EMBO/soda-nel huggingface dataset:
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 3

Figure 3. Targeting USP7 inactivates PP2A.

(A) Immunoprecipitation against IgG, USP7 and PP2A-C in whole cell extracts of RPE cells that were treated with DMSO (control) or 25 μM P22077 (USP7i). Levels of USP7, PP2A-A, PP2A-B and PP2A-C are shown. Images from two different exposure times are shown for the better inspection of saturated bands.

(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.

(C) Western blot showing the levels of PP2A-A, PP2A-B, PP2A-C and H2A in the nuclear fraction of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs and 25 μM BAY 11-7082 for 2 hrs. (D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 4

Figure 4. USP7 inhibition phenocopies the changes in the phosphoproteome induced by OA.

(A) Phosphoproteome-wide correlation between the effects observed upon UPS7i and OA treatments (4 and 2 hrs, respectively) in RPE cells. The p-value was obtained by a t-test to evaluate the Pearson correlation between both samples.

(B) Venn-diagrams representing the overlap between the peptides that show increased levels of phosphorylation upon USP7i and OA treatments. The p-value was obtained by a hypergeometric test.

(C, D) Reactome analysis illustrating the biological pathways related to the factors that show increased phosphorylation upon treatment with USP7i, or that are commonly upregulated in response to USP7i and OA.

(E) Representation of the phosphorylation levels at epitopes that follow the CDK consensus motif p(S/T)Px(K/R) upon USP7i or OA treatments from data obtained at phosphoproteomic analyses.

(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.

(G, H) Representation of the phosphorylation levels at epitopes from nucleoporins (NUPs; (G)) or Retinoblastoma (RB1; (H)) upon USP7i or OA treatments from data obtained at phosphoproteomic analyses.
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 5

Figure 5. USP7 suppresses CDK1-dependent DNA damage.

(A) HTM-dependent quantification of EdU, γH2AX, H3S10P and MPM-2 levels per individual nucleus in U2OS cells treated with 50 μM P22077 (USP7i) for the indicated times or with DMSO as a control. Quantification data derive from 3 independent experiments. Data represent mean values ± SEM. N>2000 cells analysed per condition.

(B) Flow cytometry profiles illustrating the levels of γH2AX and MPM-2 in U2OS cells in control conditions (Control, black) and after treatment with 50 μM P22077 for the indicated times (USP7i, red). Experiments were repeated 3 times and one representative result is shown.

(C) Flow cytometry profiles representing the correlation between γH2AX and MPM-2 or histone H3S10P signals in HCT-116 cells treated with DMSO (Control) or 50 μM P22077 for 8 h (USP7i).

(D) Flow cytometry profiles representing the levels of γH2AX in HCT-116 cells in control conditions or after treatment with 50 μM P22077 (USP7i) for 4 hrs alone or in combination with 10 μM RO3306 (USP7i + CDK1i) for 4 hrs. DNA content was measured with Propidium Iodide (PI).

(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 6

Figure 6. USP7 inhibitors induce CDK1-dependent cell death.

(A) Percentage of apoptotic cells in WT and CDC25aKO mESC after treatment with DMSO (CONT) and 25 μM P22077 (USP7i) for 16 hrs. This experiment was repeated 4 times using 3 different CDC25A clones. (* p<0.05, t-test). Bars represent mean values ± SEM.

(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.

(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).

EXPANDED VIEW FIGURE LEGENDS
Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells (2020)
Figure 1

Figure 1. Small PI(3,4)P2 vesicles supply PI(3,4)P2 to the back of migrating Dictyostelium cells

Confocal images of growth stage wild-type Dictyostelium AX3 cell co-expressing tPH-CynA-KikGR (green) and an F-actin biosensor LimE-RFP (red). Front and back of the cell are shown.

Time lapse confocal images of growth stage AX3 cell co-expressing tPH-CynA-KikGR (green) and an activated Ras biosensor, RBD-mCherry (red). Front and back of the cell are shown.

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells. Back region of cells was photoconverted from green fluorescence to red at t=0s. Front and back of the cell are shown, box showing the photoconverted region.

