MMR-deficient cancer cells are sensitive to cytosine-based nucleoside analogs 3
Supplementary Figure Legends
Figure S1. Duration of exposure to cytarabine modulates extent of MLH1-deficient selectivity.
Bar graphs of comparative cellular viability of HCT116+/-Chr3 cells following 50 nM or 100 nM of cytarabine versus control after (a) 24 hours of treatment or (b) 72 hours of treatment. All assay conditions were repeated in triplicate or quadruplicate. Error bars represent the SEM.
Figure S2. Silencing of MLH1 expression modulates sensitivity to cytarabine.
Survival curves from cell viability assays of HCT116 and HCT116+Chr3 cells transfected with non-targeting control siRNA or siRNA targeting MLH1, and continuously exposed to various concentrations of cytarabine, versus control, 48 hours after transfection. Assays were performed in triplicate or quadruplicate. Error bars represent the standard error of the mean.
Figure S3. MLH1-deficient cells are selectively sensitive to other cytosine-based nucleoside analogs in addition to cytarabine.
Survival curves from cellular viability assays of the effects of a) azacytidine and b) cyclocytidine on HCT116 and HCT116+Chr3 cells. Cells were treated continuously with either nucleoside analog or control 24 hours after seeding. Cellular viability was assessed after six days using the CellTiter-Glo® luminescent assay, and surviving fractions calculated. Assay conditions were repeated in triplicate or quadruplicate. Error bars represent the SEM.
Figure S4. Exposure of MLH1-deficient cells to cytarabine is associated with increased gH2AX focus formation.
Merged images of cytarabine treated HCT116 cells (top panels) and HCT116+Chr3 cells (bottom panels) at 24 hours and 72 hours after start of treatment, and cells exposed to 8Gy of ionising radiation (positive controls). Cells were stained for gH2AX (red) and DAPI (blue).
Figure S5. Inhibition of POLA is not synthetically lethal with MLH1 deficiency
Bar graphs of surviving fractions following inhibition of POLA and POLA2 with siRNA in HCT116+/-Chr3 cells. Cells were transfected with siRNA and cell viability assessed using a luminescent assay after six days. Assay conditions were repeated in triplicate or quadruplicate. Surviving fractions were calculated using non-targeting siRNA as a negative control.
Figure S6. Cytarabine exposure leads to an increase in mitochondrial oxidative damage.
Levels of 8-oxodG in nuclear and mitochondrial fractions of DNA extracted from HCT116 and HCT116+Chr3 cells following 72 hours of treatment with cytarabine or control. Cells were plated, exposed to cytarabine, and mitochondrial and nuclear DNA fractions isolated using a commercially available kit. DNA was quantified and DNA concentrations normalised for an ELISA assay performed according to manufacturer’s instructions. 8-oxodG was quantified against a standard curve. Data shown is from a representative experiment, performed in duplicate.
Figure S7. Cytarabine exposure leads to a loss of mitochondrial membrane potential in colorectal cancer cells, in a greater proportion in MLH1-deficiency
a-d) Composite bar graphs characterising HCT116 and HCT116+Chr3 cell populations sorted by FACS for positivity with Annexin V and Mitotracker stains. Cells were analysed at various time points following treatment with cytarabine. Assays were performed in duplicate or triplicate. Error bars represent the SEM.
Figure S8. Proposed mechanism for the selective sensitivity of MLH1-deficient cells to cytarabine.
Exposure of MLH1-deficient and MLH1-proficient cancer cells to cytarabine results in the destabilisation of mitochondrial permeability transition and the generation of ROS. This is likely to be initially countered by secondary upregulation of anti-oxidant defences if they are available, but if these fail in MLH1-deficient cells, may also trigger apoptosis. In MLH1-deficient cells, the unrepaired accumulation of oxidatively damaged DNA also results in an accumulation of oxidative stress and the increased potential for lethal DSB formation which triggers apoptosis, whereas in MLH1-proficient cells oxidatively damaged DNA is repaired, and the cell remains viable.