EMBO/soda-nel huggingface dataset:
HER3 targeting with an Antibody Drug Conjugate Bypasses Resistance to anti-HER2 therapies (2020)
Figure 6

Figure 6. Effect of EV20/MMAF in cells resistant to neratinib, lapanitib and T-DM1.

(A-F) Dose-response to EV20/MMAF after 5 days of treatment in BT474, (A) cells resistant to neratinib (BTRN#5 and BTRN#24), (C) cells resistant to lapatinib (BTRL, BTRL#109 and BTRL#3) and (E) cells resistant to T-DM1 (BT-TDM1R#1 and BT-TDM1R#6). Levels of HER2, pHER2, HER3, pHER3, pAKT and calnexin of (B) cells resistant to neratinib, (D) cells resistant to lapatinib and (F) cells resistant to TDM1.

Data information: In (A, C, and E), represent mean + SD of triplicates of an experiment that was repeated at least twice, and normalized to untreated controls. In (A, C, and E), when error bars are invisible, they are covered by the graphic's symbols.

Expanded View Figure legends
Porin threading drives receptor disengagement and enables active colicin import through OmpF (2021)
Figure 4

Fig 4. ColE9 threading through the subunits of OmpF disengages the toxin from its outer membrane receptor, BtuB.

A, Cartoon depicting the basis for the control where excess ColE9 R-domain (residues 348-418, orange) blocks binding of vitamin B12 (red) to BtuB (grey), impairing growth of E. coli 113/3, a B12-dependent strain Penfold et al., 2000().

B, Cartoon schematic of the two possible translocon outcomes for binding of ColE9 W39A to BtuB and their implications on vitamin B12 import in E. coli 113/3. The W39A mutation within the TBE of ColE9 (orange) impairs TolB (blue) binding thereby uncoupling the OM components of the translocon from the energized TolQ-TolR-TolA complex in the inner membrane. If threading of the ColE9 IUTD through OmpF (green) has no impact on ColE9 binding to BtuB (grey) then vitamin B12 (red) cannot enter cells to support growth. In contrast, if OmpF threading disengages ColE9 from BtuB then vitamin B12 can enter cells to support growth.

C, Growth curves for E. coli 113/3 in defined media and in the presence or absence of B12 ( and o, r­­espectively). When challenged with 40 nM isolated R-domain ( ), cell growth was largely impaired whereas growth in the presence of 40 nM ColE9 W39A (▫) a­­pproached that of the no colicin control, consistent with OmpF threading causing the disengagement of ColE9 from BtuB. Error bars represent the standard deviation across three biological replicates.
Porin threading drives receptor disengagement and enables active colicin import through OmpF (2021)
Figure 5

Fig 5. ColE9* K469CAF647 translocation across the outer membrane is PMF dependent.

ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin.

A, Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469CAF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37 °C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469CAF647 fluorescence (red). Overlays of Hoechst and ColE9* K469CAF647 fluorescence are also shown. ColE9* K469CAF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell-associated ColE9* K469CAF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.

B, Normalized fluorescence intensity profiles across E. coli JM83 cell widths for CCCP and/or trypsin treated cells. Fluorescence was measured in ImageJ for 15 cells per condition and normalised to more clearly show distribution profiles for each condition. CCCP-treated cells showed peripheral ColE9* K469CAF647 fluorescence consistent with the colicin being bound at the OM. Trypsin treatment of cells in the absence of CCCP results in loss of this peripheral fluorescence but the presence of mid-cell signal consistent with internalisation. Treatment with CCCP and trypsin removed all ColE9* K469CAF647 fluorescence from cells, as in a. Error bars represent % SEM.

C, Box and whisker plots showing mean pixel intensities for ColE9* K469CAF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1st and 3rd quartile with the median shown as a line. From left-to-right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469CAF647 cell-associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469CAF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469CAF647 fluorescence. n, number of cells used, typically from 2-4 biological replicates. ****, indicates a t value <0.05 in a student T-test as a statistically significant result, ns indicates no significant difference as determined by student T-test.
Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function (2021)
Figure 1

Figure 1 - Localization and activity of BirA*-AURKA during cell cycle

A. Experimental workflow for the BioID experiment. HEK293T cells stably expressing V5-BirA*-AURKA and V5-BirA* were treated with 50 μM biotin for 18 hours, harvested and lysed by denaturing lysis. Biotinylated proteins were captured with streptavidin affinity beads and analyzed by mass spectrometry.

B. V5-BirA*-AURKA is expressed at near endogenous levels in the stable line. HEK293T cells and HEK293T::V5BirA*-AURKA lysates were immunoblotted with antibodies against V5, AURKA and vinculin (loading control).

C. Biotinylation of the centrosome and microtubules by V5-BirA*-AURKA in different cell cycle stages. After 18 h biotin incubation, HEK293T::V5-BirA*-AURKA were fixed and stained for the fusion protein with V5 antibody, biotinylated proteins with the fluorescent streptavidin and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 5 μm.

D. Control and biotin-treated HEK293T cells stably expressing V5-BirA* and V5-BirA*-AURKA were lysed and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and captured biotinylated proteins (pulldown) were run on an SDS gel and immunoblotted with the fluorescent streptavidin and antibodies against V5 and vinculin (loading control).
Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function (2021)
Figure 3

Figure 3 - AURKA has extensive proximity interactions with centrosomes and centriolar satellites

A-B Comparison of the AURKA proximity interactome with the proximity interactome of the centrosome (Gupta et al., 2015), the proximity interactome of centriolar satellites (Gheiratmand et al., 2019) and the physical interactome of centriolar satellites (Quarantotti et al., 2019).

C. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.

D. AURKA interacts with PCM1 in centriole-less cells. GFP-PCM1 or GFP-PCM1 was co-expressed with FLAG-AURKA in HEK293T cells treated with DMSO (control) or 500 nM centrinone B for 7 days. Complexes were precipitated with GFP-Trap beads. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against GFP and FLAG.

E. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.

F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm.

G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).
Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function (2021)
Figure 4

Figure 4 - Centriolar satellites regulate AURKA localization, abundance and activity in quiescent cells

A, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μm

C. Total AURKA and p-AURKA levels in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h. Following 24 h serum starvation, cell lysates were prepared and run on an SDS-PAGE gel. Proteins were detected by immunoblotting with antibodies against AURKA, p-AURKA and GAPDH (loading control).

D. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 1

Figure 1. ESM1 is required for the process of metastasis in PCa.

(A) Graph derived from two published data available in the PubMed GEO database (GSE6919 and GSE35988). The box charts depict the relative expression of ESM1 in benign, primary, and metastatic prostate cancer patients. The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers. P value was obtained by two-tailed Student's t test.

(B) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines. P: parental cells, M: metastatic cells.

(C) Cell invasion assays of 22Rv1-M and PC3-M cells transfected with either Scramble-shRNA or ESM1-shRNA. The data are shown as the relative fold change of invasive cells compared with the shScramble group. Scale bar: 100 μm.

(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.

(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles. P value was obtained by two-tailed Student's t test.

Data information: Differences in B and C are shown as fold change compared with control cells presented as the mean ± SD of three independent experiments. * P < 0.05, ** P< 0.01 when compared to control group by two-tailed Student's t test.
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 2

Figure 2. Nuclear ESM1, instead of secreted ESM1, promotes PCa invasion.

(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells.

(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.

(C) Schematic diagram of ESM1 expression constructs.

(D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively.

(E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells.

(F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm.

(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.

Data information: Differences are shown compared with control cells presented as the mean ± SD of three independent experiments. * P < 0.05, ** P< 0.01 when compared to control group by two-tailed Student's t test.
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 3

Figure 3. Nuclear ESM1 regulates PCa metastasis by promoting cancer stem cell properties.

(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ** P< 0.01 when compared to the vechicle group of 22Rv1-P cells by two-tailed Student's t test.

(B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ** P< 0.01 when compared to vector cells by two-tailed Student's t test.

(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.

(D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments. * P< 0.05 when compared to vector cells by two-tailed Student's t test.

(E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed.

(F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. * P < 0.05, ** P< 0.01 when compared to vector cells by two-tailed Student's t test.
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 4

Figure 4. Nuclear ESM1 drives PCa metastasis through β-catenin and NFκB signaling pathway.

(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.

(B) Known regulators of differentially expressed genes in the ESM1-NLS cells giving rise to the prediction of CTNNB1 as an upstream regulator. Observed upregulation and downregulation of mRNA are shown in red and green, respectively. The predicted relationships were shown as arrow lines with different color. The detail of predicted relationships of all the genes to the upstream regulator and the color intensity scale were described in the prediction legend.

(C) GSEA of RNA seq data demonstrating the enrichment of gene sets related to Wnt signaling in 22Rv1-P cells expressing ESM1-NLS. NES, normalized enrichment score. The p values for the GSEA test statistics are calculated by permutation. The original test statistics for the features are permuted and new test statistics are calculated for each category, based on the permuted feature test statistics.

(D) GSEA analysis identified β-catenin oncogenic signatures as the top induced pathways of PRAD in the PubMed GEO datasets (GSE3325 and GSE7930). NES, normalized enrichment score. The p values for the GSEA test statistics are calculated by permutation. The original test statistics for the features are permuted and new test statistics are calculated for each category, based on the permuted feature test statistics.

(E) The transcriptional activity of β-catenin in 22Rv1-P cells with different patterns of ESM1 overexpression. Bars are the mean ± SD of three independent experiments.

(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the mean ± SD of three independent experiments.

Data information: β-catenin and NFκB promoter reporter activity were normalized by comparison with Renilla luciferase activity. ** P< 0.01 when compared to vector cells by two-tailed Student's t test.
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 5

Figure 5. Nuclear ESM1 promotes PCa stemness through β-catenin signaling pathway.

(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. * P < 0.05, ** P< 0.01 when compared to vector cells by two-tailed Student's t test.

(B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. * P < 0.05, ** P< 0.01 when compared to vector cells by two-tailed Student's t test.

(C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated.

(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.

(E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.

(F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.
Direct interaction of β-catenin with nuclear ESM1 supports stemness of metastatic prostate cancer (2020)
Figure 6

Figure 6. ESM1 associates with the ARM domain of β-catenin.

(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.

(B) Nuclear or cytosolic extracts of 22Rv1-M cells were immunoprecipitated with an ESM1 antibody.

(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.

(D) Upper, mapping β-catenin regions binding to ESM1. Schematic diagram of full-length β-catenin and deletion mutants. Lower, mapping ESM1 regions binding to β-catenin. Schematic diagram of full-length ESM1 and deletion mutants.

(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.

(F) HEK293T cells were transfected with ESM1-HA constructs and pull-down was carried out with different purified GST-ARM fragments.

(G) HEK293T cells were transfected with the indicated ESM1-GFP constructs. Cell extracts were pulled down with purified GST-ARM.