Profiles of the converted red fluorescence at the back remain almost symmetric over time. The dotted line represents the axis of symmetry of the intensity profile of the photoconverted fluorophores at t = 0.

Temporal profile of normalized intensity of total converted red fluorescence. Mean ± SEM is shown for n = 14 cells.

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells during photoconversion. Side region of cells was photoconverted at time 0. Green fluorescence was converted to red. Front and back of the cell are shown. Box shows the photoconverted region.

Profiles of the converted red fluorescence at side become skewer (steeper near the front) over time. The dotted line represents the axis of symmetry of the intensity profile of the photoconverted fluorophores at t = 0.

Confocal fluorescent images of tPH-CynA-KikGR (top) and CAR1-mcherry (bottom) Dictyostelium AX3 cell showing the bleached area and the side (blue) and middle (red) boxes where the fluorescence recovery is monitored. Front and back of the cell are shown.

The graphs depict the recovery of CAR1-mcherry (left) and tPH-CynA-KikGR (right). Mean ± SEM is shown for n = 2 cells.

Time-lapse confocal images of tPH-CynA-KikGR expressing AX3 cells showing docking events at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.

Time-lapse confocal images of PH-CynA-HALO expressing AX3 cells showing high PH-CynA vesicles accumulation at the back of a migrating Dictyostelium cell. Front and back of the cell are shown.

Time-lapse confocal images of tPH-CynA-KikGR (green) expressing AX3 cells. Back cortical region of cell was photoconverted at time 0. Green fluorescence was converted to red. Front and back of the cell is shown, box showing the photoconverted region.
Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells (2020)
Figure 2

Figure 2. PI(3,4)P2 trails F-actin and PIP3 on the leading edge macropinosomes

Time-lapse confocal images of Dictyostelium AX3 cell co-expressing tPH-CynA-KikGR (green) and LimE-RFP (red). Front and back of the cell are shown. Box shows the vesicle.

Time-lapse confocal images of Dictyostelium AX3 cell co-expressing tPH-CynA-GFP (green) and PH-Crac-RFP (red). Box shows the vesicle.

Relative intensities of tPH-CynA-KikGR on macropinosomes and LimE-RFP on macropinosomes in experiments in Fig 3A. Mean ± SEM is shown for n=5 cells.

Relative intensities of tPH-CynA-KikGR on macropinosomes and PH-Crac-RFP on macropinosomes in experiment in Fig 3B. Mean ± SEM is shown for n=3 cells.

Time-lapse confocal images of AX3 cell co-expressing PH-Crac-GFP (green) and another established PI(3,4)P2 biosensor tPH-TAPP1-RFP (red). Box shows the vesicle.

Time-lapse confocal images of tPH-CynA-KikGR (green) expressing wild-type AX3 cells. FM4-64 (red) was added at time 0 during live cell imaging. Front and back of the cell are shown. Box shows the vesicle.
Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells (2020)
Figure 3

Figure 3. PI(3,4)P2 appears in absence of PIP3

Time-lapse confocal images of wild-type AX3 cells expressing tPH-CynA-KikGR (green) and PH-crac (red) before and after PI3K inhibition by 5 µM Wortmannin.

Kymographs of PH-Crac intensity on the perimeter of one cell undergoing random migration in (A). White dotted line shows addition of Wortmannin.

Intensity plot across the white dotted line in tPH-CynA-KikGR expressing cell before and after Wortmannin treatment in (A).

Confocal images of tPH-CynA-KikGR expressing wild-type Dictyostelium AX3 cells before (left) and after (right) LY2924002 treatment.

Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wildtype Dictyostelium AX3 cells before and after LY2924002 treatment, T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus -LY2924002 group (mean±SEM, n = 7).

Confocal images of tPH-CynA-KikGR (green) and PH-Crac (red) expressing wild-type Dictyostelium AX3 cells (left) and PI3K1-2- Dictyostelium cells (right) treated with 5 µM Latrunculin A.

Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wildtype Ax3 and PI3K1-2- cells. There was no statistically significant difference of the biosensor on the membrane between the two groups (mean±SEM, n = 17).

