2013, Wu, et al. using an imaging based assay to quantify the number of cells infected during any given experiment To assess the role of DDR proteins in MuPyV replication and assembly, cell lines were normalized for infection efficiency. A) Timeline of viral titer and viral DNA accumulation assays. B) Equation used to calculate the normalized viral titer and DNA accumulation between cell lines of variable infection levels. C) Infection levels for cell lines quantified by high throughput imaging of Tag staining. D) Raw values for viral titer, quantified by immunoplaque assay and viral DNA accumulation, quantified by qPCR. E) Calculated reported values for viral titer and viral DNA accumulation for each cell line, normalized for infection efficiency. NIHMS810611-supplement-2.eps (2.7M) GUID:?7FAB7E5C-89DF-415E-9E4D-1435F44DDBC5 3: Figure S3: Localization of DDR proteins in uninfected MEFs. MEFs were fixed, permeabilized and immunostained for A) RPA32 B) pSer23 RPA32 C) pCHK1 OGT2115 D) Mre11 E) pATM or F) H2AX. NIHMS810611-supplement-3.eps (8.8M) GUID:?C3EB760B-ADFA-47A0-A16A-DE80D97E5911 4: Figure S4: Mre11 is not degraded during MuPyV infection. A) MEFs were mock infected or infected with NG59RA. Total proteins were isolated at 24 and 32 HPI and analyzed by immunoblot for Mre11 levels. B) Quantification of integrated density of Mre11 immunoblot, normalized to actin loading control. All samples were normalized to the value of the mock treated cells. NIHMS810611-supplement-4.eps (1.2M) GUID:?44E5ABEC-6D32-4874-B450-547E4304E66F 5: Figure S5: Quantification of Mre11 at MuPyV replication centers and replicating cellular DNA. MEFs were mock infected or infected for 28 hrs, 10 M EdU was then added to the media. Samples were then fixed, permeabilized, and stained for EdU, Tag, and Mre11. 2.5 m z-stacks were acquired on a spinning disc confocal using a 100X objective, NA 1.45. A) Single z-plane confocal images show DAPI, Tag, EdU, and MRE11 in OGT2115 uninfected and infected cells. B) The region of interest (ROI) for total DNA was defined by DAPI staining. The ROI that marked the viral replication centers, or viral DNA, was defined by Tag staining. The ROI that marked cellular DNA in infected cells or cellular DNA (infected) was defined by subtracting the Tag ROI from the DAPI ROI. Uninfected cells were analyzed using the total DNA ROI, only. All ROIs used for quantification are shown in white. C) Merged image of the EdU (green) and Mre11 (red) showing localization in ROIs of an infected nucleus and an uninfected nucleus. D) Co-localization analysis showing the percent of EdU co-localized with Mre11 in infected cells in the total DNA region, viral DNA region and cellular DNA region and uninfected cells. NIHMS810611-supplement-5.eps (4.6M) GUID:?D2F41541-F813-454D-A44B-2F0AEEC82961 6: Figure S6: Genotype validation for control and null cell lines. A) CHK2 control and null cell lines were analyzed using PCR primers specific for WT and neomycin alleles. WT PCR primers amplified a 440 bp OGT2115 product for the WT allele and no product for the null allele. Neo primers amplified a 900 bp product for the null allele and no product for the WT ARF3 allele. B) H2AX control and null cell lines were analyzed using PCR primers specific for WT and null alleles. WT PCR primers amplified a 547 bp product for the WT allele and no product for the null allele. Null primers amplified a 424 bp product for the null allele and no product for the WT allele. NIHMS810611-supplement-6.eps (3.8M) GUID:?73AD9A0B-1BC9-470E-AED3-F3179E8FF40D 7: Figure S7: CHK1 autophosphorylation is inhibited by PF477736 treatment during infection. A) MEFs were mock infected or infected with NG59RA and at 2 HPI DMSO or 1 M PF477736 were added to the media. Total proteins were isolated at 28 HPI and analyzed by immunoblot for pSer296 CHK1 levels. B) Quantification of integrated density of pSer296 CHK1 levels in control and infected MEFs normalized to GAPDH loading control. All samples were normalized to the value of the uninfected control cells. NIHMS810611-supplement-7.eps (1.2M) GUID:?EBEA28D7-4E60-4781-9E06-5B087C256EBF 8: Figure S8: Cell viability and infection levels following treatment with ATR inhibitor (VE-821) A) MEFs were treated with DMSO or ATR inhibitor for 32 hrs, and cell viability was measured by propidium iodide uptake using high throughput microscopy. The data were normalized to DMSO control, with live cell counts shown in black and dead cell counts shown in gray. B) MEFs were infected with NG59RA. At 16 HPI DMSO or the ATR.
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