Fig. 2: In vitro and in vivo effects of pemetrexed on human UC based on MTAP protein status.

a Western blot verification of MTAP protein loss in four MTAP^def vs four MTAP^prof human UC cell lines paired with UHPLC-ESI/triple quadrupole mass spectrometry measuring MTA concentration in cell media of human UC cell lines (ng/mL). MTA levels are significantly higher in MTAP^def compared to MTAP^prof cell lines P value was calculated by Welch T test. b Sub-G1 analysis of MTAP^def and MTAP^prof cell lines when treated with 0.5 μM and 20 μM of pemetrexed (PEM). P value was calculated by Mann-Whitney test. c Cell viability after treatment with increasing concentrations of pemetrexed is significantly higher in MTAP^prof compared to MTAP^def cell lines P value was calculated by two-way ANOVA. d, e Xenograft tumor volume in HT-1376 and UM-UC-3 when treated with pemetrexed vs solvent. P value was calculated by two-way ANOVA. f Western blot and UHPLC-ESI/triple quadrupole mass spectrometry confirmation of MTAP knockdown in two HT-1376 cell lines: shMTAP2 and shMTAP3. g Pemetrexed resulted in significantly lower viability in the transfected HT1376 compared to the parental cell lines, with or without transduction of the lentivirus control vector. P value was calculated by two-way ANOVA. Data in a (bottom panel), b–e, f (bottom panel), and g are presented as mean + /− SD.