Confocal images of tPH-CynA-KikGR (green) and LimE (labeling F-actin) expressing PI3K1-2- cells. Front and back of the cell are shown and white arrow showing the back localization.

Confocal images of tPH-CynA-KikGR (green) expressing PI3K1-2- cells, white box showing the vesicle, and white arrow showing the back localization.
Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells (2020)
Figure 4

Figure 4. Macropinocytic vesicle processing is necessary for PI(3,4)P2 enrichment at the back of migrating cells.

Confocal images showing localization of PI(3,4)P2 ( tPH-CynA-KiKGR ; green) and α-tubulin (red) in fixed wild type Dictyostelium AX3 cells in 3D. Arrow shows a vesicle attaching to the α-tubulin labelled microtubules.

Confocal images showing localization of PI(3,4)P2 (green) and α-tubulin (red) in fixed AX3 cells after Nocodazole treatment in 3D.

Time-lapse confocal images showing dynamic localization of PI(3,4)P2 (green) and F-actin (red) in wild-type Dictyostelium cells. Nocodazole was added at time 0 during live cell imaging.

Color-coded tracing of vesicle at 10-sec intervals from experiment in C.

Time-lapse confocal images showing dynamic localization of PI(3,4)P2. Nocodazole was added at time 0 during the live cell imaging.

Quantification of vesicle number, vesicle number/min, and vesicle lifetime (s) from experiment in E. T-test was carried out by GraphPad Prism, **P ≤ 0.01 versus After Nocodazole group; mean±SEM (n = 10).

Time-lapse confocal images of individual cells of growth-stage wild-type Dictyostelium AX2 or RacH- strain highlighting tPH-CynA-KikGR-coated macropinocytic vesicles and back-to-front membrane gradient. Images were captured every 7 sec.

Intensity plot across wild-type Dictyostelium AX2 or RacH- strain in image "147 s" shown in (G).

(I-K) (I) Lifetime, (J) number or (K) area of tPH-CynA-KikGR coated macropinocytic vesicles in wild-type AX2 and RacH− cells. T-test was carried out by GraphPad Prism, *P ≤ 0.05, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 20).

(L) Representative confocal images of tPH-CynA-KikGR in wild-type Dictyostelium AX2 or RacH− cells treated with 5 μM latrunculin A Scale bar: 5 μm.

(M) Ratio of membrane to cytosol intensity of tPH-CynA-KikGR in wild-type Dictyostelium AX2 and RacH− cells. T-test was carried out by GraphPad Prism, ****P ≤ 0.0001 versus AX2 group; mean ± SEM (n = 33).
Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells (2020)
Figure 5

Figure 5. A link between PI(3,4)P2 on front macropinosomes and the back to front gradient of PI(3,4)P2

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. Below are zoomed in images from images above. Yellow arrows point to small satellite vesicles.

Time-lapse confocal images of tPH-CynA-KikGR expressing Dictyostelium AX3 cells. The macropinosome at the front of the cell was photoconverted at time 0. White arrow points to PI(3,4)P2 localization at the back of the membrane. Cell front and back are shown, box showing the photoconverted region.

Pie chart quantification of percentage of relative intensity of total green fluorescence (not converted) and converted red fluorescence (converted), showing 4.97% of total fluorescence was converted to red (top). Bottom: 4.97% of total fluorescence was converted to red, and around 50% of converted red fluorescence end up at the back of membrane (red), less than 50% of the converted red fluorescence not at back (orange). n=7

Schematic representation of two potential working alternative possibilities. Red dots show photoactivated tPH-CynA, green dots show not converted tPH-CynA. Left shows that if the biosensor remains associated with the vesicles, a larger proportion of the red signal will appear at the back. Right shows that if the biosensor disassociates from the vesicles, a much smaller amount of the red signal will appear at the back.

Time-lapse confocal images of tPH-CynA-KikGR in giant Dictyostelium AX3 cells. Arrows point to vesicles.
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 1

Figure 1. Mutations in upstream activating sequence (UAS) reduces burst size, but not burst frequency.

(A) Transcription at GAL3 is visualized by addition of PP7 loops at the 5' of GAL3 in budding yeast. Example trace of the quantified fluorescence intensity of the transcription site. Traces are binarized to determine on (active) and off (inactive) times.

