EMBO/soda-nel huggingface dataset:
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 4

The Wnt/β‐catenin/TCF pathway controls p62 expression. (A) Relative p62/SQSTM1 mRNA levels by quantitative real‐time‐PCR (qRT–PCR) increased following β‐catenin knockdown in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐Catenin knockdown was confirmed by western blotting. (B) Relative p62 mRNA levels by qRT–PCR decreased with β‐cateninS33Y overexpression after 24 h of starvation in HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). β‐CateninS33Y overexpression was confirmed by western blotting. (C) The increase in p62 protein expression induced by β‐catenin siRNA was attenuated in HT29 cells treated with 10 μg/μl cycloheximide and starvation for 8 and 24 h compared to vehicle control. A complementary experiment using the transcription inhibitor actinomycin D is shown in Supplementary Figure S1C. (D) Overexpression of Fzd6 (green, left panel) increased p62 (red, right panel) protein expression in HEK293T cells. As an internal control, cells not overexpressing Fzd6 are shown in the field of view. (E) Doxycycline induction (1 μg/ml) of DNTCF4 increased p62 and LC3‐II protein expression in doxycycline‐inducible DNTCF4 LS174T‐L8 cells. LGR5 downregulation by DNTCF4 confirmed inhibition of β‐catenin/TCF4 signalling. (F) Relative p62 mRNA levels (48 h) by qRT–PCR increased following doxycycline induction of DNTCF4 in LS174T‐L8 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). (G) Upper panel: Wnt3a (200 ng/ml) treatment of HCT116 β‐cateninWT/− cells (24 h) decreased LC3‐II and p62 protein expression by western blotting. Lower panel: quantification by densitometry of the LC3‐II/β‐actin ratio (mean±s.e.m. of three independent experiments).Source data for this figure is available on the online supplementary information page.
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 5

β‐Catenin and TCF4 associate with the p62/SQSTM1 gene promoter. (A) Chromatin immunoprecipitation (ChIP) demonstrates β‐catenin and TCF4 binding to the p62 promoter. HT29 cells were grown in normal or starvation conditions for 2 h and subjected to ChIP analysis with the indicated antibodies (IgG was used as a negative control). Binding of RNA Pol II, β‐catenin and TCF4 to the p62 promoter region was measured by quantitative PCR and expressed as percent enrichment relative to the input chromatin. During nutrient deprivation, binding of RNA Pol II to the p62 promoter increased; binding of β‐catenin to the p62 promoter decreased; TCF4 binding did not change (data are from one representative experiment of at least three independent experiments performed in triplicate). PCR products subjected to agarose gel electrophoresis are shown in Supplementary Figure S2A. (B) Relative p62 mRNA expression by qRT–PCR increases ∼14‐fold after 24 h starvation in HT29 cells (mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). (C) Binding of acetyl‐Histone H3 to the p62 promoter increased under starvation conditions, suggesting p62 gene derepression (data are from one representative experiment of at least three independent experiments performed in triplicate). (D) Binding of β‐catenin to the p62 promoter (relative to β‐catenin binding to a non‐target gene promoter, GAPDH) significantly decreased under starvation conditions (mean±s.e.m., three independent experiments performed in triplicate, **P0.01).
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 6

