EMBO/soda-nel huggingface dataset:
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 2

Figure 2. Phosphorylation of FoxM1 by GSK3β at the S474 promotes the ubiquitination of FoxM1.

A. Schematic of Flag-FoxM1 deletion mutants in a mammalian expression system. FL: full length; NT: NH2-terminal domain; FB: forkhead box domain; CT: COOH-terminal domain.

B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel).

C. Sequence analysis of human FoxM1 identified three putative GSK3β target sites at S228, T309, and S474.

D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.

E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.

F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.

G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.

All data are representative of three independent experiments.
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 3

Figure 3. Wnt activation inhibits the phosphorylation of FoxM1 mediated by Axin-GSK3 complex and the ubiquitination of FoxM1 mediated by FBXW7.

A. Cell lysates of LN229 cells were subjected to IP using Axin antibody or control IgG, followed by IB with indicated antibodies.

B. Cell lysates of LN229 cells with or without Wnt-3a treatment for 4 hours were subjected to IP using Axin antibody or control IgG, followed by IB with FoxM1 and Axin antibodies.

C. Axin siRNA or control siRNA were transfected into LN229 cells, after 36 hours, lysate of the cells was performed with co-IP using FoxM1 antibody followed by IB with indicated antibodies.

D. Axin siRNA or control siRNA were transfected into LN229 cells for 36 hours. Lysates of the cells were subjected to IB with FoxM1 phospho-S474 antibody.

E. Axin siRNA or control siRNA with HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.

F. Wild type Flag-FoxM1 or S474A mutant was transfected into 293T cells after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.

G. HA-Ubiquitin and/or FBXW7, GSK3β CA, Flag-FoxM1 were co-transfected into 293T cells, after 36 hours, cells were treated with MG132 for six hours. Then lysate of the cells was performed with IP using Flag antibody followed by IB with indicated antibodies.

H. FBXW7 siRNA or control siRNA and HA-Ubiquitin were transfected into 293T cells, after 36 hours, cells were treated with MG132 with or without Wnt-3a (50 ng/ml) for six hours. Then lysate of the cells was performed with IP using FoxM1 antibody followed by IB with indicated antibodies.

All data are representative of three independent experiments.
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 4

Figure 4. USP5 deubiquitylates and stabilizes FoxM1 in response to Wnt stimulation.

A. 293T cells were transfected with USPs cDNA or control plasmid. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a for 4 hr. FoxM1 protein levels in the cells were then determined by Western blotting.

B. 293T cells were transfected with 1 µg control plasmid or USP5 cDNA in 6-well plates. After 36 hr of transfection, cells were treated with 100 µg/ml CHX for 0, 2, 4, or 6 hr. FoxM1 protein levels in the cells were then determined by Western blotting.

C. GSK3-β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 4 hr. Cell lysates were subjected to IP with USP5 antibody and followed by IB with Flag or USP5 antibody.

D. GSK3βCA, USP5, or control plasmid was co-transfected with Flag-FoxM1 into 293T cells. After 36 hr of transfection, cells were treated with 50 ng/ml Wnt-3a or control PBS for 6 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.

E. USP5 and Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A or Flag-FoxM1S474E plasmids were co-transfected into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.

F. GSK3β CA, USP5, or control plasmid was co-transfected with Flag-FoxM1 (WT) or mutants Flag-FoxM1S474A into 293T cells for 36 hr. Cell lysates were subjected to IP with Flag antibody and followed by IB with Flag or USP5 antibody.

G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.

H. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without siUSP5 into U87 cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a and 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody followed by IB with HA or FoxM1 antibody.

All data are representative of three independent experiments.
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 5

Figure 5. Wnt promotes FoxM1 nuclear translocation to enhance interaction ofβ-catenin/TCF4 via FoxM1 competing with ICAT.

A. Expression of FoxM1 and ICAT was examined via IHC staining in 30 GBM and paired adjacent normal tissues. Representative ICAT and FoxM1 expressions are shown in a section of a GBM tumor with adjacent normal brain. Scale bar: 50 µm

B. ICAT stainings were scored as 1–12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.

C. FoxM1 stainings were scored as 1–12 according to the intensity and percentage in the 30 GBM and paired adjacent normal tissues. Boxes indicate interquartile range. Bars from each box extend to largest and smallest observations.