(B) Histogram of GAL3 on time (burst duration) for cells with wt and mutated UAS, respectively, reveals shorter on times for mutated UAS. Errors indicate SE of 3 experiments, n = 324 cells UASwt, n = 250 cells UASmut.

(C) Histogram of GAL3 off times. Errors indicate SE of 3 experiments.

(D) Similar to (A), but from cells were the UAS was mutated to a lower affinity UAS. Also see Figure EV1.

(E). Average on time for UASwt and UASmut from exponential fit. Errors indicate SD of fit.

(F) Average off time for UASwt and UASmut from exponential fit. Errors indicate SD of fit.

(G) Example traces of quantified fluorescence intensity at the transcription site for 5'PP7 and 3'MS2 at GAL3.

(H) Cross correlation of the PP7 and MS2 signals shows a peak at 37.9s±1.8s delay, n = 137 cells. Shaded area and errors indicate SEM.

(I) Distribution of number of nascent RNAs at the transcription site determined by smFISH. The distribution of UASwt does not fit a Poisson distribution (grey line), supporting that GAL3 is not constitutively transcribed, but is transcribed in bursts. Example image in shown, yellow arrows indicate TSs. Scale bars: 5 μm. n = 11,839 cells.

(J) Same as (G) for cells with UASmut. The distribution of UASmut fits a Poisson distribution, indicating that GAL3-UASmut is transcribed with random initiation of individual polymerases, similar to constitutive genes. n = 10,616 cells. See also Figure EV1B and C.
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 2

Figure 2. Mutations in UAS reduce residence time of Gal4 on nucleosomal DNA in vitro.

(A) Competitive binding experiments were performed to determine relative affinity of Gal4 to UASwt (green) and UASmut (blue) motifs. Gal4 occupancy on UASwt Cy3/Cy5 DNA was determined by measuring protein induced fluorescence enhancement (PIFE) on 51 bp oligos containing either Gal4 UASwt or UASmut site 1 bp away from Cy3 fluorophore).

(B) Titrating unlabeled UASwt or UASmut competitor DNA reduces UASwt Cy3/Cy5 DNA occupancy. IC50UASwt = 4.0 ± 0.6 nM, IC50UASmut = 17.3 ± 3.5 nM. n = 3. Error bars indicate SD.

(C) Comparison between relative affinities of Gal4 UASwt versus UASmut in naked or nucleosomal DNA shows 4.3 ± 1.1x difference in KD from naked DNA and 6.8 ± 1.7x from nucleosomal DNA. n = 3. Error bars indicate SD.

(D) Experimental setup for smFRET experiments to measure Gal4 binding at nucleosomal DNA. Gal4 binds to site 8 bp into nucleosome. A FRET pair in the entry/exit region provides readout on binding events (one fluorophore on DNA, one on histone). In the absence of Gal4, nucleosomes are in the high FRET state. A Gal4 binding event traps nucleosome in low FRET state.

(E) Gal4 affinity to nucleosomes containing UASwt or UASmut sequences gives S1/2 of 7.2 ± 0.8 nM and 48.9 ± 10.8 nM, respectively. n = 3. Error bars indicate SD.

(F) Example smFRET traces showing Gal4 binding to UASwt in nucleosomal DNA at two different Gal4 concentrations. States are determined using HMM fit.

(G) Same as F but for UASmut.

(H) Binding-rates of Gal4 are concentration dependent, but are similar for UASwt and UASmut: kon UASwt = 0.011 ± 0.002 s-1 nM-1, kon UASmut = 0.009 ± 0.001 s-1 nM-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.

(I) The dissociation-rate of Gal4 at UASwt is ~5-fold slower compared to the UASmut: koff UASwt = 0.20 ± 0.01 s-1, koff UASmut = 0.92 ± 0.05 s-1. n = 2 for all, except n = 4 for UASwt 10 nM Gal4 and n = 3 for UASwt 20 nM Gal4. Error bars indicate SD.

