EMBO/soda-nel huggingface dataset:
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 4

Figure 4. PTPN2-deletion enhances CAR-T cell efficacy in vivo.

a, b) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl or Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.

c-d) TILs isolated from HER-2-E0771 mammary tumour versus lymphocytes isolated from the contralateral mammary fat pad were analysed for c) CD11b+ F4/80hiLy6ChiLy6Glo myeloid derived suppressor cells (MDSC) and d) CD4+CD25+FoxP3+ regulatory T cells (Tregs) by flow cytometry.

e) Tgfb and Il10 mRNA levels in HER-2-E0771 tumours and mammary fat pads were assessed by quantitative real time PCR.

f, g) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. f) 13 or g) six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with f) IL-2 (50,000 IU/day) or g) saline on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth.

Data information: Representative flow cytometry profiles and results (means ± SEM) from three independent experiments are shown. In (a, b, f, g) significance was determined using 2-way ANOVA Test. In (c-e) significance was determined using 2-tailed Mann-Whitney U Test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 5

Figure 5. PTPN2-deficiency prevents the re-emergence of tumours.

a) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified Lck-Cre;Ptpn2fl/fl CD8+CD44hiCD62Lhi central memory HER-2 CAR-T cells. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for survival.

b) HER-2-E0771 tumours at day 10 post adoptive CAR-T cell transfer or from HER-2-E0771 tumours that had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analyzed for HER-2 expression by immunohistochemistry. Scale bars: 200 μm (full size) and 70 μm (zoom).

c) Normal mammary tissue or HER-2-E0771 tumours at day 10 post-adoptive CAR-T cell transfer, or from those had re-emerged after being cleared by Lck-Cre;Ptpn2fl/fl CD8+ HER-2 CAR-T cells were analysed for HER-2 expression by quantitative PCR.

d-g) HER-2-E0771 versus HER-2 negative E0771 breast cancer cells (2x105) were injected into the contralateral fourth inguinal mammary fat pads of female HER-2 TG mice 50 days after adoptive CAR-T cell transfer and mice were monitored for tumour growth. e) At day 28 the number of CD45+CD3+CD8+ mCherry+ CAR-T cells in HER-2-E0771 versus E0771 tumours and dLNs were determined by flow cytometry.

f-g) Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells isolated from the spleens of HER-2 TG mice at f) 70 days and g) 10 days post adoptive transfer were stained for CD8, CD44 and CD62L and analysed by flow cytometry.

Data information: Representative flow cytometry profiles and results (means ± SEM) from three independent experiments are shown. In (a) significance was determined using Log-rank (Mantel-Cox) test. In (d) significance was determined using 2-way ANOVA Test. In (c, e-g) significance was determined using 2-tailed Mann-Whitney U Test. *p<0.05, **p<0.01, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 6

Figure 6. PTPN2-deficiency enhances CXCR3 expression and promotes CAR-T cell homing.

HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer.

a) Lymphocytes were isolated from the tumours and dLN at day 10 post adoptive transfer and mCherry+CD45+CD3+CD8+ CAR-T cell numbers were determined by flow cytometry.

b) CTV-labelled CD8+ HER-2 CAR-T cells were incubated with 24JK-HER-2 or 24JK sarcoma cells and CTV dilution assessed by flow cytometry to monitor proliferation.

c, d) CXCR3 MFIs on CD8+CD44hiCD62Lhi versus CD8+CD44hiCD62Llo CAR-T cells (c) and CXCR5, CCR7, CCR5 MFIs on CD8+CD44hiCD62Lhi CAR-T cells (d) were determined by flow cytometry.

e) Cxcl9 and Cxcl10 mRNA levels in HER-2-E0771 tumours were assessed by quantitative real time PCR.

f) Lymphocytes were isolated from HER-2-E0771 tumours at day 3 post adoptive CAR-T cell transfer and mCherry+CD45+CD3+CD8+ CAR-T cell numbers were determined by flow cytometry.

g) Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Lck+/- or Lck-Cre;Ptpn2fl/fl;Stat5fl/+ HER-2 CAR-T cells were incubated with plate-bound α-CD3 and CXCR3 MFIs on CD8+CD44hiCD62Llo CAR-T cells determined by flow cytometry.

h) Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl;Stat5fl/+ HER-2 CAR-T cells were incubated with plate-bound α-CD3 and stimulated with murine recombinant IL-2 and IL-15 for the indicated time points. Intracellular p(Y694)-STAT-5 MFIs in CD8+CD44hiCD62Llo were determined by flow cytometry.

Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (a, b, c, e, f, g) significance was determined using 2-tailed Mann-Whitney U Test. *p<0.05, **p<0.01, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 7

Figure 7. PTPN2-deficiency enhances CAR-T cell efficacy in vivo by promoting STAT-5-mediating homing to CXCL9/10-expressing tumours.

a-c) HER-2-E0771 mammary tumours cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 6x106 FACS-purified CD8+CD62LhiCD44hi central memory HER-2 CAR-T cells generated from Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl, Lck-Cre;Ptpn2fl/fl;Stat5fl/+ or Lck-Cre;Ptpn2fl/fl;Lck+/- splenocytes. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and monitored for tumour growth. a, c) Lymphocytes were isolated from the tumours on a) day 3 or c) day 16 post adoptive transfer and mCherry+CD45+ CD8+ CAR-T cell numbers were determined by flow cytometry. In (c) TILs were stained for intracellular IFNγ and TNF after PMA/Ionomycin treatment.

d) HER-2-E0771 cells generated to inducibly overexpress PTPN2 in response to doxycycline (E0771-HER-2-PTPN2hi) were pre-incubated (24 h) with vehicle or doxycycline (DOX) subsequently stimulated with IFNγ for the indicated times. STAT-1 Y701 phosphorylation (p-STAT-1) and PTPN2 levels were assessed by immunoblotting.

e) Cxcl9 and Cxcl10 mRNA levels in vehicle versus DOX-treated and IFNγ-stimulated HER-2-E0771 cells were assessed by quantitative real time PCR.

f-h) E0771-HER-2-PTPN2hi mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Five days after tumour injection mice were administered vehicle or DOX in drinking water followed by irradiation (4 Gy) on day 6 and the adoptive transfer of 6x106 FACS-purified central memory Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl HER-2 CAR-T cells. Mice were then injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and (h) tumour growth was monitored. In (g) CD45+CD8+mCherry+ TILs were quantified by flow cytometry at day 4 post adoptive transfer.

Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (a, e) significance was determined using 1-way ANOVA Test. In (b, g, h) significance was determined using 2-way ANOVA Test. In (c) significance was determined using 2-tailed Mann-Whitney U Test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR-T cell efficacy in solid tumours (2019)
Figure 8

Figure 8. PTPN2 targeting enhances murine and human CAR-T cell responses.

a) CD8+ LY CAR-T cells generated from human PBMCs were treated with PTPN2-inhibitor (+) or vehicle (-) followed by incubation with plate-bound α-LY, LY-negative MDA-MB-435 cells and LY-expressing OVCAR-3 cells. The proportion of IFNγ+ versus IFNγ+TNF+ CD8+CAR-T cells was determined by flow cytometry.

b) HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs and intracellular PTPN2 levels were determined by flow cytometry.

c-d) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 HER-2 CAR-T cells generated from C57BL/6 splenocytes transfected with GFP versus Ptpn2 siSTABLE™ FITC-conjugated siRNAs two days before adoptive CAR-T cell transfer. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 after adoptive CAR-T cell transfer and tumour growth was monitored. d) CD45+CD3+CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 positive tumours and spleens by flow cytometry 21 days post adoptive transfer.

e) HER-2 CAR-T cells generated from C57BL/6 and Lck-Cre;Ptpn2fl/fl splenocytes were transfected with Cas9 and control or Ptpn2 sgRNAs using the Lonza 4D-Nucleofector and after two days intracellular PTPN2 levels determined by flow cytometry.

