(A) RT-PCR expression analysis of indicated Wnt and GAPDH mRNAs in Population Doubling (PD)31 or PD58 WI38 cells, or PD31 cells infected with Ras or control retrovirus. (B) WI38 cells infected with control (young) or Ras (senescent) retrovirus and stained with DAPI to visualize DNA and SAHF and with antibodies to GSK3β. (C) Cell extracts from (B) were immunoprecipitated with indicated antibodies and then western blotted to detect GSK3β. (D) Cell extracts from (B) were immunoprecipitated with indicated antibodies and then incubated with Mg²⁺ATPγ³²P. (E) Immunoprecipitates from (D) were incubated with the substrate phosphopeptide YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE and Mg²⁺ATPγ³²P. Mean of 4 experiments with standard deviation (SD). (F) Soluble extracts from WI38 cells, PD35 or PD58, were western blotted with antibodies to β-catenin.

(A) WI38 cells were passaged from PD33 to PD49 and assayed for the number of cells with HIRA localized to PML bodies by immunofluorescence and Wnt2 and GAPDH mRNAs by RT-PCR. (B) WI38 cells were infected with Ras or control retrovirus, selected in puromycin, total RNA prepared at indicated days after infection and mRNA abundance determined by RT-PCR. (C) WI38 cells were infected with SV40 large T-antigen or control retrovirus, selected and passaged in neomycin, total RNA prepared and mRNA abundance determined at indicated PD after infection. (D) Western analysis of SV40 large T-antigen in cells from (C). (E) Images of cells from (C). Percent of cells with SAHF in each case (scored by epifluorescence of DAPI stained cells) is indicated. Control cells at PD #12 are senescent. SV40 Tag-expressing cells at PD #18 are in crisis.

(A) Primary WI38 cells were treated without or with 50mM LiCl or 30μM kenpaullone and scored for HIRA in PML nuclear bodies. Mean of 3 experiments with SD. (B) Primary WI38 cells were infected with indicated retroviruses and then stained with antibodies to HIRA and with DAPI. (C) WI38 cells were infected with retroviruses encoding GSK3βS9A, Myc-HIRA(520–1017) (HIRA-C) or control. 8 days later cells were stained with DAPI and antibodies to HIRA. Monoclonal antibody, WC119, used to stain HIRA does not recognize HIRA-C. (D) Quantitation of results from (C). Mean of 3 independent experiments with SD.

(A) U2OS cells were transfected with plasmids encoding HA-GSK3β or Myc-HIRA (wild type, full length), extracts prepared and immunoprecipitated and then western blotted with anti-HA or anti-Myc antibodies. (B) Purified recombinant GST or GST fused to residues 421-729 of HIRA (GST-HIRA(421-729)) was incubated with purified GSK3β or buffer. Bound proteins were isolated on glutathione beads and western blotted to detect HIRA and GSK3β. (C) Purified recombinant GST-HIRA(421-729)WT or GST-HIRA(421-729)ΔP was incubated with purified GSK3β and Mg²⁺ATPγ³²P. (D) Purified, recombinant variants of GST-HIRA(421-729) were incubated in absence or presence of purified GSK3β and Mg²⁺ATPγ³²P. (E) Purified GSK3β was incubated in absence or presence of a S697 substrate phosphopeptide (RRTLQVSSDPpSMYI) and Mg²⁺ATPγ³²P. (F) WI38 cells were infected with control or HA-HIRA(421-729)WT retrovirus, extracts immunoprecipitated with anti-HIRA antibodies and treated with or without λ-phosphatase. (G) WI38 cells were infected with control, HA-HIRA(421-729)WT or HA-HIRA(421-729)ΔP retrovirus, cells were metabolically labeled with ³³P-orthophosphate and lysates immunoprecipitated with anti-HA antibodies. (H) WI38 cells were infected with control or indicated HIRA retroviruses and western blotted with a phosphospecific antibody to phosphoS697 of HIRA. (I) WI38 cells were infected with HA-HIRA(421-729) retrovirus in absence or presence of a GID or control virus. 7 days later, lysates were prepared and western blotted with anti-phosphoS697HIRA.

(A) WI38 cells were infected with HA-HIRA(421-729)WT, HA-HIRA(421-729)S697A, HA-HIRA(421-729)S511A, S515A or control retroviruses. 8 days later cells were pre-extracted in buffer containing NP40, fixed and stained with anti-HA and anti-PML antibodies. (B) Quantitation of (A). Mean of 3 independent experiments with SD. (C) Cell extracts from (A) were immunoprecipitated and western blotted with anti-HA antibodies and anti-ASF1a antibodies.

(A) Primary WI38 cells were infected with lentiviruses encoding 5 different shRNAs to Wnt2, or a control lentivirus. The cells were selected in puromycin and 4 days after later Wnt2 and GAPDH mRNAs were assayed by RT-PCR. (B) 10 days after infection the cells from (A) were stained with antibodies to HIRA and with DAPI. Bars are color coded as in (A). Mean of 3 independent experiments with SD. (C) Images of the cells from (B). (D) Cells from (B) (Control and shWnt2–5) were stained for SA β-gal activity. (E) Cells from (D) were stained with DAPI and antibodies to macroH2A1.2.

(A) Primary WI38 cells were grown from PD#43 to PD#63 in control L-medium, Wnt3a-conditioned medium or standard medium (none) and scored for localization of HIRA to PML nuclear bodies and formation of SAHF. (B) WI38 cells were cultured under standard conditions, in Wnt3a-conditioned medium (Wnt3a-CM) or 50ng/ml recombinant Wnt3a (rWnt3a) and scored for localization of HIRA to PML bodies after 48 hours. Mean of 3 independent experiments with SD. (C) Primary WI38 cells were infected with a Ras retrovirus, grown in control L-medium or Wnt3a-conditioned medium, labeled with 5′-BrdU for 24 hours and assayed for 5′-BrdU positive cells by FACS, 8 days after virus infection. Shown are histograms of 5′-BrdU positive cells and the percent 5′-BrdU positive cells is indicated. (D) As (C), but the cells were labeled with 5′-BrdU for 24 hours or 1 hour (results for the 24 hour pulse are the same results as in (C)). Mock are uninfected cells in standard growth medium. (E) As (C), but the Ras-infected cells were grown in the absence or presence of Wnt3a-conditioned medium (Wnt3a-CM) or 75ng/ml recombinant Wnt3a (rWnt3a). (F) Human RPE cells were infected with a control or Ras retrovirus or a virus and then grown under standard conditions, in control L-medium or Wnt3a-conditioned medium for 8 days. (G) As (F), but the cells were pulse-labeled with 5′-BrdU for 1 hr and the percent 5-BrdU positive cells determine by FACS.