Fig 2: GABPa regulates DNA replication and cell cycle progression. a Naive (CD62Lhi CD44lo) CD4+ or CD8+ T cells from Gabpa f/f (WT) and CD4 Cre Gabpa f/f (KO) mice were purified by flow cytometric sorting, and were subjected to anti-CD3 and anti-CD28 stimulation in the presence of IL-2. After 22 h, cells were pulsed with EdU for 2 h. Flow cytometric analysis of DAPI and EdU was shown. b GABPa-bound regions in the Mcm3 and Mcm5 gene loci. Gene structure, chromosomal location, and sequence homology were shown below the binding peaks. The Y-axis represent read per million (RPM). c Alignment of the conserved GABPa-binding sites in mouse and human Mcm3 and Mcm5 proximal promoter regions. The consensus GABPa-binding sequences were marked in red, and nucleotides were numbered relative to the transcription start site. d ChIP-qPCR validation of GABPa binding to the promoter regions of Mcm3 and Mcm5 genes in WT CD4+ T cells and CD8+ T cells. IgG was used as a control for the GABPa antibody. Hprt1 locus was used as a negative control. Bars are mean ± SEM with unpaired t-test, ns = not significant, *P < 0.05, **P < 0.01. e Naive CD4+ or CD8+ T cells from WT and KO mice were purified by flow cytometric sorting, and were stimulated with CD3 and CD28 antibodies in the presence of IL-2 for 0, 12, or 24 h. Immunoblotting analysis of Mcm3, Mcm5, CDK6, and Cyclin D2 was shown. GADPH was used as a loading control. The relative amounts of proteins were normalized to the 0-h time point of each individual group. Uncropped western blotting images were included in Supplementary Fig. 10

Fig 3: pSTAT5, but not NS1, interacts with the MCM complex.(A) Immunoprecipitation (IP) assay. Cell lysates of NS1Flag-expressing UT7/Epo-S1 cells were prepared for pull-down assays with either anti-Flag-conjugated beads or control beads. Immunoprecipitated proteins were examined for the presence of MCM2 by Western blotting. Blots were reprobed with rabbit anti-pSTAT5(Y694), anti-E2F5, and anti-Flag antibodies. Detection of E2F5 was used as a positive control for NS1 IP. (B) Co-IP assay. UT7/Epo-S1 cells were collected, washed, and lysed with RIPA buffer. After centrifugation, the supernatant was incubated with either rabbit anti-pSTAT5(Y694) or control IgG antibody. Immunoprecipitated proteins were blotted for the presence of the MCM complex with an anti-MCM5 antibody and for pSTAT5 with rabbit anti-pSTAT5(Y694). (C) Reverse Co-IP assay. Reverse Co-IP was performed with an anti-MCM5 antibody. Immunoprecipitated proteins were examined for pSTAT5, MCM2, and MCM5, respectively. (D) Co-IP of lysates treated with DNase. UT7/Epo-S1 cell lysates, either treated or untreated with DNase (750 units of Benzonase) were incubated with anti-pSTAT5(Y694) or control IgG antibodies for Co-IP assay, and immunoprecipitated proteins were examined for MCM2 by Western blot analysis. (E-H) Immunofluorescence analysis. (E&F) Mock- or B19V-infected CD36+ EPCs were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies, followed by (E) incubation with respective secondary antibodies, or by (F) proximal ligation assay, which produces amplified signal for labeled molecules in close proximity. (G) CD36+ EPCs were incubated with either DMSO or pimozide (at 30 µM) for 2 days. And then the cells were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies for proximity ligation assay. (H) Infected EPCs were stained with an anti-capsid antibody. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.