EMBO/SourceData huggingface dataset:
Figure 7A. Survival of patients with colorectal cancer from Kaplan-Meier database was analyzed Left panel of A were representative of 150 technical replicates, and right panel of A were representative of 499 technical replicates.representative histology images in human colorectal cancer specimens (1#-5#) and human colon polyp specimens (6#-7#) were shown. Number of cells expressed BTNL2 or IL-22 was analysed in high power field (HPF), arrows indicate cells expressed BTNL2 or IL-22 (B).D-E. Expression of BTNL2 in normal colon tissue or DSS+AOM induced tumour tissue were measured by immunoblot (D) and real-time PCR (E) (n=5). Data information: All data are mean ± s.e.m. NS, not significant., **P < 0.01 based on two-tailed Student's t test for (E). Each dot represents one mouse, n=5.
Figure 1D HCT116, Lovo and RKO cells were transfected with control or p53 siRNAs. 48 h after transfection, total protein was then analyzed by western blot analysis. E HCT116, Lovo and RKO cells were transfected with empty vector and p53 plasmid. 48 h after transfection, total protein was then analyzed by western blot analysis.F Representative immunofluorescence staining of the p53 (green) and METTL14 (red) proteins in p53-WT HCT116, Lovo and RKO cells and p53-MT HT29 (p53R273H), SW480 (p53R273H/P309S), SW620 (p53R273H) and SW1116 (p53A159D) cells. Nuclei were stained with DAPI (blue). Scale bars = 10 μm. The relative mean fluorescence density was analyzed by ImageJ.I Western blot analysis of METTL14 levels after transfection with empty vector, wild-type or mutant p53 plasmids in HCT116 (p53-/-) cells.J Representative IHC images, and statistical analysis of immunoreactive score (IRS) of METTL14 expression in p53-WT (n = 63) and p53-MT (n = 41) CRC samples. The horizontal lines represent the median; the bottom and top of the boxes represent the 25% and 75% percentiles, respectively; and the vertical bars represent the range of the data. The insets show enlarged images of indicated p53-WT and p53-MT CRC tissues, respectively. Scale bars = 20 μm and 2 μm (inset).K ChIP assay verified the potential p53 binding site in the METTL14 promoter region in HCT116 and Lovo cell lines. Input fractions and IgG were used as controls.
Figure 2A Colony formation assay of p53-WT (HCT116, Lovo and RKO) and p53-MT (HT29, SW620, SW1116 and SW480) cells stably infected with shNC or shMETTL14. Data are presented as mean ± SD (biological replicates, n = 3; *P < 0.05, **P < 0.01, ns = no significance).B, C Representative images and analysis of tumors in nude mice bearing stably transfected shNC or shMETTL14 p53-WT (HCT116 and Lovo) and p53-MT (HT29 and SW620) cells. Data are presented as mean ± SD (biological replicates, n = 7, ns = no significance).G Comparison of colorectum length between METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models, respectively. Data are expressed as mean ± SD.I Representative HE staining images of colorectum in METTL14WT (n = 14, n = 24) and METTL14ΔIEC (n = 13, n = 21) mice from AOM/DSS-induced and AOM-induced CRC models. Lower panels show enlarged images of indicated normal or CRC tissues. Scale bars = 2 mm (upper) and 40 μm (lower). Black dashed line refers to the border of tumor (T) and normal (N) tissues. Tumors are classified as adenomas with low to focal high-grade dysplasia. The percentages of mice with dysplasia are shown (right).
Figure 3C Lactate production, ATP level, glucose uptake and pyruvate level in stably transfected Lv-vector and Lv-METTL14 or shNC and shMETTL14 HCT116 (p53-WT) cells. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01).H Western blot analysis of METTL14, SLC2A3 and PGAM1 protein levels of intestinal epithelial cells from AOM/DSS-induced Mettl14WT (n = 4) and Mettl14ΔIEC (n = 4) and mice.I Representative IHC staining images and quantitative analysis of SLC2A3 and PGAM1 in tumor tissues and non-tumor tissues from AOM/DSS-induced Mettl14ΔIEC and Mettl14WT mice CRC models. The insets show enlarged images of tumor tissues and non-tumor tissues, respectively. Scale bars = 40 μm and 4 μm (inset). Data are presented as mean ± SD (biological replicates, n = 6; **P < 0.01, ***P < 0.001).