f-g) HER-2-E0771 mammary tumour cells (2x105) were injected into the fourth inguinal mammary fat pads of female HER-2 TG mice. Six days after tumour injection HER-2 TG mice received total body irradiation (4 Gy) followed by the adoptive transfer of 10x106 control HER-2 CAR-T cells or those in which PTPN2 had been deleted by CRISPR RNP. Mice were injected with IL-2 (50,000 IU/day) on days 0-4 post adoptive CAR-T cell transfer and tumour growth monitored. g) CD45+ CD8+ mCherry+ CAR-T cells numbers were determined in HER-2-E0771 tumours by flow cytometry 19 days post adoptive transfer.

Data information: Representative flow cytometry profiles and results (means ± SEM) from two independent experiments are shown. In (a, d, g) significance was determined using 2-tailed Mann-Whitney U Test. In (c, f) significance was determined using 2-way ANOVA Test. *p<0.05, ****p<0.0001.

EXPANDED VIEW FIGURE LEGENDS
Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
figf1

a, Immunoblot analysis of Atg5 and LC3. Brain homogenates were prepared from six-week-old control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice. Immunoblot analysis was performed using antibodies against Atg5 and LC3. GSK-3β was used as a loading control. The positions of the Atg12–Atg5 conjugate, LC3-I and LC3-II (LC3–PE conjugate) are indicated. Atg5 monomer was not detected in either lane (data not shown). b, A representative male control (Atg5flox/+; nestin-Cre) and an Atg5flox/flox; nestin-Cre (Atg5flox/flox) littermate at three weeks of age. c, Left, paw placement records of eight-week-old mice. Right, stride lengths corrected for paw base widths (stride/width ratio) of Atg5flox/flox; nestin-Cre and control (Atg5flox/+; nestin-Cre) littermate mice. Values represent means ± s.d. of four mice. Asterisk, P 0.01 (Students t-test). d, Abnormal limb-clasping of an Atg5flox/flox; nestin-Cre mouse compared with a control mouse (Atg5flox/+; nestin-Cre) when suspended by its tail. e, Rotarod testing of Atg5flox/+; nestin-Cre (open symbols) and Atg5flox/flox; nestin-Cre (closed symbols) mice. One male and one female mouse were analysed for each genotype. The time until drop from the rod (rotating at 20 r.p.m.) is shown.
Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
figf2

a, HE-stained sections of the cerebral cortex, the gracile nucleus and cerebellum from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at three months of age. Purkinje cells are indicated with arrowheads. Arrows indicate eosinophilic spheroids, which represent axon swelling. Scale bar, 10 µm. b, Immunohistochemistry using an anti-calbindin antibody on cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age. Scale bar, 25 µm. c, Loss of Purkinje cells in Atg5flox/flox; nestin-Cre mice. Purkinje cells were counted in comparable areas for each mouse, and three fields were counted in each area for each mouse. Data are normalized against values from control mice (Atg5flox/+; nestin-Cre). Values represent mean ± s.d. of three mice. Asterisk, P 0.01 (t-test). d, Apoptotic death of granular cells. Cerebellum sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre mice at six weeks of age were subjected to TUNEL staining. Nuclei were stained with Hoechst 33258. Scale bar, 500 µm.
Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
figf3

a, Immunohistochemistry of brain sections from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice at six weeks of age, stained with an anti-ubiquitin antibody (1B3). Scale bar, 10 µm. b, Ubiquitin-positive inclusions in neurons. Sections of medulla from Atg5flox/flox; nestin-Cre mice at six weeks of age were stained with an antibody against NeuN (a neuron-specific nuclear protein) and ubiquitin. Nuclei were stained with Hoechst 33258. Scale bar, 10 µm. c, Immunoelectron microscopy of ubiquitin-positive inclusion bodies. DRG neurons isolated from Atg5-/- neonates were analysed by immunoelectron microscopy using an anti-ubiquitin antibody. Scale bars, 500 nm.
Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
figf4

