EMBO/soda-nel huggingface dataset:
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 1

Fig. 1. MYB supports the survival and metabolic adaption of pancreatic cancer cells under hypoxia.

(A) MYB knockdown (MiaPaCa-shMYB and Panc1-shMYB) and forced MYB-overexpressing (BxPC3-MYB) PC cells along with their respective control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were subjected to hypoxia (1% O2) treatment for different time intervals (0-96 h). Their growth was measured by viable cell counting using a trypan blue exclusion assay.

(B) Apoptosis indices of genetically engineered MYB-silenced or -overexpressing pancreatic cancer cells were analyzed by flow cytometry after staining with Annexin V and 7-AAD.

(C, D) MYB-modulated PC cells were cultured under hypoxia and normoxia for 48 h. Conditioned media was collected and analyzed for consumed glucose (C) and lactate secretion (D). The amount of glucose uptake and lactate secretion was normalized for the number of cells.

(E, F) ECAR (E) and OCR (F) in control and MYB silenced MiaPaCa cells cultured under normoxia or treated with hypoxia mimetic CoCl2 (100 μM for 4 h). Thereafter, ECAR was measured in PC cells with sequential injection of glucose, oligomycin, and 2-DG, while OCR was measured with the serial addition of oligomycin, FCCP, and antimycin A/rotenone.

(G) MiaPaCa-Scr and MiaPaCa-shMYB cells were cultured under normoxia and hypoxia for 48 h and harvested for metabolites extraction. The mean area covered by each metabolite's mass spectrum from the triplicate set of LC-HRMS experiments was analyzed and normalized with protein concentration. The heat map was prepared after calculating the relative fold changes of metabolite levels in cells cultured under hypoxia (H) versus normoxia (N).

Data information: Data are shown as means ± SD. The number of technical (A, C-F) and biological (B, G) replicates were 3. The reproducibility of the results (A, C-F) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test was used for statistical analyses. n.s., not significant, *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 2

Fig. 2. MYB upregulation under hypoxia is HIF1α-independent and does not occur at the transcriptional level.

(A) MYB expression was analyzed by immunoblotting in pancreatic cancer cells cultured under hypoxia for different time intervals. HIF1α accumulation was examined as a positive control.

(B) MYB transcript levels were determined by qRT-PCR in hypoxia-treated cells.

(C, D) MYB protein (C) and transcript (D) levels were measured in the prostate (LNCaP), lung (A549), breast (MB231), and colon (HCT116) cancer cells cultured under normoxia and hypoxia (6 h) by immunoblotting and qRT-PCR, respectively. β-actin/ACTB served as an internal control. N- normoxia and H- hypoxia.

(E) Pancreatic cancer cells were transiently transfected either with non-targeted control siRNA (siCtrl) or HIF1α -targeting siRNA (siHIF1α) for 24 h and then incubated under hypoxia for 6 h. MYB expression was analyzed at protein and mRNA levels.

(F) Cells were treated with either vehicle or CoCl2 (100µM) for 6 h, and total protein and RNA were isolated. The expression of HIF1α and MYB was examined. β-actin was used as an internal control.

(G) Cells were co-transfected with a plasmid expressing Gaussia luciferase under the control of MYB promoter and secreted alkaline phosphatase (SEAP) expression plasmid under a constitutive CMV promoter. After 24 h, media was replaced and cells were cultured under normoxia or hypoxia for 6 h. Luciferase and SEAP activities were analyzed and data presented as relative luciferase activity normalized with SEAP activity.

Data information: Data are shown as means ± SD. The number of technical (B, D-G) replicates was 3. Reproducibility of the results (A-G) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test was used for statistical analyses. n.s., not significant, *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 3

Fig. 3. MYB is induced under hypoxia via a HuR-mediated post-transcriptional mechanism. (A) Cells were pre-treated with Actinomycin D (10 µg/ml) for 1 h followed by incubation under normoxia or hypoxia for indicated time points. Total RNA was collected and MYB mRNA levels were examined by q-RT PCR. 18s rRNA was used as an internal control.

(B) HuR localization was examined by immunofluorescence microscopy in cells exposed to hypoxia for 1 h. HuR (green), and nucleus (blue). Scale bar, 25 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles.

