EMBO/soda-nel huggingface dataset:
Centromere Localization and Function of Mis18 Requires 'Yippee-like' Domain- Mediated Oligomerization (2016)
Figure 4

Figure 4. Dimerization mediated by the MeDiY domain promotes spMis18 function and centromere association.

A. spMis18fl-Myc co-immunoprecipitates with both GFP-tagged spMis18fl and spMis18MeDiY, but shows reduced association when MeDiY-mediated dimerization is disrupted in spMis18flI31A. The asterisk (*) in the top panel denotes the IgG heavy chain.

B & C. Dimer-II interface mutations I31A and Y114A affect the ability of ectopically expressed spMis18fl to rescue the temperature sensitivity of mis18-818 and mis18-262 cells, while expression of spMis18MeDiY alone confers a dominant negative effect on growth in a MeDiY dimerization-dependent manner. Five-fold serial dilutions of cells expressing the indicated spMis18 constructs integrated at the leu1 locus in the genome, spotted on complete PMG + Phloxine B media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink.

D. Mutations that disrupt MeDiY dimerization lead to reduced levels of spMis18fl association with centromeres. qChIP analyses of spMis18fl-GFP association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates.
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level (2015)
Figure 1

Figure 1: An UnaG-based hypoxia sensor efficiently indicates hypoxic growth conditions in CHO cells.

a) Schematic representation of sensor constructs for the hypoxia-inducible expression of fluorescent proteins. Five HRE-sequence motifs derived from the enhancer region of the Vegfa gene confer hypoxia-specific activity upon the CMV minimal (mCMV) promoter. Fluorescent proteins are destabilized by in frame fusion of a PEST sequence from mouse ornithine carboxylase gene. The EGFP-encoding construct was obtained from Addgene (#46926).

b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.

b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.

c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.

d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm

e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.

f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.

b, c, e, f ) flow cytometric measurements, all experiment represent 3 independent repeats, bars show averages ± standard errors. MFI, mean fluorescence intensity
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level (2015)
Figure 2

Figure 2: UnaG-based hypoxia sensor kinetics can be tuned by fusion to different protein destabilization domains.

a) Schematic representation of the different variants of UnaG used in the four indicated hypoxia-reporter constructs. dUnaG is identical to Fig. 1a, oUnaG is destabilized by in frame fusion with the oxygen-dependent degradation domain sequence (ODDD) from HIF1a (aa338-608), doUnaG is destabilized by both degradation motifs.

b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.

c) Flow cytometric analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.

d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level (2015)
Figure 3

Figure 3: An UnaG-based hypoxia sensor faithfully marks areas of HIF-1α stabilization in a mouse model of glioblastoma growth.

a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCID mouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).

b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.

c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).

d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α . The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.

e) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCID mouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.

f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level (2015)
Figure 4

Figure 4: Retrospective assessment of the recent HIF-1 activity in individual cells by an UnaG-mOrange hypoxia-reoxygenation fusion sensor.

a) Schematic representation of the dUnOHR hypoxia-reoxygenation sensor. A PEST-destabilized fusion protein of UnaG and mOrange is expressed from the hypoxia sensitive HRE-mCMV promoter. Under hypoxic conditions only the UnaG component of the fusion protein is capable of adopting the fluorescent state, while mOrange is expressed and folds, but fails to mature, which requires higher oxygen concentrations.

b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.

c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.
A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level (2015)
Figure 5

Figure 5: Preferential localization of cells that have undergone recent reoxygenation to the edge of hypoxic areas.

a) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.

b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.

c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.

d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.

e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.
Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry (2020)
Figure 1

Figure 1: FKK5 and FKK6 activate PXR.

A, B (A) Histogram (mean, 95 %CI) of fold induction of PXR activity reporter assay (luc, luciferase) in LS180 cells transiently transfected with wild-type PXR and p3A4-luc reporter plasmids. (B), same as (A), in HepG2 cells stably expressing pCYP1A1 luciferase plasmid (AhR reporter). Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. Indole is colored blue. (A, B), The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.