a, Immunohistochemistry of DRG neurons from Atg5+/+ and Atg5-/- mice at different developmental stages (embryonic day (E)13.5, E15.5 or postnatal day (P)0), stained with an antibody directed against ubiquitin. Scale bar, 25 µm. b, Accumulation of Triton-X-100-soluble polyubiquitinated proteins in the brains of Atg5flox/flox; nestin-Cre mice. Brain homogenate prepared at the indicated times from control (Atg5flox/+; nestin-Cre) and Atg5flox/flox; nestin-Cre (Atg5flox/flox) mice were separated into Triton-X-100-soluble (S) and -insoluble (P) fractions and analysed by immunoblotting using anti-ubiquitin antibodies. Arrowhead indicates the stacking gel. c, Immunohistochemistry of liver sections from control (Atg5flox/+; CAG-Cre) and Atg5flox/flox; CAG-Cre (Atg5flox/flox) mice at four months of age, using an anti-ubiquitin antibody. Scale bar, 25 µm. d, Immunohistochemistry of liver sections from six-week-old Atg5flox/flox; Mx1-Cre mice at the indicated time points after pIpC injection, using anti-ubiquitin antibodies. Arrowheads indicate ubiquitin-positive inclusion bodies. Scale bar, 25 µm. e, Five thousand hepatocytes were randomly selected, and the number of cells with diffuse cytosolic ubiquitin signals, with (black) or without (white) inclusion bodies, were counted.
CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle (2021)
Figure 1

Figure 1

Mouse ES cells provide a model system for studying the mammalian CMG helicase. (A) Guide RNAs used to target the 5' end of exon 1 of the SLD5 gene in mouse ES cells. Each of the targeted sites contains 20 nt homology to the corresponding gRNA, followed by a 3 nt 'Protospacer Adjacent Motif' of PAM that has the form 'NGG' and is also required for cleavage by Cas9. Note that the predicted PAM site of gRNA1 has a polymorphism in E14TG2a ES cells, which prevents cleavage by Cas9 (see below). (B) The TAP or GFP tag was inserted after the initiator methionine of SLD5. The donor DNA also contained silent mutations in the first few residues of SLD5 (indicated in red), to prevent the modified locus being cut by Cas9. (C) PCR analysis of a pool of transfected cells after treatment with Cas9-D10A 'nickase' and the indicated combination of gRNAs from (A). The ~200 bp product was centred around the start of exon 1 of the SLD5 gene. Efficient cutting of both template strands by Cas9-nickase was indicated by the production of a smeary PCR product (gRNAs 2+3), reflecting DNA repair that mostly produced small deletions around the nicking site (e.g. see Figure EV4D below). The combination of gRNAs 1+3 did not produce a smeary PCR, due to a polymorphism within the PAM motif of the genomic locus (see A above). (D) Outline of the construction of donor vectors with TAP or GFP tags flanked by 1 kb of 5' and 3' homology sequences. (E) Strategy for generation of mouse ES clones with tagged SLD5 (TAP or GFP). (F) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP. (G) Asynchronous cultures of the indicated cells were used to generate cell extracts, which were then incubated with agarose beads coupled to anti-GFP nanobodies. The indicated factors were monitored by immunoblotting. (H) Extracts of TAP-SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 °C, before immunoprecipitation of TAP-SLD5. (I) TAP-SLD5 cells were synchronised as indicated (further details in Materials and Methods). DNA content was monitored throughout the experiment by flow cytometry. (J) Samples from (C) were taken at the indicated times and used to isolate TAP-SLD5 by immunoprecipitation on IgG beads. The indicated proteins were monitored by immunoblotting. For reasons of space, samples A, S, M and 1-6 were resolved in a separate gel to samples 7-8.
CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle (2021)
Figure 2

