) detecting human LIF in culture medium of Panc2.03, which expressed inducible shRNA against human LIF. The cells were treated with doxycycline (1 µg/mL) for 14 days before ELISA assay. Reduced sphere formation efficiency (right panel) of cells where LIF had been knocked down by inducible sh-LIF in the presence of doxycycline (N = 6, **P < 0.01, ****P < 0.0001). Error bars from a to d represent the standard deviation and P-value was generated by t-test. d Representative western blots (left panel) showing LIF protein expression in Panc6.03 cell line which expressed inducible shRNA against human LIF. Induced expression of shRNA against LIF by doxycycline reduced the growth of the established Panc6.03 xenograft tumors when compared with the control (right panel). e Representative western blots (left panel) showing LIF protein expression in Panc1.0, which expressed inducible shRNA against human LIF. Tumor growth curve (right panel) of Panc1.0 xenografts where inducible LIF shRNA was expressed in response to gemcitabine at 50 mg/kg with or without doxycycline