Figure 4E Western blot analysis of SLC2A3 and PGAM1 protein levels in HCT116 and Lovo (p53-WT) cells transfected with control or miRNA mimics.F Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HCT116 (p53-WT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scale bars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; **P < 0.01). G Representative IF staining and quantitative analysis of the SLC2A3 (green) and PGAM1 (green) proteins in HT29 (p53-MT) cells transfected with control or miRNA mimics. Nuclei were stained with DAPI (blue). Scalebars = 20 μm. Data are presented as mean ± SD (biological replicates, n = 4; ns = no significance).J qRT-PCR analysis of the pri-miR-6769b and pri-miR-499a levels in stably transfected Lv-vector and Lv-METTL14 HCT116 cells with control, YTHDF1-3, YTHDC1-2 or IGF2BP1-3 siRNAs. Data are presented as mean ± SD (biological replicates, n = 3; **P < 0.01). The dotted line represents basal mRNA expression of pri-miR-6769b and pri-miR-499a in HCT116 cells, acting as a control.N Representative images of tumors and analysis in nude mice intervened with control, miR-6769b-3p or miR-499a-3p expression p53-WT (HCT116 and Lovo) cells. Data are presented as mean ± SD (biological replicates, n = 7). O Representative images of tumors and analysis in nude mice intervened with control, miR-6769b-3p or miR-499a-3p expression p53-MT (HT29 and SW620) cells. Data are presented as mean ± SD (biological replicates, n = 7, ns, no significance).
Figure 5A Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-6769b-3p mimics or inhibitor for 48h. B Glucose uptake, Lactate, ATP, pyruvate levels were determined in HCT116 (p53-WT) cells treated with control, miR-499a-3p mimics or inhibitor for 48h.E Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture inhibitors for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment. F Glucose uptake, Lactate, ATP and pyruvate levels were determined in stably transfected Lv-vector and Lv-METTL14 HCT116 (p53-WT) cells treated with control or mixture mimics for 48h. Western blot analysis of the corresponding METTL14, SLC2A3 and PGAM1 protein levels in indicated treatment.I, J Representative images and analysis of tumors in nude mice generated by stably transfected shNC and shMETTL14 or miR-6769b-3p and miR-499a-3p overexpressing HCT116 (p53-WT) cells with or without METTL14 knockdown. Data are presented as mean ± SD (biological replicates, n = 7, ***P < 0.001).
Figure 6A Representative IHC and ISH staining images and corresponding quantitative analysis of miR-6769b-3p/SLC2A3 IRS and miR-499a-3p/PGAM1 IRS in p53-WT (n = 63) and p53-MT (n = 41) samples from Cohort 3. The insets show enlarged images of indicated p53-WT and p53-MT CRC tissues, respectively. Scale bars = 200 μm and 20 μm (inset). The horizontal lines represent the median; the bottom and top of the boxes represent the 25% and 75% percentiles, respectively; and the vertical bars represent the range of the data.B Representative IHC and ISH images of METTL14, SLC2A3, PGAM1, miR-6769b-3p and miR-499a-3p in CRC tissues with higher or lower METTL14 expression in p53-WT (n = 63) samples from Cohort 3. Right panels show enlarged images of indicated p53-WT CRC tissues. Scale bars = 200 μm (left) and 20 μm (right).F Kaplan-Meier survival curves of OS in CRC patients with wild type p53 (n = 63) and mutant p53 (n = 41) from Cohort 3 database based on expression levels of METTL14.
Figure 1C) Immunofluorescence image of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in C maximum intensity projections taken across the depth of the EGFP foci.E) Quantification of CENP-T signal intensity at MUC4 ectopic foci vs. endogenous centromeres, normalised to CENP-T signal intensity at endogenous centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).H) Quantification of KNL‑1 signal intensity at MUC4 foci, normalised to KNL‑1 signal intensity at centromeres (=1, red line). LinesLines = Mean ± SD, 3 experiments, each with ≥ 10 metaphases analysed per condition. Each point = 1 focus or centromere. ns = p>0.05, *** = p<0.01, **** = p<0.001 (Kruskal Wallis test with Dunn's multiple comparison correction).
Figure 2Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against CENP-T. Images in are maximum intensity projections taken across the depth of the EGFP foci.Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. F) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against KNL-1. Images in ,F are maximum intensity projections taken across the depth of the EGFP foci.Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. I) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted cells stained with antibodies against Ndc80. Images in I) are maximum intensity projections taken across the depth of the EGFP foci.
