Fig 1.
Skin fibroblasts expressing iafgp demonstrate increased cold resistance.
Full-thickness skin punches of iafgp-transgenic mice and wildtype controls (C57Bl/6) were cut into small pieces and stored at 4°C for up to 4 days before shifting them to 37°C to determine fibroblast survival inside the samples. Fibroblast migration and replication was monitored microscopically and 7 days (48h cold storage) or 14 days (72h and 96 h cold storage) later the number of attached cells in each sample was quantified after extensive washing using the Guava ViaCount. A: Absolute number of recovered cells was pooled from 3–4 independent experiments with 1–2 mice per group and timepoint (*: p<0.05, Student's T-Test). The detected cell quantity comprises fibroblasts that migrated from the skin punch and cells replicating after attachment. B: The ratio of iafgp-transgenic cells to controls was calculated for each independent fibroblast migration experiment and pooled per timepoint (*: p<0.05, Student's T-Test).
Fig 2.
Iafgp-transgenic mice are resistant to frostbite.
The tails of homozygous iafgp-transgenic mice were immersed into a -22°C liquid for 4 minutes. Tissue autoamputation following frostbite was determined 7 to 10 days later. A: Shown data were pooled from 9 independent experiments comprising up to two batches (n corresponds to the number of animals; Fisher's exact test, ***: p<0.0001). B: Representative tail images from wildtype (WT) and iafgp-transgenic (TG) mice at 24 hours, 72 hours and 7 days post thermal injury inflicted on the distal 5 cm of the murine tails. Tail injury from fighting (*) occurred sporadically at the tail base and these sites were excluded from evaluation. About 35% of the transgenic mice showed macroscopic changes consistent with thermal injury (***) but less severe than control mice. Scale bars = 1 cm. C: Representative histopathology images of tails from untreated controls,wildtype (WT) and iafgp-transgenic (TG) mice at 24 hours and 7 days post thermal injury. Complete loss of nuclei in skeletal muscle and bone with empty blood vessels (*) in the intervening connective tissue in WT tails and viable tissue or red blood cells in TG tails can be observed (**). TJ = tail joint numbered starting from the most distal joint. Tails are oriented proximal left and distal right. Scale bars = 500 μm.
Fig 3.
Iafgp-expression reduces inflammation.
Peritoneal macrophages (PMO) isolated from iafgp-transgenic mice and controls were stimulated ex vivo with E. coli LPS and cytokine production was quantified. A: In silico quantified cytokine array intensities of samples stimulated with 1 ng/ml LPS in comparison to unstimulated controls are shown for C57Bl/6 control mice and transgenic animals. The corresponding cytokine array raw data is depicted in S1 Fig. Overall cytokine release is significantly reduced between the two mouse lines (Two-Way-ANOVA; p = 0.003). This experiment was performed once. B: IL-6 - or C: TNF-α release determined by ELISA using PMOs stimulated with 1 ng/ml LPS. Shown data represents the average TNF-α concentration of three independent stimulations ± SEM.