Autophagy induction reduces Wnt/β‐catenin signalling. (A) Western blotting showing that autophagy was induced after 2 h starvation, as evidenced by decreased p62 protein and increased LC3‐II protein expression. β‐Catenin protein expression decreased over a 24‐h period of starvation in HT29 cells. (B) Autophagy decreased following Atg7 knockdown as shown by increased p62 and decreased LC3‐II protein expression. β‐Catenin protein expression increased following Atg7 siRNA. Reduction of LC3 puncta with Atg7 siRNA was confirmed by immunofluorescence and is shown in Supplementary Figure S2B. (C, D) Reduction of TopFlash activity in HT29 cells after 24 h of autophagy induction using (C) starvation or (D) 100 nM mTOR inhibitor PP242 (C and D, mean±s.e.m., three independent experiments performed in triplicate, ***P0.001). Autophagy induction was confirmed by western blotting and is shown in Supplementary Figures S2C and S2D). (E, F) qRT–PCR shows reduction of Wnt‐target gene expression (E) Axin2 (*P=0.017) and (F) Cyclin D1 (**P=0.0036) after 8 h starvation (mean±s.e.m., three (Axin2) or six (Cyclin D1) independent experiments performed in triplicate). (G–I) Inhibition of Wnt‐induced TopFlash activity by autophagy induction in (G) HCT116 β‐cateninWT/− cells (mean±s.e.m., three independent experiments performed in triplicate, **P0.01) and (H) RKO cells (mean±s.e.m., three independent experiments performed in triplicate, *P0.05). (I) Reduction of Wnt3a‐induced Cyclin D1 gene expression RKO cells after 12 h treatment with autophagy induction using starvation or PP242 (mean±s.e.m., two independent experiments). (J, K) TopFlash activity following Atg7 knockdown in (J) HT29 cells (mean±s.e.m., four independent experiments performed in triplicate, *P=0.0488) and (K) RKO cells with 24 h Wnt3a treatment (mean±s.e.m., three independent experiments performed in triplicate, *P=0.0282). (L) A representative western blot of RKO cells following Wnt3a treatment and Atg7 siRNA. (M) Relative β‐catenin mRNA levels did not change after 8 h starvation by qRT–PCR (mean±s.e.m., three independent experiments performed in triplicate). (N) Western blot showing prevented β‐catenin protein degradation during starvation in the presence of lysosomal autophagy inhibitors chloroquine (10 μM) and bafilomycin A1 (100 nM) compared to starvation alone. (O) Inhibition of autophagy using wortmannin (50 nM) prevented β‐catenin protein degradation during starvation. (P) Western blot analysis of RKO cells expressing a myc–tagged β‐catenin mutant (S33A, S37A, T41A, S45A) resistant to proteasomal degradation (myc–β‐cateninAAAA). RKO cells were subject to nutrient starvation for 2, 8 and 24 h, and autophagy induction was confirmed by increased LC3‐II and decreased p62 protein expression. Both endogenous β‐catenin and myc–β‐cateninAAAA protein expression decreased during starvation. (Q) Proteasome inhibition with MG132 (10 μM) did not prevent starvation‐induced decrease in β‐catenin protein expression.Source data for this figure is available on the online supplementary information page.
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 7

LC3 and β‐catenin co‐localise in the mouse intestinal epithelium. (A and B) Immunofluorescence of LC3 (red) and β‐catenin (green) expression in the intestinal epithelium following 2 days tamoxifen treatment in control (A: β‐catenin+/lox‐villin‐creERT2) and β‐catenin deleted (B: β‐catenin−/lox‐villin‐creERT2) mice. (C and D) Magnified areas from B revealing co‐localisation of LC3 (red) and β‐catenin (green). Arrowheads indicate co‐localisation of LC3 and β‐catenin. (E) Linescan analyses from C and D showing staining intensity of indicated co‐localised puncta. Red and green channel levels were adjusted post acquisition (equal changes applied across the entire figure) and the blue channel was removed for clarity.
Autolysosomal β‐catenin degradation regulates Wnt‐autophagy‐p62 crosstalk (2013)
Figure 8

β‐Catenin directly interacts with the autophagy protein LC3. (A) Co‐immunoprecipitation of YFP–LC3 or negative control YFP in HT29 cells. Binding of endogenous β‐catenin to YFP–LC3 was detected after 8 h of autophagy induction by starvation and starvation in the presence of lysosomal autophagy flux inhibitor chloroquine (10 μM). Input and immunodepleted lysates are shown. (B) Immunoprecipitation of endogenous LC3 in HT29 cells. IgG was used as a negative control. (C) β‐Catenin contains a W/YXXI/L motif (putative LC3‐interacting region) at amino‐acid positions 504–507. β‐Catenin wild‐type (green highlight) and W504A/I507A double point mutant (blue highlight) sequences used in D–F are shown. (D) Pulldown assays using recombinant GST or GST–LC3B and lysates from HEK293 cells expressing HA–tagged wild‐type β‐catenin (HA–β‐cateninWT) or W504A/I507A β‐catenin (HA–β‐cateninW504A/I507A). HA–β‐cateninW504A/I507A exhibited reduced GST–LC3B binding compared to HA–β‐cateninWT. (E) Recombinant myc–tagged His–LC3 (His–LC3–myc) interacted with recombinant GST–β‐cateninWT in vitro, but binding to GST–β‐cateninW504A/I507A was reduced (upper panel). Input recombinant proteins visualised with Coomassie blue are shown (lower panels). (F) Co‐immunoprecipitation experiments using lysates from HEK293 cells transiently expressing YFP–LC3 with β‐cateninWT or β‐cateninW504A/I507A starved for 8 h with chloroquine (10 μM). YFP–LC3 immunoprecipitated β‐cateninWT but not β‐cateninW504A/I507A. Input lysates are shown and immunoprecipitated p62 and YFP–LC3 served as positive controls. (G) HA–β‐cateninS33A/S37A/T41A/S45A/W504A/I507A (HA–β‐catenin6A) was more resistant to the starvation‐induced reduction in β‐catenin protein expression than HA–β‐cateninS33Y. (H) Relative cell death in HT29 cells subject to nutrient starvation for 24 h. Atg7 knockdown increased cell survival. The increase in cell survival following Atg7 knockdown was significantly reversed by simultaneously depleting β‐catenin (double knockdown of Atg7 and β‐catenin). Data are the mean±s.e.m. of three independent experiments performed in triplicate, ***P0.001; **P0.01.Source data for this figure is available on the online supplementary information page.
Chromatin accessibility landscape of pediatric T-lymphoblastic leukemia and human T-cell precursors (2020)
Figure 1

