Fig 1: Intracellular CDA protects cells from the DHODHi+cytidine combination.A HCC44 or NCIH358 NSCLC cells were treated as indicated and analyzed by the IncuCyte method. B Effect of the Bay2402234+cytidine combination on clonogenic assays with NCIH358 cells. C–E CDA-GFP expression protects IMR32, NCIH2030 and NCIH82 cells from the DHODHi+cytidine combination. Cells were treated as indicated and cell growth (confluency) was measured by the IncuCyte method. A western blot demonstrating the expression of CDA-GFP is shown using an antibody against CDA.
Fig 2: Human serum allows low-CDA expressing cancer cells to proliferate in the presence of the DHODHi+cytidine combination.IMR32, NCIH82 and NCIH2030 cells were grown in medium containing FBS or HS and the indicated compounds or recombinant human CDA. The effect of the treatment was measured by the IncuCyte system.
Fig 3: Identification of cancers with alterations in the expression of pyrimidine nucleotide metabolism enzymes.A Left: CDA mRNA expression levels in cell lines (Depmap portal). Right: CDA mRNA expression levels in SCLC and NSCLC cell lines (Depmap portal). B, C CDA protein expression analyzed by Western blots in neuroblastoma cell lines B and lung cancer cell lines C. See Figure S15 for full size blots. D CDA protein levels in non-activated and activated (48 h) T cells treated as indicated. See Figure S15 for full size blot. E Left: CDA expression levels in tumors (R2 Genomics Analysis and Visualization Platform). Number of samples in each study is indicated. Studies on neuroblastoma, lung cancer and AML containing more than 50 tumor samples are included. The denBoer data on childhood AML is highlighted in green. Right: Analysis of the Plamadeala data on NSCLC shows that high risk tumors have lower levels of CDA expression.
Fig 4: Supplementation with cytidine sensitizes low CDA expressing cells to DHODH inhibitors.A, B IMR32 (neuroblastoma), NCIH82 (SCLC) or NCIH2030 (NSCLC) cells were treated as indicated and cell growth (confluency) and death (YOYO-3 red/confluency) were measured by the IncuCyte method (See Materials and Methods). C Effect of the Bay2402234+cytidine combination on clonogenic assays with NCIH82 cells. D NCIH82 cells were grown as spheroids and treated as specified. Green colour indicates caspase3/7 activity.
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