D. Increased amounts of FoxM1 plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.

E. Increased amounts of ICAT plasmids were transfected into 293T cells. After 36 hr, cells were treated with 50 ng/ml Wnt-3a for 6 hr. Cell lysates were then subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.

F. FoxM1 siRNA or control siRNA was transfected into U87 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.

G. ICAT siRNA or control siRNA was transfected into LN229 cells, lysates were subjected to IP with β-catenin antibody, followed by IB with β-catenin, ICAT, FoxM1 or IgG antibodies.

H. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, FoxM1- or/and ICAT plasmid-transfected U87 cells. Fold was calculated relative to that in cells transfected with the vector, which was set as 1.

I. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in FoxM1 siRNA-, ICAT siRNA-, control siRNA- or combination of FoxM1 siRNA and ICAT siRNA-transfected U87 cells as described in (H).

J. ChIP analyses of endogenous β-catenin binding at the TCF-binding site of the Axin2 promoter were performed in vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmid-transfected U87 cells as described in (H).

Data shown in (E)-(G) are representative of three independent experiments. Data shown in (H)-(J) are the means ± SD of two independent qPCR quantitative experiments with triplicate samples in each experiment.
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 6

Figure 6. FoxM1 regulates β-catenin transcription activity and promotes Wnt target gene expression

A. FoxM1 or/and ICAT plasmids were co-transfected with Axin-2 promoter luciferase and TK-Renilla plasmid into U87 cells. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the vector group. Values are mean ± SD for triplicate samples.

B. FoxM1 siRNA#1, #2 or control siRNA were co-transfected to U87 cells with Axin-2 promoter reporter. Luciferase activity was measured with the Dural Luciferase reporter assay, and relative luciferase activity was normalized to that of the control siRNA group. Values are mean ± SD for triplicate samples.

C. ICAT siRNA or control siRNA were co-transfected into LN229 cells with Axin-2 promoter reporter. Luciferase activity was measured as in (C).

D. FoxM1 or/and ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.

E. ICAT, FoxM1 or FoxM1-∆FB (FoxM1 mutant lacking the forkhead box domain) plasmid or their combinations were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.

F. FoxM1 siRNA and/or ICAT siRNA were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into LN229 cells, and the cells were treated with Wnt3a (50 ng/ml) or control PBS. Luciferase activity of TOP-Flash in the cells was measured as in A. Values are mean ± SD for triplicate samples.

G. Vector-, β-catenin-NLS-, sh-FoxM1-, FoxM1-shR- and/or ICAT plasmids were co-transfected with TOP-Flash reporter and TK-Renilla plasmids into U87 cells. Luciferase activity of TOP-Flash in the cells was measured as in (A). Values are mean ± SD triplicate samples.

H. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 siRNA#1 or #2 were determined by Western blotting.

I. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with ICAT siRNA were determined by Western blotting.

J. Levels of Axin-2 and cyclin D1 protein in LN229 cells transfected with FoxM1 or/and ICAT plasmids were determined by Western blotting.

Data shown in (A)-(G) are representative of three independent experiments with three triplicate samples in each experiment. Data shown in (H)-(J) are representative of three independent experiments.
Wnt-Induced Deubiquitination of FoxM1 Ensures Nucleus β-catenin Transactivation (2016)
Figure 7

Figure 7. USP5-FoxM1 axis regulated Wnt/β-catenin signaling-mediated cell proliferation.

A. A total of 1 × 104 LN229 cells were transfected with control vector, FoxM1, and/or ICAT plasmids and treated with Wnt3a (50 ng/ml) and were then plated and counted 48 hr after seeding. The proliferation percentage was calculated relative to that in cells transfected with the control vector, which was set as 100%. Data represent the mean ± SD of three independent experiments.

B. LN229 cells were transfected with vector control, FoxM1, FoxM1 plus sh-β-catenin or FoxM1 plus ICAT plasmid. The proliferation percentage was determined as described in (A).

C. LN229-shFoxM1 cells were transfected with FoxM1-shR (siRNA-resistant FoxM1), or FoxM1-shR plus ICAT plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A).

