Fig. 2

Development and characterization of transgenic FS hESC. A FS hESC were genetically engineered with a piggyBac transposon to overexpress either luciferase or sCX3CL1, each with an mCherry fluorescent reporter. B Phase contrast and mCherry images of parental and transgenic FS subclones that express luciferase (FS-Luc) or sCX3CL1 (FS-CX3-3, CX3-5, CX3-9) were derived, scale is 65 µm. C In vitro bioluminescence imaging assay of FS and luciferase-expressing FS-Luc. All comparisons are significant, ****p < 0.0001, unless stated otherwise (n = 3 biological replicates, each consisting of 3 technical replicates). D RT-qPCR analysis of sCX3CL1 RNA expression relative to housekeeping gene, YWHAZ (n = 4 biological replicates, each consisting of 3 technical replicates). Gene expression normalized to GAPDH. (E) Western blotting analysis of hESC conditioned media—two different biological replicates of media from each FS-CX3 clone were loaded. sCX3CL1 was identified by C-terminal 8×-His tag (n = 2–3). Full length western blot represented in Supplementary Fig. 1. One-way ANOVA followed by Tukey’s post-hoc analysis was performed on data in panels C and E. Error bars represent SD. nd, not detected; ns, not significant