Fig.1

Chromatin accessibility by ATAC-Seq differentiates maturation stages of healthy T-cell precursors

A Thymi collected from healthy donors (n=6) were sorted by FACS to obtain seven populations of double negative (DN2, DN3, and ISP), double positive (DPCD3- and DPCD3+), and single positive (SPCD4+ and SPCD8+) stages. Sorted populations were subjected to the ATAC-sequencing.

B Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test).

C Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).

D Unsupervised learning by PCA of all distal peaks. Color represents developmental stage. The gray triangle indicates the projection of the bulk thymus sample on the PCA space of sorted populations.
Chromatin accessibility landscape of pediatric T-lymphoblastic leukemia and human T-cell precursors (2020)
Figure 2

Fig.2

Signature open chromatin regions distinguish T-cell developmental stages

A Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages (see Fig. EV2 for 5 stages).

B ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database (Fishilevich et al., 2017).

C Functional enrichment analysis of the signature OCRs allocated to three developmental groups by GREAT. Top five unique GO-terms which have the lowest FDR q-val are shown. In parenthesis: number of uniquely enriched GO-terms / total number of enriched GO-terms.
Chromatin accessibility landscape of pediatric T-lymphoblastic leukemia and human T-cell precursors (2020)
Figure 3

Fig.3

Open chromatin regions contain maturation specific transcription factor binding motifs.

A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.

B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.
Chromatin accessibility landscape of pediatric T-lymphoblastic leukemia and human T-cell precursors (2020)
Figure 5

Fig.5

The integration of differential chromatin accessibility and expression analyses reveals recurrently dysregulated genes

A Number of unique and shared OCRs (TSS and distal) in T-ALLs and in healthy T-cell precursors.

B Unsupervised learning by PCA of all TSS and distal peaks collected in the combined analysis of healthy and leukemic T-cells.

C log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4.

D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.

EXPANDED VIEW FIGURE LEGENDS
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 1

Figure 1: CPAP-Tubulin interaction is a cancer target.

(A) Proof-of-principle experiment. Time-lapse images show overexpression of Myc-tagged CPAP-WT (i) or CPAPΔT (ii) has no effect on two centrosome-containing MCF10A cells (-Dox, two centrosomes). The cells were imaged from interphase to Cytokinesis. Bar graph at right quantifies mitotic duration of cells overexpressing CPAP-WT and CPAPΔT. We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis. Number of cells (n) analyzed in each condition is indicated at the top of each bar. Number of independent experiments (N), (N)=3. Error bars, mean ± SEM. Unpaired t-test. CPAP-WT (iii) or CPAPΔT (iv) expressing extra centrosome-containing MCF10A cells (+Dox, extra centrosomes). Note compared to CPAP-WT expressing centrosomes (iii) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase-like stage (iv). White arrows indicate centrosomal dots. Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles. Scale bar, 2µm. Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP-WT and CPAPΔT. (N)=3. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001.

(B-D) Fixed cell images showing the expression of CPAP-WT (top panel) and CPAPΔT (bottom panel) in extra centrosome containing MCF10A (+Dox, extra centrosomes), MDA-MB-231 and H1975T790M cells. Note, in CPAP-WT expressing cells, clustered extra centrosomes remain inactive with no or reduced microtubule nucleation. On the other hand, CPAPΔT expression enhances microtubule nucleation in extra centrosomes (arrows) and preventing them from clustering at G2-like interphase and mitosis. Cells were stained with CPAP (GFP), microtubules (α-tubulin, red), Cep152 (magenta). Scale bar, 2µm.

(E) Bar graph quantifies percentages of mitotic cells exhibiting multipolar spindles upon CPAP-WT or CPAPΔT expression. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001.

(F) Subcutaneous xenograft tumor volume measurements of MDA-MB-231 cells expressing CPAP-WT (red) as control or CPAPΔT (blue) in nude mice. (i) Bar graph at right shows total tumor volume reduction at day 22. (ii) Each condition used 4 mice each containing 4 xenograft tumors. Error bars, mean ± SEM with n = 16 for CPAP-WT and n = 16 for CPAPΔT. Unpaired t-test ***P < 0.0001.
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 2

Figure 2: CCB02 binds tubulin, perturbing CPAP-Tubulin interaction.