Figure 2

The p97 ATPase is required for chromatin extraction and disassembly of ubiquitylated CMG helicase in mouse ES cells. (A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7. (B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103). (C) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA (random integration of mCherry-PCNA as described in Materials and Methods). Arrows indicate co-localisation on heterochromatic patches, which was observed in 14 % asynchronous cells (n = 238). GFP-SLD5 appeared and disappeared with similar kinetics to mCherry-PCNA (100 % cells, n = 101). (D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125). (E) Immunofluorescence of the replisome components CLASPIN (i) and CTF4 (ii) in GFP-SLD5 cells treated with p97i. Arrows indicate co-localisation of GFP-SLD5 patches with CLASPIN (95 % cells, n = 107) or CTF4 (91 % cells, n = 45). Scale bars in all panels correspond to 10 µm.
CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle (2021)
Figure 3

Figure 3

CUL2LRR1 is required for CMG ubiquitylation and replisome disassembly during S-phase in mouse ES cells. (A) Cell extracts were generated after the indicated treatments and then incubated with anti-GFP beads. Factors co-purifying with GFP-SLD5 were monitored by immunoblotting. (B) TAP-SLD5 cells were subjected to the indicated treatments. The level of LRR1 and VHL was assessed by immunoblotting. The asterisk denotes a non-specific band. (C) For the experiment described in (B), TAP-SLD5 was isolated from cell extracts and the indicated factors monitored by immunoblotting. (D) The activity of CUL2LRR1 was inhibited in GFP-SLD5 cells as described in Materials and Methods, by treatment with a combination of LRR1 siRNA and MLN4924 (the combined treatment was important to block CMG disassembly during DNA replication termination, and Figure EV2B shows combining LRR1 siRNA with MLN4924 produces a tighter block to CMG-MCM7 ubiquitylation than either individual treatment). Fixed cells were analysed as above. Arrows indicate the accumulation of GFP-SLD5 on constitutive heterochromatin (49 % cells, n = 125). (E) Cells were treated as in (D) and the localisation of GFP-SLD5 was compared with CLASPIN by immunofluorescence. Arrows indicate the co-localisation of GFP-SLD5 and CLASPIN on constitutive heterochromatin (in 90 % cells with GFP-SLD5 patches, n = 103).

Data information: Scale bars in all panels correspond to 10 µm.
CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle (2021)
Figure 4

Figure 4

Upon inhibition of CUL2LRR1, CMG persists on chromatin throughout interphase and is removed during early mitosis. (A) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA, following inhibition of CUL2LRR1. Arrows illustrate the persistence of GFP-SLD5 on heterochromatin after disappearance of mCherry-PCNA. (B) Similar time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-Histone H2B (from the CAG promoter at the ROSA26 locus - see Materials and Methods), after inhibition of CUL2LRR1. Arrows indicate heterochromatic patches of GFP-SLD5 that disappear during entry into mitosis (98 % cells, n = 97). (C) Equivalent data to (B) but for untreated cells (100 % of untreated cells lacked GFP-SLD5 on mitotic chromatin, n = 149).

Data information: Scale bars in all panels correspond to 10 µm.
CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle (2021)
Figure 5

Figure 5

Mitotic CMG disassembly in mouse ES cells is dependent upon TRAIP E3 ligase activity but does not require association of TRAIP with PCNA. (A) TRAIP co-purifies with the CMG helicase from extracts of mouse ES cells. The asterisk indicates a non-specific band. (B) Mouse ES cells were processed as in Figure 3B. Arrows in (i) indicate accumulation of GFP-SLD5 on heterochromatin during interphase, whereas arrows in (ii) denote GFP-SLD5 on chromatin during mitotic metaphase. Scale bars correspond to 10 µm. (C) Immunoblot illustrating the stable expression from the CAG promoter of the indicated forms of TRAIP at the ROSA26 locus in TRAIP -/- mouse ES cells. Asterisks indicate non-specific bands. (D) Quantification of the percentage of mitotic cells with GFP-SLD5 on mitotic chromosomes, after inhibition of CUL2LRR1 as above, in cells with the indicated genotypes. Cells were fixed before analysis.

Data Information: In (D), data are presented as mean of three experiments +/- SD.

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