C, Histogram (mean, 95 %CI) of fold mRNA expression, CYP1A1 (top panel), CYP3A4 (middle panel) and MDR1 (bottom panel) in LS180 cells with or without (mock) transfected PXR plasmid, HepaRG hepatic progenitor cells (PXR-knockout, PXR-KO; AhR-knockout, AhR-KO; parental control 5F clone) and primary human hepatocytes (HEP) from four donors is shown. The bar graph represents one experiment of a series of experiments (n > 3) performed in four consecutive passages of LS180 cells; n = 2 independent experiments with one well/compound and RT-PCR performed in triplicate for each HepaRG genotype; for each donor hepatocyte, n = 1 well/compound and RT-PCR performed in triplicate. *, #p < 0.05, two-way ANOVA with Tukey's post hoc test. *significant over vehicle control. #significant over the same treatment in corresponding mock transfected or knock-out cells.

D, Chromatin Immunoprecipitation (ChIP) assay in LS174T cells. Top panel, PCR products cells exposed to vehicle or FKK6 and run on a 2% agarose gel. DNA 100 base pair marker; Vehicle,10% DMSO; FKK6 (10 μM); 1/10 Input - 0.2 million cells before IP; IgG - IP with polyclonal rabbit IgG; PXR - IP with PXR antibody. Bottom panel, quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH (fold expression). Dash line, 2-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biological replicates, n = 4 technical replicates). Mean fold expression is expressed above each histogram (mean + SD). E, PXR TR-FRET assay. TR-FRET ratio 520/495 nm are plotted against concentration of compound(s). Half-maximal inhibitory concentrations IC50 were obtained from interpolated standard curves (sigmoidal, 4PL, variable slope); error bars show standard deviation of n = 2 independent experiments each with four technical replicates.

F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.
Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry (2020)
Figure 2

Figure 2: FKK5 and FKK6 inhibit NF-κB activation in a PXR-dependent manner.

A, PXR (luciferase) reporter assay in HEK293T cells transiently transfected with PXR plasmids (wild-type and ligand binding domain mutant C285I/C301A). RLU, relative light units are shown normalized to beta-galactosidase (β-Gal) expression. The histogram represents one experimental data (of n > 2 independent experiments each performed in quadruplicate) mean (95 %CI); n.s. not significant; * p < 0.05, two-way ANOVA.

B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates).

(C-E) Points and error represent mean (95 %CI) values for control (TNFα 5 ng/ml) normalized NF-κB (luciferase) reporter activity in LS180 cells after C, Passage 13 (P13) and D, Passage 8 (P8) and in E, LS174T PXR-KO (knockout) cells transiently transfected with pNL3.2.NF-κB-RE vector. C, D, Line graph depicts one representative experiment of a series of experiments (n > 4) performed in four consecutive passages of cells. Data was expressed as mean with 95% CI of four technical replicates. E, mean (+ SD). *, ** p < 0.05, two way ANOVA with Tukey's multiple comparisons test.
Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry (2020)
Figure 3

Figure 3: FKK5 and FKK6 inhibit cytokine and salmonella infection induced NF-κB nuclear translocation.

A In Caco-2 monolayer cells, Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations.

B, Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation.

C, confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm.

D-F In human intestinal organoids (HIO), D-Fare identical to A-C. Scale bars (x-y, bottom left) = 70-100 µm

Data information: The histogram, mean with 95% CI, depicts combined data from biological replicates (n = 3) with three consecutive passages of cells. Each biological replicate has three technical replicates. Results were expressed as. * p < 0.05 , two-way ANOVA with Tukey post hoc test. * for cytokines versus no cytokines, compares either the 12 hr, 6hr, 4hr or 2 hr equivalent time-point(s) as indicated; * for cytokines versus cytokines + FKK compound, compares either the 12hr, 6hr, 4hr or 2hr equivalent time-point(s) as indicated. c, Arrows represent localization of NFĸB (red, cytoplasmic; purple, nuclear). Confocal images shown are representative images from (n = 3) biological replicates.
Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry (2020)
Figure 4

Figure 4: FKK6 inhibits cytokine mediated IL8 induction in human colonic organoids and induces PXR target genes in mice.

A-C Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without A, FKK5 (n = 9 paired samples). B, FKK6 (n = 3 paired samples; one removed due to poor inflammatory response). C, FKK9 (n = 5; 3 removed due to poor response and tissue integrity). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.

