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. 2021 Jun 8;12:3460. doi: 10.1038/s41467-021-23546-6

Fig. 7. Diphtheria toxin induces experimental secondary lymphedema development in Flt4-CreERT2; iDTRfl/fl mice.

Fig. 7

a Monitoring paw thickness and paw clinical score in tamoxifen-treated Flt4-CreERT2; iDTRfl/fl mice after treatment with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM for paw thickness and median and IQR for clinical score from 4–18 mouse paws in each group (two-tailed, paired T-test for paw thickness P = 4.21 × 10−12 on day 8 for 16 mice, P = 5.05 × 10−6 on day 16 for 14 mice, P = 1.62 × 10−5 on day 30 for 18 mice, P = 0.0008 on day 60 for 13 mice, and P = 0.0112 on day 75 for 4 mice. Two-tailed Wilcoxon signed-rank test for clinical score P = 3.05 × 10−5 on day 8 for 16 mice, P = 0.0002 on day 16 for 14 mice, P = 6.10 × 10−5 on day 30 for 18 mice, P = 0.0020 on day 60 for 13 mice, and P > 0.9999 on day 75 for 4 mice). b Haematoxylin and Eosin staining of the paws of Flt4-CreERT2; iDTRfl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 5 mouse paws per group (bars, 200 µm). c Paw fibroadipose area of tamoxifen-treated Flt4-CreERT2; iDTRfl/fl mice 30 and 75 days after intradermal paw injection with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM from Haematoxylin and Eosin stained slides of 3–4 mouse paws in each group (two-tailed, paired T-test, P = 0.0469 on day 30 for 4 mice and P = 0.0172 on day 75 for 3 mice). d Representative images of anti-LYVE1 and anti-Podoplanin immunostaining of the paws of Flt4-CreERT2; iDTRfl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 4–5 mouse paws in each group (bars, 50 µm). Arrows indicate LYVE1 and Podoplanin double positive lymphatic vessels. e Number of lymphatic vessels of tamoxifen-treated Flt4-CreERT2; iDTRfl/fl mice 30 and 75 days after intradermal paw injection with PBS or Diphtheria toxin. Quantitative data are represented as mean and SEM from 4–5 mouse paws in each group (two-tailed, paired T-test, P = 0.0319 after 30 days for 4 mice and P = 0.0434 after 75 days for 5 mice). f Representative images of anti-Collagen I immunostaining of the paws of Flt4-CreERT2; iDTRfl/fl tamoxifen-treated mice 30 and 75 days after treatment with PBS or Diphtheria toxin. Representative images are shown of 5 mouse paws in each group (bars, 50 µm). Asterisks indicate P < 0.05 compared with control. All cell nuclei are labeled with DAPI (blue) in paraffin-embedded tissues.