(C) Cytosolic and nuclear protein fractions were prepared from cells cultured under hypoxia for 1h and HuR expression was examined by immunoblotting. α-Tubulin (nuclear) and Lamin A (cytoplasm) were used as loading controls.

(D) RNP-IP assay was performed using the cytoplasmic fractions of cells exposed to hypoxia for 3 h. Following DNase I treatment, cytoplasmic fractions were sonicated and subjected to immunoprecipitation using an anti-HuR antibody. MYB mRNA was checked by qRT-PCR from isolated RNA fragments and normalized with IgG isotype control. 18s rRNA was used as an internal control.

(E) Following transfection with siHuR or control siRNA for 24 h, cells were co-transfected with MYB 3′-UTR-firefly luciferase reporter and Renilla luciferase-expressing control plasmid. After 24 h, cells were incubated under hypoxia for 6 h. Data is presented as Firefly luciferase activity normalized with Renilla luciferase activity.

(F) Cells transfected with HuR or control siRNA for 48 h were pretreated with Actinomycin D (10 µg/ml) for 1 h and then cultured under normoxia and hypoxia for various time points (0-6 h). Total RNA was collected, and MYB mRNA levels were checked in HuR-silenced cells by q-RT PCR. 18s rRNA was used as an internal control.

(G) Cells transfected with HuR or control siRNA for 48 h were exposed to hypoxia for 6 h, and MYB and HuR expression was determined by immunoblotting or qRT-PCR.

Data information: Data are shown as means ± SD. The number of technical (A, D-G) replicates was 3. Reproducibility of the results (A-G) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test was used for statistical analyses. *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 4

Fig. 4. MYB regulates HIF1α expression at the transcriptional level by directly binding to its promoter.

(A) MYB-silenced (MiaPaCa-shMYB and Panc1-shMYB) and MYB-overexpressing (BxPC3-MYB) PC cells, along with their control cell lines (MiaPaCa-Scr, Panc1-Scr, and BxPC3-Neo) were exposed to hypoxia for 6 h. Protein lysates were made, and the expression of MYB and HIF1α was examined by immunoblotting. β-actin served as a loading control.

(B) Total RNA was also isolated from MYB-modulated pancreatic cancer cells incubated under hypoxia (6 h). Levels of HIF1α transcripts were measured by qRT-PCR. β-actin served as an internal control. Bars represent relative fold change in HIF1α transcripts.

(C) The transcriptional activity of the HIF1α promoter was determined by performing the promoter-reporter assay. MYB-modulated PC cell lines were transfected with the reporter plasmid, and after 24 h of transfection, cells were cultured under hypoxia or normoxia for 24 h, and the supernatant was collected. HIF1α promoter-driven Gaussia luciferase activity was normalized with SEAP activity, and the data was presented as a bar diagram.

(D) In silico analysis of the HIF1α promoter was performed to identify MYB binding sites using PROMO- ALGGEN and TFBIND. Three putative binding regions, P1 (-36 to -140), P2 (-668 to -799), and P3 (-842 to -960) preceding the transcription start site, were predicted.

(E) MYB-silenced or -overexpressing PC cell lines were exposed to normoxia or hypoxia for 6 h and subjected to chromatin immunoprecipitation using anti-MYB antibodies or IgG control followed by qPCR analysis using site-specific primers.

Data information: Data are shown as means ± SD. The number of technical (B, C, E) replicates was 3. The reproducibility of the results (A-C, E) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test was used for statistical analyses. n.s., not significant, *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 5

Fig. 5. Forced expression of HIF1α in MYB-silenced pancreatic cancer cells fails to rescue their survival and metabolic shift under hypoxia.

(A) Immunoblot analysis was performed to analyze HIF1α and MYB expression in MYB-silenced (shMYB) and those having a forced overexpression of HIF1α (shMYB-HIF1αOE) along with their respective controls (Scr and shMYB-Neo) cultured under hypoxia. β-actin served as a loading control.

