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. 2016 Jul;186(7):1825–1836. doi: 10.1016/j.ajpath.2016.03.009

Figure 3.

Figure 3

Expression of LEC markers in AML cells with or without TSC2 correction supports an LEC lineage derivation. A: Western blot showing expression of LEC and LEC precursor markers PROX1, COUP-TFII, SOX18, LYVE1, VEGFR-3, podoplanin, and CD34 in both TSC2− and TSC2+ cells with increased expression in TSC2+ cells for all the markers, except peripheral LEC/vascular endothelial precursor cell marker CD34, which is decreased. Peripheral LEC/vascular endothelial precursor cell marker CD133 is negative in both cell lines. GAPDH detection indicates equal loading. Histograms show density of Western blot signals in TSC2− cells relative to TSC2+ cells, as analyzed by ImageJ software version 1.47 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). B: Western blot showing absence of VEC marker CD31 and VEC precursor marker VEGFR-2. C: Real-time PCR revealing increased PROX1, SOX18, LYVE1, and podoplanin mRNA levels in TSC2+ cells along with decreased VEGFR-3 mRNA. Real-time PCR showing no difference in COUP-TFII and CD34 mRNA levels. D: Luciferase reporter assays consistent with TSC2 transcription regulation of Lyve1 (control), additionally suggest that the increase in Vegfr-3 expression in TSC2+ cells occurs posttranscriptionally. Real-time PCR data are expressed as means ± SD. ∗P < 0.05, ∗∗P < 0.01, by t-test. AML, angiomyolipoma; COUP-TFII, COUP transcription factor 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LEC, lymphatic endothelial cell; LYVE1, lymphatic vessel endothelial hyaluronan receptor-1; PROX1, prospero homeobox-1; SOX18, SRY-related HMG-box-18; TSC2, tuberous sclerosis complex-2; VEC, vascular endothelial cell; VEGFR, vascular endothelial growth factor receptor.