(J) Histogram of Gal4 dwell time to nucleosomal DNA containing UASwt and UASmut sequences. n = 11 for UASwt, n = 8 for UASmut. Error bars indicate SE.

(K) Scatter plot showing the average number of nascent RNA at TS (from smFISH) vs affinity of Gal4 to different UAS sequences in nucleosomal DNA (from in vitro measurements). In vivo transcription levels correlate with in vitro affinity of Gal4, but transcription saturates above wildtype sequence. UAS sequences are shown in the box. For mean number of nascent RNA from smFISH, n = 2 for UASconsensus, UAS-2C, UAS-8T and n = 8 for UASwt, UASmut and errors indicate SEM. For in vitro affinity measurements, n = 3 and errors indicate SD.
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 3

Figure 3. Gal4 binds at the edge of a fragile nucleosome in galactose in vivo. Profiles of nucleosome midpoint positions by MNase-seq experiments. Samples were digested with the indicated MNase concentrations in both raffinose (raf) and galactose (gal) containing media. Midpoints of nucleosomes are smoothed by 31 bp. In galactose the stable nucleosomes move away from the Gal4UAS, creating space for an additional fragile nucleosome (indicated by arrow).
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 4

Figure 4. Measurements of the Gal4 residence time in vivo shows two Gal4 populations, with long residence times colocalizing with target gene.

(A) Simultaneous imaging of Gal4 binding kinetics in vivo using single-molecule tracking and RNA imaging of the GAL10 target gene. Gal4 is tagged with a HALO-tag, which covalently binds to the dye JF646. Transcription of the target gene GAL10 is visualized by PP7-loops.

(B) Example image of a cell, showing the GAL10 TS (arrow, left panel), single molecules of Gal4 (arrows, middle panel) and an overlay (right panel). Scale bar: 2 μm.

(C) Example kymograph of a cell. Upper panel (PP7-GAL10) shows the position of the TS over time. Middle panel (Gal4-HALO) show tracks of Gal4. Data is in white, colored lines show analyzed tracks. Lower panel shows overlay, showing colocalization of some the Gal4 tracks to the GAL10 TS. Scale bar: 1 μm.

(D) Survival probability of the duration of Gal4 tracks (after displacement thresholding) at 200 ms interval from cells grown in raffinose (n = 258 tracks in 30 cells) or galactose (n = 346 tracks in 25 cells). Lines show bi-exponential fit, indicating 2 Gal4 populations. Inset shows data in semi-logarithmic plot.

(E) Residence time of the fast and slow component of the fits from (D). The slow component changes between conditions. Error bars indicate 95% CI.

(F) Survival probability of Gal4 residence times that colocalize with GAL10 TS (<250nm), showing that Gal4 with long residence time colocalizes with GAL10. Data was taken at 1s interval and shows overlap between 267 Gal4 tracks and 83 GAL10 tracks, representing 458 bound molecules. Line shows exponential fit, with mean of 12.3s ± 0.83s. Errors indicate 95% CI.
Live-cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting (2019)
Figure 5

Figure 5. Real-time correlation of Gal4 binding and transcription in vivo using orbital tracking.

(A) Schematic of 3D orbital tracking of GAL10 TS. Scale bar: 2 μm.

(B) Example trace of Gal4 binding and GAL10 transcription in the same cell.

(C) Autocorrelation of Gal4. The exponential fit shows an average dwell time of 34.8 ± 0.5s. n = 19 traces. Shaded area and errors indicate SEM.

(D) Autocorrelation of GAL10. The fit of 2 linear lines reveals a burst duration of 152.4 ± 2.3s. n = 19 traces. Shaded area and errors indicate SEM.

(E) Cross correlations for Gal4-GAL10 RNA (blue) and Gal4-RNR2 RNA (grey, negative control). Asymmetry and positive temporal shift of the cross correlation function is consistent with a 79.5 ± 0.2 second delay between the middle of Gal4 and GAL10 signals (n = 19 traces), which is not seen in the negative control (n = 8 traces). Shaded areas and errors indicate SEM.

(F) Schematic of the different signal durations of Gal4 binding and GAL10 transcription. Gal4 binding overlaps with the GAL10 transcription for 14.1s.