Fig. 1

The doxycycline-inducible IKFM model of NF-κB activation in macrophages. a Schematic of generation of IKFM double transgenics. Mice harboring an rtTA transgene gene under the control of a macrophage-specific c-fms promoter (FMR) are crossed with mice harboring a FLAG-cIKK2 transgene under the control of a doxycycline-inducible rtTA-responsive Tet operon (IKK). b Representative DNA genotyping gels for double transgenic IKFM mice (FLAG-cIKK2 and c-fms-rtTA positive) mice and control mice (c-fms-rtTA positive only). IKFM and control mice injected with tumor cells were treated with 1 g/L dox using schedules for treating established TBR5 and ID8-Luc tumors as defined in Methods. FLAG-cIKK2 transgene expression was confirmed in IKFM macrophages by c qRT-PCR analysis of mRNA expression from TBR5 ascites cellular fraction in FVB IKFM mice. Equal loading was confirmed by corresponding GAPDH levels. d Western blot analysis of cytoplasmic FLAG and nuclear p65 protein levels in macrophages isolated from ID8-Luc ascitic fluid from C57Bl/6 IKFM mice. Equal loading was confirmed by probing for cytoplasmic α-tubulin and nuclear histone H3. Brightness was reduced uniformly across the gel image to increase visualization of faint bands. e Representative images from immunofluorescence analysis of TBR5 omental tumor tissue from FVB IKFM mice showing tumor-infiltrating macrophages; nuclei (blue); F4/80 (green); FLAG-IKK2 (red). Figure 1a was created by the authors with BioRender.com and Fig. 1b-e were generated with Microsoft PowerPoint 2016 (Version 16.16.26) and Adobe Photoshop CC 2018 (Version 19.0.1)