D. LN229 cells were transfected with USP5 and/or FoxM1 siRNA. The proliferation percentage was determined as described in (A).

E. LN229 cells were transfected with USP5 siRNA and/or FoxM1 plasmid. The cells were then treated with Wnt3a (50 ng/ml). The proliferation percentage was determined as described in (A). Data shown in (A)-(E) represent the mean ± SD of three independent experiments.

F-H. Expression of FoxM1, Cyclin D1 and USP5 proteins was examined via IHC staining in 50 GBM specimens. Staining of FoxM1, Cyclin D1, or USP5 was scored as 1–12 according to the intensity and percentage. The significance of the correlations was determined by Pearson’s correlation test.

I-J. Axin2 mRNA levels in the above 50 GBM specimens were examined through RNAscope technology. The staining was scored as 1–12 according to the intensity and percentage. The significance of the correlations was determined by Pearson’s correlation test.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 1

Figure 1. The cortical localization and spindle orienting function of Mud both rely on Pins.

A. GFP:Mud is observed at spindle poles and at the lateral cell cortex in control mitotic follicle cells (A), but is only seen at the spindle poles in pinsp62/pinsp62 cells (A'). Scale bars = 5 microns. B. Interphase localization of Mud at the cortex is lost in pinsp62/pinsp62 follicle cells. Scale bar = 20 microns.

C-E. Full length GFP:Mud shows the wild type pattern of localization in mud3/mud4 mitotic follicle cells (C) and rescues spindle orientation (D, E). Scale bars = 5 microns. GFP:Mud lacking the Pins binding domain localizes only at spindle poles (C') in mud3/mud4 mitotic follicle cells and fails to rescue spindle orientation (D, E). Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001, *** p<0.001.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 2

Figure 2. Expression and localization of Pins:Tom and GFP:Mud in interphase and mitotic follicle cells.

A. Pins:Tom and GFP:Mud both appear enriched at the apical surface of follicle cells, and can be weakly detected at lateral surfaces. Enrichment of Mud at the oocyte nucleus has been reported previously (Zhao, Graham, Raposo, & St Johnston, 2012). Unlike GFP:Mud, Pins:Tom can be seen past Stage 6, the developmental point at which follicle cell division ceases. The red arrow points to a later (~Stage 8) egg chamber. Scale bars = 20 microns.

B. Pins and actin overlap extensively, though actin is observed at the basal surface (red arrow in B) whereas Pins is not (red arrow in B''). Scale bars = 5 microns.

C, D. Pins:Tom and GFP:Mud have overlapping but not identical localization patterns in mitotic follicle cells. Representative pictures (C) and quantification from 9 cells (D) are shown. Scale bars = 5 microns.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 3

Figure 3. Pins does not require a Pins-specific cue to relocalize at mitosis.

A-C. Pins-myr-GFP and myr-RFP show a similar localization pattern to Pins:Tom during mitosis. Representative pictures are shown (A, B); a heatmap lookup table (A', B') emphasizes the relative enrichments of Pins-myr-GFP and myr-RFP along the cortex. Quantification (Pins-myr: 4 cells, myr-RFP: 2 cells) (C).

D-F. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full-length Pins or Pins-myr-GFP. Representative pictures (D, E) and quantification (F) are shown. Scale bars = 5 microns. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 4

Figure 4. Dlg and Pins interact to promote spindle orientation in the follicle epithelium, but Dlg is not required for cortical localization of Pins.

A. Spindle orientation is randomized in Dlg1P20/Dlg1P20 and Dlg1P20/Dlg2 mutant follicle cells.

B. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full length Pins or phosphomimetic (S436D) Pins. Unphosphorylatable (S436A) Pins fails to rescue.

C. Quantification of spindle orientation phenotypes. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001, *** p<0.001.

D. Pins:Tom and GFP:Mud are cortical in Dlg1P20/Dlg2 mutant follicle cells.

E. Pins:Tom and Pins-myr are both laterally enriched at mitosis in Dlg1P20/Dlg2 mutants. Wild-type Pins:Tom signal quantification data as in Fig 2D (9 cells). Quantification of Pins:Tom in Dlg1P20/Dlg2 is 5 cells, Pins:Myr in Dlg1P20/Dlg2 is 8.

