• A new mechanism for gemcitabine resistance via stemness induction is proposed.• Gemcitabine induces pancreatic CSCs and stemness-associated genes.• Gemcitabine enhances cell migration, chemoresistance, and tumorigenesis.• STAT3 pathway partially mediates gemcitabine-induced CSC properties and tumor relapse.• Nox/ROS/NF-κB participates in gemcitabine-induced STAT3 activation and stemness.
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