Fig 1: Tubular structures contain VP1.PyV-infected 3T3 cells or PML−/− MEFs (MOI of 10–20 pfu/cell) at 32 hpi were frozen by high pressure and processed by cryo-substitution for immunoelectron microscopy. Thin sections (45 nm or 70 nm) of Lowicryl-embedded samples were stained with either anti-PML or anti-VP1 antibodies followed by a secondary antibody conjugated to 10 or 15 nm colloidal gold. Top panels: 45 nm sections stained for the PML protein; white arrows, anti-PML staining, black arrow, tubular structures. Bottom panels: 70 nm sections stained for VP1; white arrows, anti-VP1 staining, black arrows, tubular structures.
Fig 2: PyV DNA and T-antigen localization in PyV-infected PML−/− MEFs.PML−/− MEFs were infected with PyV at an MOI of 30–40 pfu/cell. At 22 or 24 hpi cells were fixed, permeabilized, and co-stained with either anti-Tag and/or anti-MRE11a antibodies followed by AlexaFluor-conjugated secondary antibodies, a fluorescently-labeled PyV DNA FISH probe, and DAPI staining of nuclei. A) FISH for PyV DNA at 24 hpi followed by antibody staining for Tag. B) Infected cells were stained by FISH for PyV DNA at 22 hpi followed by antibody staining for MRE11 or co-stained for MRE11 and Tag. All images represent a 0.1 mm z-stack slice.
Fig 3: Dach1 Gene Deletion Reduces the Abundance of Mammary Gland Stem Cell and Ductal Cell Lineage Gene Targets(A) Immunofluorescent double staining for cytokeratin 5 (CK5) and CK8 showed that Dach1 gene deletion decreases the relative amount of CK5-positive cells versus CK8-positive cells.(B) Immunohistochemical staining for differentiation factors (Gata3, Pml, and Pygo2) in the mammary gland of Dach1+/+ versus Dach1−/− (Cre deletion mice) (Dach1wt/wtROSA26CreERT2/mTmGfl versus Dach1fl/flROSA26CreERT2/mTmGfl). The red arrowheads indicate basal epithelial cells.(C) Quantitation of immunohistochemical staining shown as mean ± SEM for n = 3 separate mice.
Fig 4: PyV DNA and T-antigen are localized near PML-NBs.C57 MEFs were infected at an MOI of 30–40 pfu/cell. At 24 or 28 hpi cells were fixed, permeabilized, and co-stained with either anti-PML, anti-Tag and/or anti-MRE11 antibodies followed by AlexaFluor-conjugated secondary antibodies, a fluorescently-labeled PyV DNA FISH probe, and DAPI staining of nuclei. A) PyV-infected C57 MEFs at 24 hpi stained for either PML (top) or Tag (bottom) followed by fluorescent in situ hybridization (FISH) for PyV DNA. B) PyV-infected C57 MEFs at 28 hpi co-stained for PML and Tag. C) C57 MEFs at 24 hpi co-stained for either MRE11 and PyV DNA (by FISH) or MRE11 and Tag. All images represent a 0.1 mm z-stack slice. Insets show enlarged regions from image to illustrate the localization of proteins in relation to each other (B) or PyV DNA (C).
Fig 5: DACH1 Occupies the Promoter Regions of Mammary Gland Stem Cell and Ductal Cell Lineage Gene Targets(A–F) qRT-PCR analysis of mRNA for genes participating in mammary gland stem cell and ductal development, (A) Dach1, (B) Gata3, (C) Elf5, (D)Pml, (E) Notch1, and (F) Pygo2, in the mammary gland of Cre deletion mice (Dach1wt/wtROSA26CreERT2/mTmGfl versus Dach1fl/flROSA26CreERT2/mTmGfl) with quantitation shown as mean ± SEM for n = 3 separate mice. Notch1 mRNA levels were modestly reduced in the Dach1−/− mammary gland.(G–L) Analysis of DACH1 binding to these target genes was assessed using chromatin immunoprecipitation sequencing assays conducted on DACH1 stable MDA-MB-231 cells (G). (H) Gata3, (I) Elf5, (J) Pml, (K) Notch1, and (L) Pygo2.
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