F. GFP-tagged Pins variants (control, phosphomimetic, unphosphorylatable) are observed at the mitotic cell cortex in pinsp62/pinsp62 null mutant follicle cells.

G. Pins-myr-GFP fails to rescue spindle randomization in Dlg1P20/Dlg2 mutant follicle cells. Quantification in (C).

H. Quantification of spindle orientation phenotypes in the Stage 9/10 embryonic ectoderm. n = 17 cells from 2 embryos in w1118 and n = 18 cells from 2 embryos in Dlg1P20/Dlg2 embryos. Statistical significance in spindle orientation was determined using the Mann-Whitney test. ** p=0.0015.

I. Pins:Tom is cortical in the early embryonic ventral ectoderm from a Dlg1P20/Dlg2 mother. Representative image from an embryo imaged live.

Data information: scale bars = 5 microns.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 5

Figure 5. Putative microtubule-capturing factors are not required for spindle orientation in the follicular epithelium.

A-C. Multiple genetic approaches were used to test a role for either Khc73 or GUKHolder in metaphase spindle orientation. Representative pictures (A-B) and quantification (C) are shown. Scale bars = 5 microns. Spindle angle randomization in pinsp62/pinsp62 null mutant follicle cells is included as a positive control for comparison.
The Drosophila mitotic spindle orientation machinery requires activation, not just localization (2022)
Figure 6

Figure 6. Inscuteable and Dlg function as distinct activators of the pulling machinery.

A-D. Two distinct patterns of Inscuteable localization are observed in the follicular epithelium when UAS-Inscuteable is driven by Traffic jam-GAL4. (A, C) In the first pattern (InscA), Inscuteable is highly enriched at the apical cell surface. (B, D) In the second pattern (InscB), Inscuteable is observed at both the lateral and apical cortex. Mud and Pins localizations are also shown. Representative pictures (A, B) and quantifications (InscA: average of 5 cells, InscB: average of 7 cells) (C, D) are shown.

E. aPKC localization in InscA and InscB follicle cells.

F. Quantification of spindle orientation phenotypes shows that mitotic spindles are reoriented in InscA cells, but not InscB cells. Reorientation relies on Mud but not on the interaction between Pins and Dlg.

G. The ratio of InscA to InscB is impacted by temperature and by the interaction between Pins and Dlg. Significance was determined using the Chi-square test. ** p<0.01.

Data information: scale bars = 5 microns.

Expanded View Figure legends
CRISPR/Cas9 mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse (2016)
Figure 2

Figure 2. Generation and characterization of variant F9 mutant mouse strains.

A Schematic diagram of the strategy to generate F9Y381D and F9Y381S mouse strains. Introduced mutant oligonucleotide is in red and the corresponding amino acids are in green.

B Sanger sequencing showing the point mutations in F9 mutant strains. Mutated oligonucleotides are indicated by arrows and the premature stop codon is underlined.

C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears.

D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.

E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.

F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.

G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.
CRISPR/Cas9 mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse (2016)
Figure 3

Figure 3. Amelioration of HB in F9 mutant mice through naked DNA injection of Cas9 components.

A Schematic diagram of plasmids used for treatment in vivo. Cas9 protein and sgRNA were from pX458 vector. The plasmid donor was 800 bp in length in pEASY vector. The ssODN donor was 120 oligonucleotides.

B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.

C Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14.

D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).

E HDR donor design for correction of Y381D and representative Illumina sequencing reads (rn) in DNA treated HB mice. Red text indicates the correction of mutation, whereas blue text indicates the synonymous mutations.

F The genome editing efficiency of individual mice is presented.

G Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5).

H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real-time PCR. Data represent means ± SE.
CRISPR/Cas9 mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse (2016)
Figure 4

Figure 4. Genetic correction of F9 mutation via the Cas9 system delivered through AdV.

A Schematic of AdVs used for in vivo genome editing. AdvCas9: humanized spCas9 is fused to mCherry through 2A peptide. AdvG/T: an 800 bp corrective template and sgRNA targeting the mouse F9 Y381D mutation was inserted into an AdV containing GFP.

B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm.

C T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as ‘%’ below the lane.

D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE.

E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouse liver tissues.