Fig. 5: HLF regulates cell migration via the LPXN-PXN axis.

a Immunoblotting analysis of 786-O cells following HLF knockout or overexpression. Each experiment has been repeated at least two times with similar results. For the left panel, the samples derive from the same experiment but different gels for LPXN, another for p-PXN-Tyr118, another for PXN and another for FLAG, vinculin. For the right panel, the samples derive from the same experiment but different gels for LPXN, Vinculin, another for p-PXN-Tyr118, another for PXN and another for V5 were processed in parallel. b Representative fluorescence images in cells following HLF knockout and cultured on stiff surface (50 µg/ml collagen coating). Nucleus were stained blue, n = 6 independent cell cultures, scale bar 10 µm. c Representative images and quantification (n = 7 independent cell cultures) of 3D collagen invasion assay in 786-O cells with HLF overexpression followed by infection with Paxillin Y118E (phosphomimetic version) or Y118F (non-phosphorylatable version). Scale bar, 200 µm. d–f Schematic showing single cells migrating on surface coating with stiff collagen I (50 µg/ml) and representative wind-rose plots showing cell tracks (d), migration velocity (e), and Euclidean distance (f) of the tracked 786-O cells cultured on stiff collagen surface, cells from three independent cell cultures were analyzed. Created in BioRender. Zhou, J. (2025) https://BioRender.com/x5scj3g. g-i Representative wind-rose plots showing cell tracks (g), migration velocity (h) and Euclidean distance (i) of the tracked 786-O cells overexpressed with HLF and cultured on surface with or without stiff collagen coating. The analyzed cells were from three independent cell cultures. j–l Representative fluorescence images of F-actin and filaments ( j), intensity of F-actin (k) and filament number per cell (l) in 786-O cells with HLF knockout following by LPXN overexpression and cultured on stiff surface. Scale bar, 10 µm. Cells from three independent cell cultures were analyzed. m Schematic showing the functional mechanism by which HLF loss decreases LPXN expression, enhancing the integration of collagen stiffness and the actin cytoskeleton via paxillin, ultimately potentiating cancer cell migration. Created in BioRender. Zhou, J. (2025) https://BioRender.com/x5scj3g. Data are mean ± s.e.m. (e, f, h, i, k, l), box plots show the median and interquartile range, and whiskers show the data range (b, c). One-way ANOVA followed by a post hoc Dunnett-t-test (c, e, f, h, I, k, l) or unpaired two-tailed t-test (b). Source data are provided as a Source Data file.