Figure 2. Cell-specific RNA metabolic labeling in mouse xenograft WT and (+)-3xUPRT LM2 primary tumors.

3-week xenografted mice were IP-injected with 500 mM of each indicated substance for 3 hours in biological triplicates for uracil analog treatments. a. RNA was extracted from tumors, reacted with biotin-N₃ with ethynyl-RNA and biotin-tetrazine with vinyl-RNA prior to dot blot analysis. b. ImageJ quantification of chemiluminescence was used to calculate signal-to-noise ratios. Statistical significance relative to WT signal was determined using a one-tailed Student’s t test indicated as follows: P<0.01; **. 5-eu= 5-ethynyluracil, 5-euD= 5-ethynyluridine, 5-vu= 5-vinyluracil, 5-vuD=5-vinyluridine. c. Streptavidin bead enrichment of biotin-RNA:cDNA for qPCR analysis. Biotinylated RNA was reverse transcribed to make intact RNA:cDNA which were subsequently enriched with streptavidin beads, eluted by RNase hydrolysis and quantified with qPCR. Fold enrichment was determined through 2^-dCT standardized to the untreated mouse for vimentin and GFP labeled RNA. Statistical significance relative to enrichment from untreated mice was determined using a one-tailed Student’s t test indicated as follows: P<0.1; *. WT=wild-type, UPRT=3xUPRT, 5-vu=5-vinyluracil, 5vuD=5-vinyluridine. d. LC-MS/MS for analysis of vinyl-U modification from tumors and metastatic lungs. Purified RNA was analyzed for % of vinyl-U substation of total U normalized to untreated mouse tissue. 3-week xenograft mouse lungs did not produce metastases and were not included in the analysis. Biological triplicates were used in this data set. Statistical significance relative to WT tumors and metastases was determined using a one-tailed Student’s t test indicated as follows: P<0.05; **, P<0.5 *. WT=wild-type, UPRT=3xUPRT, 5-vu=5-vinyluracil, 5vuD=5-vinyluridine, ND=not detected.