Inescence (Pierce). Antibodies used include Prox1 (07-537, Upstate), VEGFR-2 (sc-504, Santa Cruz Biotechnology), and bactin (AC15, Sigma). Quantifying endothelial cell content by Title Loaded From File DilAc-LDL generated a ratio that related endothelial cell content within the mixed smooth muscle cell culture. This number 1326631 was then used to normalize Prox1 levels, derived by densitometry, within each experimental condition.VP16 Title Loaded From File expression and the developing lymph sacs. Tie2 tTA:tetOS prox1 E13.5 double transgenic mice display VP16 staining, a surrogate marker for driver expression, on the dorsal aorta and the jugular vein (arrows) but not on the lymph sac (arrowheads). This is in agreement with previous results from Srinvasin et al, who demonstrated using a tie2-Cre system that early LECs were Tie2 negative by way of in situ hybridization, immunohistochemical GFP and by FACS (Srinivasan et al., 2007). Scale bar = 50 mm. JV: jugular vein; DA: dorsal aorta; LS: lymph sac. (TIF)Figure S4 Figure S5 Prox1 expression on the jugular vein of E13.5 embryos. (A) Control E13.5 embryos display no Prox1 expression on the jugular vein. (B) In contrast, the jugular vein of Prox1 double transgenic embryos is Prox1 positive. Scale bar = 100 mm. JV: jugular vein; DA: dorsal aorta. (TIF) Figure S6 Expression of Prox1 on early dorsal aortas of wild type and double transgenic embryos. Our model suggests that the support cells associated with endothelial cells can regulate Prox1 expression. (A) We find early examples of Prox1 expression on the dorsal aorta of control E9.5 embryos that correlate with no SMA expression (arrowheads). Moreover, we also observe diminished Prox1 expression correlating with SMA expression (arrows). (B) On double transgenic E10.5 embryos we find examples of Prox1 expression that correlate with no SMA (arrowheads) as well as with SMA (arrows). Thus we believe that a continuum of Prox1 regulation likely exists that is influenced by SMCs over the developmental period of E9.5 to E11.5. Scale bar = 100 mm. DA: dorsal aorta. (TIF)Supporting InformationFigure S1 Overexpression of Prox1 results in theexpression of the lymphatic marker Podoplanin on the jugular vein. (A) Normally, the expression of Podoplanin (FITC) on the jugular vein is downregulated by E13.5 and upregulated on lymph sacs, along with Prox1 (arrowheads, Cy3). (B) Prox1 overexpression results in its’ expression on the jugular vein as well as the lymph sac. Furthermore, Podoplanin is now found expressed on the jugular vein (arrows). (C ) Single channel for Prox1 and Podoplanin. Scale bar = 25 mm. JV: jugular vein; LS: lymph sac. (TIF)Figure SAcknowledgmentsD.J.D. is the Canada Research Chair in Angiogenic and Lymphangiogenic Signaling. Many thanks to Dr. Paul Van Slyke for critical reading and valuable input.Author ContributionsConceived and designed the experiments: HK DJD. Performed the experiments: HK AB. Analyzed the data: HK DJD. Contributed reagents/ materials/analysis tools: MC. Wrote the paper: HK.Prox1 is not found on the dorsal aorta in DT embryos at E11.5. (A and B) Expression from E11.5 DT
Infectious disease diagnostics traditionally rely heavily on pathogen detection [1,2,3]. However, the development of reproducible means for extracting RNA from whole blood, coupled with advanced statistical methods for analysis of complex datasets, has created the possibility of classifying infections based on host gene expression profiling. We recently developed a robust whole blood mRNA expression cla.Inescence (Pierce). Antibodies used include Prox1 (07-537, Upstate), VEGFR-2 (sc-504, Santa Cruz Biotechnology), and bactin (AC15, Sigma). Quantifying endothelial cell content by DilAc-LDL generated a ratio that related endothelial cell content within the mixed smooth muscle cell culture. This number 1326631 was then used to normalize Prox1 levels, derived by densitometry, within each experimental condition.VP16 expression and the developing lymph sacs. Tie2 tTA:tetOS prox1 E13.5 double transgenic mice display VP16 staining, a surrogate marker for driver expression, on the dorsal aorta and the jugular vein (arrows) but not on the lymph sac (arrowheads). This is in agreement with previous results from Srinvasin et al, who demonstrated using a tie2-Cre system that early LECs were Tie2 negative by way of in situ hybridization, immunohistochemical GFP and by FACS (Srinivasan et al., 2007). Scale bar = 50 mm. JV: jugular vein; DA: dorsal aorta; LS: lymph sac. (TIF)Figure S4 Figure S5 Prox1 expression on the jugular vein of E13.5 embryos. (A) Control E13.5 embryos display no Prox1 expression on the jugular vein. (B) In contrast, the jugular vein of Prox1 double transgenic embryos is Prox1 positive. Scale bar = 100 mm. JV: jugular vein; DA: dorsal aorta. (TIF) Figure S6 Expression of Prox1 on early dorsal aortas of wild type and double transgenic embryos. Our model suggests that the support cells associated with endothelial cells can regulate Prox1 expression. (A) We find early examples of Prox1 expression on the dorsal aorta of control E9.5 embryos that correlate with no SMA expression (arrowheads). Moreover, we also observe diminished Prox1 expression correlating with SMA expression (arrows). (B) On double transgenic E10.5 embryos we find examples of Prox1 expression that correlate with no SMA (arrowheads) as well as with SMA (arrows). Thus we believe that a continuum of Prox1 regulation likely exists that is influenced by SMCs over the developmental period of E9.5 to E11.5. Scale bar = 100 mm. DA: dorsal aorta. (TIF)Supporting InformationFigure S1 Overexpression of Prox1 results in theexpression of the lymphatic marker Podoplanin on the jugular vein. (A) Normally, the expression of Podoplanin (FITC) on the jugular vein is downregulated by E13.5 and upregulated on lymph sacs, along with Prox1 (arrowheads, Cy3). (B) Prox1 overexpression results in its’ expression on the jugular vein as well as the lymph sac. Furthermore, Podoplanin is now found expressed on the jugular vein (arrows). (C ) Single channel for Prox1 and Podoplanin. Scale bar = 25 mm. JV: jugular vein; LS: lymph sac. (TIF)Figure SAcknowledgmentsD.J.D. is the Canada Research Chair in Angiogenic and Lymphangiogenic Signaling. Many thanks to Dr. Paul Van Slyke for critical reading and valuable input.Author ContributionsConceived and designed the experiments: HK DJD. Performed the experiments: HK AB. Analyzed the data: HK DJD. Contributed reagents/ materials/analysis tools: MC. Wrote the paper: HK.Prox1 is not found on the dorsal aorta in DT embryos at E11.5. (A and B) Expression from E11.5 DT
Infectious disease diagnostics traditionally rely heavily on pathogen detection [1,2,3]. However, the development of reproducible means for extracting RNA from whole blood, coupled with advanced statistical methods for analysis of complex datasets, has created the possibility of classifying infections based on host gene expression profiling. We recently developed a robust whole blood mRNA expression cla.