(B) The cell viability was determined by trypan blue exclusion method in MYB knockdown cells or with ectopic expression of HIF1α in MiaPaCa and Panc-1 along with their control cells exposed to hypoxia at different time intervals (0-96 h).

(C) Live/Dead staining was performed in MYB-silenced MiaPaCa and Panc-1 or MYB silenced- HIF1α overexpressed cells cultured under hypoxia for 48 h. Cells were visualized under a fluorescence microscope. Representative fluorescence micrographs are presented. The number of live and dead cells was counted from the captured random images. Scale bar, 150 μm (lower right corner).

(D) Relative levels of metabolites were calculated in MiaPaCa-Scr, -shMYB, and -HIF1α-overexpressing shMYB cells exposed to hypoxia, and data normalized with protein concentration. (E) A schematic diagram shows metabolic pathways with altered changes in metabolites in genetically engineered cell lines having a differential expression of MYB and HIF1α.

(F) MYB-silenced and HIF1α overexpressing MYB knockdown PC cells and control cells were cultured under hypoxia for 48 h. Conditioned media was collected and analyzed to assess glucose consumption and lactate secretion by measuring their levels. The data was normalized with the number of cells and presented as relative fold changes.

Data information: Data are shown as means ± SD. The number of technical (B-D, F) replicates was 3. The reproducibility of the results (A-D, F) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test (B, F) and one-way ANOVA (C) were used for statistical analyses. *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 6

Fig. 6. MYB alters the expression of a select set of metabolic genes by binding to their promoters and enhancing the recruitment of HIF1α and p300

(A) MYB modulated PC cells (MiaPaCa Scr and shMYB) were cultured under normoxia/hypoxia for 12 h. Afterward, total RNA was isolated, and relative fold change in the mRNA expression of genes associated with glucose metabolism was examined by qRT PCR. ACTB was used as an internal control.

(B) Heat map showing the relative fold changes in glucose metabolism-associated gene expression in Scr control, shMYB, and shMYB-HIF1αOE MiaPaCa and Panc-1 cells cultured under hypoxia for 12 h.

(C) Quantitative ChIP-qPCR was performed to analyze the occupancy of MYB under normoxia and hypoxia at promoter regions of selected glycolytic genes (GLUT3, HK2, PFKL, ENO2, and MCT4) using anti-MYB or IgG control antibodies. The enrichment of promoter regions was compared between cells cultured under normoxia or hypoxia.

(D-F) Genomic occupancy of HIF1α (D), p300 (E), and H3K9ac (F) on the promoter regions were also confirmed by ChIP- qPCR using specific antibodies and fold enrichment calculated and presented as a bar graph after normalization with control IgG.

Data information: Data are shown as means ± SD. The number of technical (A-F) replicates was 3. The reproducibility of the results (A-F) was confirmed at least three times in independent experiments. Two-tailed unpaired student's t-test was used for statistical analyses. *P < 0.05.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 7

Fig. 7. MYB interacts with HIF1α and p300 and exhibits nuclear co-localization.

(A, B) Interaction of MYB with HIF1α (A) and p300 (B) was examined by co-immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h. Immunoprecipitation with IgG isotype antibody was used as a control. 10% of total cell lysate was used as input for western blot analysis.

(C, D) Co-localization of MYB with HIF1α (C) and p300 (D) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy. Scale bar, 30 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles. For ease of visualization, a pseudo color (purple) was adopted for p300 staining.

Data information: Reproducibility of the results (A-D) was confirmed at least three times in independent experiments.
MYB sustains hypoxic survival of pancreatic cancer cells by facilitating metabolic reprogramming (2022)
Figure 8

Fig. 8. Tumorigenicity of MYB-silenced PC cells remains significantly reduced despite restored HIF1α expression.

(A) Luciferase tagged-MiaPaCa control, MYB knockout, or HIF1α overexpressed MYB knockout cells were implanted into the tail of the pancreas of athymic nude mice (n=6 per group).

(B, C) Tumor growth was monitored by non-invasive bioluminescence imaging. Tumors were resected at the experimental endpoint, and their weights (B) and volumes (C) were recorded.

(D-F) The levels of glucose (D), lactate (E), and citrate (F) were also analyzed in the tumor tissues and normalized with the tissue weight used for metabolite extraction.

