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. 2019 Mar 28;104(11):2164–2177. doi: 10.3324/haematol.2018.208660

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Figure 6. — Both Notch1 and Tie2 signaling are activated during endothelial niche recovery after chemotherapy. (A) Expression kinetics of Tie2 and Notch1 after chemotherapy. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) expression of Tie2 and Notch1 in sorted CD31⁺ primary bone endothelial cells (pBEC) from wildtype (WT) mice before (CTL) and at days 3, 5 and 7 after 5-fluorouracil (5-FU) treatment. GAPDH was used as an internal expression control at each time-point. (B) Representative western blot of protein level analysis for phosphorylated Tie2 (p-Tie2), total Tie2, cleaved Notch1 and GAPDH from sorted pBEC before (CTL) and at days 3, 5 and 7 after 5-FU treatment (left panel). The band intensities of Tie2 and p-Tie2 in pBEC were quantified by ImageJ software. The ratios of pTie2/Tie2 at days 3, 5 and 7 after 5-FU treatment were normalized to that of the control (CTL) group. The fold changes of pTie2/Tie2 ratio are presented (n=3, right panel). (C) Experimental design of 5-FU treatment for pBEC analysis (phosphate-buffered saline, PBS, n=6; 5-FU, n=15). Five-to six-week old mice were treated intraperitoneally (IP) with PBS or 5-FU. CD31⁺Ter119⁻CD45⁻cells (pBEC) were sorted from digested bones 5 days after 5-FU treatment. (D) RT-qPCR expression of indicated genes from pBEC treated as described in (C). Values are normalized to PBS-treated pBEC. GAPDH was used as an internal expression control. (E) Protein expression of Dll4 and Jag1 was measured in pBEC from mice treated with PBS or 5-FU. GAPDH was used as a loading control (left panel). The band intensities of Dll4 and Jag1 in pBEC were quantitated by ImageJ software (n=3, right panel). (F) Experimental design of 5-FU injection of Notch1^f/f;VE-CadherinCre^ERT2+ mice (left panel). Notch1^f/f;VE-CadherinCre^ERT2 mice were injected IP with tamoxifen daily for 5 days. One week later, 5-FU was injected IP into Notch1^f/f;VE-Cadherin Cr^eERT2⁺ or CreERT2⁻littermates. pBEC were analyzed at day 5 after 5-FU injection (n=3). (G) Expression of Tie2, Dll4 and Jag1 was measured by RT-qPCR in pBEC as described in (F). Absolute expression values were compared to those of PBS-injected mice of each genotype. GAPDH was used as an internal expression control.