D-F Fold induction (mRNA) of genes in D, C57BL/6 mice, E, pxr -/- mice, and F, hPXR mice (mice expressing the human PXR gene) gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks (D, E) basal fold expression and (F) 2-fold gene induction. * p < 0.05, two-way ANOVA with Tukey's post hoc test.
Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry (2020)
Figure 5

Figure 5: FKK6 abrogates Dextran Sulfate Sodium induced colitis in mice expressing the human PXR gene.

A-F After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration.

A, % weight loss from baseline (day 1 vs day 10) B, colon length (cm) C, Inflammation score (see methods) D, Fecal lipocalin 2 (pg/ml) E, serum FITC-dextran (μg/ml).

Data information:

A-C, mean (95 %CI).

D-E, median (interquartile range). F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate). (A-E), The entire experiment was repeated three independent times and one representative experiment is shown. *(A, B, C) p < 0.05, Welch's t-test ; *( D, E) p < 0.05, Mann-Whitney test; ns, not significant; (F), *compares FKK6 versus vehicle control for each gene, p < 0.05, two-way ANOVA
Amyloid aggregates accumulate in melanoma metastasis modulating YAP activity (2020)
Figure 3

Figure 3. Oxysterols quantification in primitive and metastatic melanoma cells and PMEL expression in IGR37 and IGR39 melanoma cell line.

A. Absolute quantitation of 24, 25 and 27-hydroxycholesterol in primitive and metastatic melanoma cells as indicated. (T-test analysis, ***p-value<0.001, N=3 biological replicates, data are mean ±SD).

B. Histogram representing R square of correlation analysis between absolute quantitation of 24, 25 and 27-hydroxycholesterol and label free quantitation of APOE in a cohort of primitive (A375, IPC298) and metastatic (SKMEL5, SKMEL28) melanoma cell lines.

C. Confocal fluorescence images of anti-HMB45 PMEL antibody signal (green) and DAPI staining (blue) in IGR37 and IGR39. Scale bar is 10µm.

D. Western blot on IGR37 and 39 cellular lysates probed with anti-HMB45 PMEL antibody and anti HDAC2 to check the loading of similar amount of total lysates.
Amyloid aggregates accumulate in melanoma metastasis modulating YAP activity (2020)
Figure 5

Figure 5. Secretome analysis of IGRs upon BACE inhibition.

A. Unsupervised hierarchical clustering of the proteins identified and quantified in IGR37 and IGR39 upon treatment with DMSO or NB-360.

B. Volcano plot of the proteins secreted by IGR37 cells treated with DMSO or NB-360.

C. KEGG enrichment pathway analysis of the significantly regulated proteins upon BACE inhibition in both IGRs. D. Confocal fluorescence images of Proteostat signal (1:2000, red spots) and DAPI staining (blue), scale bar is 10µm.

E. Quantitation of protein aggregates in IGRs by immunofluorescence analysis using Fiji software. (T-test analysis, ** = p-value<0.01, N=3 biological replicates, data are mean ±SD).

F. Quantitation, by immunofluorescence analysis, of YAP in different cellular compartments (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD). Images were quantified by subdividing cells into mostly cytosolic YAP (Cytosol), mostly nuclear YAP (Nuclear), or equal distribution (Total) from three biological replicates.

G. mRNA levels of CTGF measured by real-time PCR in IGR37 treated with DMSO or NB-360 (T-test analysis, * = p-value<0.05, N=3 biological replicates, data are mean ±SD).

H. CTGF mRNA level measured by real-time PCR in IGR39 treated with IGR39 conditioned medium (CTRL) or with IGR37 conditioned medium (CM), N=4 biological replicates. T-test analysis, *** p-value <0.001, data are mean ±SD.

I. CTGF mRNA level measured by real-time PCR in IGR39 supplemented with recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis: *** p-value <0.001, data are mean ±SD.

J. CTGF mRNA level measured by real-time PCR in IGR37 treated with DMSO, NB-360 or NB-360 plus recombinant PMEL amyloid fibrils (0.5µM), N=3 biological replicates. T-test analysis* = p-value<0.05, ** = p-value<0.01. Data are mean ±SD.