(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.

(H) Schematic figure showing MYB expression is induced under hypoxia via HuR, which then transcriptionally upregulates HIF1α leading to its more significant accumulation under hypoxia. Further, MYB interacts with HIF1α and p300 and enhances their recruitment to glycolytic gene promoters. Transcriptional upregulation of glycolytic genes via coordinated action of MYB, HIF1α, and p300 helps in the metabolic adaptation of cancer cells to sustain their growth and survival.

Data information: Data are shown as means ± SD (n=6). The number of technical (D-F) replicates was 3. A one-way ANOVA test was used for statistical analyses. *P < 0.05, ** p < 0.01.

EXPANDED VIEW FIGURE LEGENDS
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 1

Figure 1. Identification of BCL9 as a novel mitotic Wnt signalling regulator to stabilise proteins and promote oncogenesis.

A Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.

B ELISA validation of selected Wnt/STOP signalling regulators (n=3).

C-D (C) Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).

E Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.

F Venn diagram analysis of the number of Axin1, APC, and Wnt3a signalling and/or BCL9-regulated transcriptional targets.

G Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.

H The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.

I Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.

Data information: Significance was measured by two-tailed unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001. All data are the mean ± SD. See also Figure EV1 and Table EV1-3.
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 2

Figure 2. The novel role of BCL9 in maintaining mitotic Wnt signalling to regulate proper cell division.

A Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.

B Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase.

C Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment.

D Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm.

E Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm.

F Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments.

G Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.

Data information: Significance was measured by two-tailed unpaired t-test, *p < 0.05, **p < 0.01, ***p < 0.001. All data are the mean ± SD. See also Figure EV2.
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 3

Figure 3. Dissecting the mitotic interactome of BCL9.

A Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2.

B DAVID functional annotation analysis of enriched biological process (BP) by the potential mitotic BCL9 interactome (normalised SILAC H/L ratio > 1.5).

C An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot.

D Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.

See also Fig EV3 and Table EV4.
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 4

Figure 4. BCL9 regulates the mitotic destruction complex and competes with clathrin for Axin1 complex binding.

A-B Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm. Significance was measured by two-tailed unpaired t-test, ***P < 0.001, the horizontal lines in boxplot indicate the medium reading of the data, box ranges from Min to Max, all data are the mean ± SD.

C Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.

D Schematic diagram of 20 truncated BCL9 forms designed for subsequent immunoprecipitation analysis.

E Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line.

F Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes.

G Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.

H Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.

See also Fig EV4.
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 5

Figure 5. BCL9 stabilises LRP6 signalosome components to maintain oncogenicity of the cancer cells by inhibiting clathrin.

A Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX.

B Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.

C Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.

D Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.

E Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.

F Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.

G A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.

H A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.

See also Fig EV5.
CDK1-Mediated BCL9 Phosphorylation Inhibits Clathrin to Promote Mitotic Wnt Signalling (2018)
Figure 6

Figure 6. CDK1-driven T172 phosphorylation stabilises BCL9 and regulates its interaction with clathrin.

A Venn diagram analysis of BCL9 mitotic phosphorylation sites in HeLa cells by mass spectrometry. B Schematic diagram of the position of two phosphorylation sites on the BCL9 N-terminal domain, and protein alignment analysis of the sequence around T172 in different species from http://www.uniprot.org.

C In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.

D Immunoprecipitation analysis of transiently-expressed BCL9-MF or T172A-MF interactors in synchronised cells, from G1S to G2 M phase. The relative level of BCL9-MF or T172A-MF immunoprecipitation is determined; red numbers indicate up-regulation, and green numbers indicate down-regulation compared to the starting time point.

E Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.

F Immunoprecipitation analysis of the interaction of the T172 inactive mutant T172A, the activation mutant T172D, and the double inactive mutant T172\T144 (2A) or activation mutant (2D) transiently-expressed in mitotic cells.

G Quantification for the inactive or activation mutant interactions indicated in Fig 6F. The wild type BCL9 interaction is normalised to 1, indicated by a green dashed line with an arrow.

H A schematic diagram of BCL9 phosphorylation regulation of its interactions.

See also Fig EV6.