Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase- and Prevents DNA Damage during Replication

Supplemental Material

This article contains the following supporting material:

• Figure S1 - Mcm10 siRNA1 treated HeLa cells arrest in late S/G2 phase and show an increase in apoptosis. Cells treated with Mcm10-specific or control siRNA were labeled with BrdU for 22 h and harvested at indicated time points. The DNA was stained with 7-amino-actinomycin D (7-AAD) and DNA content was measured by flow cytometry.
• Figure S2 - Lamin depletion in HeLa cells does not cause defects in cell cycle progression. (A) Profiles of HeLa cells incorporating BrdU at various time intervals are shown. a-i show profiles of cells that were untreated (a-c), or treated with either control (d-f) or lamin (g-i) siRNA and then BrdU labeled for 22 h. Cells were harvested at the indicated time points for cell cycle analysis. (B) The fraction of cells (BrdU postive and BrdU negative) in various phases of the cell cycle was quantified based on DNA content and plotted.
• Figure S3 - Mcm10/p180 co-depleted cells undergo apoptosis but not p180-depleted cells. HeLa cells were untreated, lipofectamine treated or treated with Mcm10-, p180- or control siRNA as described previously and then stained with Annexin V and propidium iodide (PI) to measure apoptosis. These profiles were quantified by CellQuest Pro software. The fraction of cells undergoing early or late apoptosis or both are indicated for various treatments. A total of 25000 cells were sorted for each sample and then cells were quantified in early or late apoptosis based on their staining with Annexin V and PI. The threshold values for Annexin V or PI were set based on untreated samples stained with either Annexin V or PI only. A cartoon showing the threshold for early and late apoptosis is depicted in the top left corner of the figure.
• Figure S4 - p180 knockdown causes an increase in γ-H2AX foci 96 h post transfection. (A) Fluorescence microscopy was applied to detect γ-H2AX foci in HeLa cells treated with p180 siRNA or lamin siRNA 96 h post transfection. Quantification of the results is shown in Figure 3B.