(A) 1H NMR spectra of free CCB02 (black) and bound to tubulin in 50:1 ratio (orange). Broadening of CCB02 proton peaks indicate its binding ability to tubulin.

(B) NOESY spectrum recorded for a solution comprising tubulin (6.5µM), CPAP peptide (400µM) and CCB02.1 (200µM) with 70ms mixing time. The signals colored in green and (highlighted by boxes) correspond to inter-ligand trNOE signals arising due to protein-mediated spin-diffusion (INPHARMA NOEs), while the black peaks represent intra-ligand trNOE signals.
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 3

Figure 3: CCB02 prevents proliferation of cells with centrosome amplification.

(A) 14 day CCB02 treatment of MDA-MB-231, HCC827-GR, Calu-6 and MCF10A (+Dox, extra centrosomes) mostly eliminated cells with extra centrosomes. At the end of the treatment, cells were fixed and stained for centrosomes. Bar graph shows the percentage of cells displaying two- and more than two centrosomes. "Vehicle" at the bar graph indicates percentages of cells with extra centrosomes (also refer to Appendix Fig S1A). Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02-treatment (open bars). Data represent mean ± SEM of three independent experiments. (N)=3, (n=70). p values were obtained using unpaired t test **P < 0.001, ***P < 0.0001.

(B) CCB02 treatment prevents centrosome clustering in HCC827-GR cells. Activated centrosomes (arrows) fail to cluster leading to multipolar mitosis. Cells were stained with Cep152 (green), CPAP (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm, Insets, 0.5µm.

(C, D) Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression of MCF10A (+Dox, extra centrosomes) (C), HCC827-GR (D) cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment. Cartoons of a cell with extra centrosomes below to the panel (C) represent various events occurring upon vehicle or CCB02 treatment. Scale bar, 2µm. Bar graphs at right to panel (C) and (D) quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv). At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 4

Figure 4: siRNA depletion of CPAP abrogates the effects of CCB02 in extra centrosome containing cells

(A, B) (i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A) and MDA-MB-231 cells (B). Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP.

(C) Fractions of cells containing centrosomes after siRNA treatment. 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments. Bar graph shows the percentage of cells with extra centrosomes in MCF10A (+Dox, extra centrosomes) in the presence of control siRNA or CPAP siRNA. Data represent mean ± SEM of three independent experiments. (N)=3, (n=300 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01, **P < 0.001.

(D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm.

(E) Bar graph shows the percentage of cells with multipolar mitosis. Higher percentage of multipolar mitotic cells is observed in control siRNA-treated cultures. Bar graph on right shows the percentage of cells with multipolar mitosis. Data represent mean ± SEM of three independent experiments. (N)=3, (n=680 cells). p values were obtained using unpaired t test. ***P < 0.0001.

(F) Bar graph shows the percentage of living (TUNEL-negative) and dyeing (TUNEL-positive) cells under various conditions tested. Higher percentage of TUNEL-positive cells is observed in CCB02-treated cultures. Data represent mean ± SEM of three independent experiments. (N)=3, (n=500 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01.
Inhibition of CPAP-tubulin interaction prevents proliferation 
of centrosome amplified cancer cells (2018)
Figure 5

Figure 5: Chemical inhibition of CPAP-Tubulin interaction causes interphase centrosomes to recruit enhanced levels of PCM.

(A) Interphase centrosomes of CCB02-treated two-centrosome containing MCF10A cells display an enhanced level of PCM proteins. CPAP (green) intensity remains same. Cells are stained for CPAP (green), PCM proteins (magenta) Cep152 (i), PCNT (ii) and CDK5RAP2 (iii) and microtubules (α-tubulin, red). Scale bars, 2µm for left panels and 0.2µm for right panels. Heat map images show intensity saturation (blue) for CPAP, Cep152, PCNT and CDK5RAP2 upon CCB02 treatment. Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells (given in boxes at heat map panels) treated with vehicle (blue line) or CCB02 (red line). Images were recorded with low laser excitation and high zoom factor.

(B) Bar graph quantifies relative intensity of CPAP-interacting proteins in interphase centrosomes compared to vehicle treatment. At least 150 centrosomes (for vehicle); 130 centrosomes (for CCB02) were analyzed in each condition. Error bars, mean ± SEM. (N)=3. Ordinary Two-way ANOVA test ***P < 0.0001.

(C) Immunopurification of CPAP complexes from CCB02-treated cell extracts. (i) CCB02 specifically prevents CPAP-Tubulin interaction. This allows CPAP to bind an enhanced amount of Cep152 and γ-tubulin. Control IP experiment is shown below. (ii) Plot showing PCM protein intensities co-immunoprecipitated with CPAP at various concentrations of CCB02 treatment. CPAP amount does not change with CCB02 treatment. Data represent mean ± SEM. (N)=3.