Figure 3Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against alpha-tubulin. B) Immunofluorescence images of mitotic cells ­­­with CENP‑T∆C‑dCas9‑EGFP targeting after nocodazole treatment. C) Length measurements of individual EGFP foci in µm. Example measurements are indicated on zooms in A-B) in orange. Data is ≥20 cells per condition, from 1 experiment. Lines = Mean ± SEM. * = p<0.05, ** = p<0.01,**** = p<0.001 (Kruskal-Wallis with multiple comparison correction). Images in A,B are maximum intensity projections taken across the depth of the EGFP foci. In A,B) Orange bars = measured lengths in C.Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. D) Immunofluorescence images of metaphase cells with CENP-T∆C-dCas9‑EGFP targeting after cold treatment, stained with antibodies against alpha-tubulin. E) Percentage of metaphase cells showing microtubule (MT) recruitment. ≥50 metaphases per condition, from 1 experiment.. are maximum intensity projections taken across the depth of the EGFP foci.Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN in HEK293T cells. F) Frames from live cell imaging of cells with dCas9 targeted to Chr9-CEN, or an EGFP negative cell. are maximum intensity projections taken across the depth of the EGFP foci.
Figure 4Targeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. A) Immunofluorescence images of paired EGFP foci for each attachment status category. Images were chosen where both the EGFP foci and any of their attachments lay in the same Z-plane for clarity of presentation. B) Percentage of paired EGFP foci showing each attachment status. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment forTargeting of dCas9-EGFP or CENP‑T∆C‑dCas9‑EGFP to Chr9-CEN and Chr1‑TELO in HEK293T cells. C) Immunofluorescence images of CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2. D) Percentage of individual EGFP foci showing EGFP and Mad2 signal co-localisation, in metaphase cells with CENP‑T∆C-dCas9-targeting. 2 experiments for (D). Image in C) is a maximum intensity projection taken across the depth of the EGFP signal. Scale bars = 1µm on small images and zooms, 5µm on larger images in C).Immunofluorescence images from CENP‑T∆C‑dCas9‑EGFP targeted metaphase cells stained with antibodies against Mad2 and alpha-Tubulin, showing examples of microtubule attached (G) EGFP foci. H) Percentage of individual EGFP foci with EGFP and Mad2 signal co‑localisation attached (H) EGFP foci. are from ≥75 EGFP foci pairs from n≥15 metaphases per condition 1 experiment for H) are single Z-slices.
Figure 5C,D) Example images of chromosome mis-segregation events involving the target locus (EGFP signal) in anaphase HEK293T (C) or HCT116 (D) cells with CENP-T∆C-dCas9‑EGFP targeting after Mps1i pulse. E,F) Mis‑segregation rate in fixed dCas9-targeted HEK293T (E) or HCT116 (F) cells after Mps1i pulse, involving only EGFP+ chromosomes, only EGFP- chromosomes or both. Bars = mean ± SD, 3 experiments for each cell line, each with ≥25 anaphase cells per condition. * = p<0.05, **= p<0.01, *** = p<0.001, **** = p<0.0001 (Oneway ANOVA with Šidák's multiple comparison correction, in E/F) comparing the mis-segregation rate for any events involved EGFP).G-H) Frames from live cell imaging of HEK293T H2B-RFP cells after treatment with Mps1i (t=0), with CENP‑T∆C‑dCas9‑EGFP targeted to Chr9-CEN or Chr1-TELO. Frames are either across the duration of the 4 h movie (G) or show examples of the different categories of fate (H). I) Percentage of CENP-T∆C-dCas9‑EGFP targeted cells that made an EGFP+ error in anaphase, categorised by subsequent fate of EGFP signal in daughter cells 3 h after Mps1i addition. Assessed by live cell imaging. ≥22 cells with error per condition, combining data from ≥3 independent experiments.
Figure 1(B) Slice through a tomogram showing EBOV virions inside a late endosomal compartment.(I-J) Magnified areas highlighted in (F) and (H), respectively, showing the viral membrane and VP40 densities at the luminal side. For comparison, line profiles at 3 nm distance from the inner membrane monolayer, visualized by dotted profiles (magenta and blue, respectively), were determined. (K) Line profiles adjacent to the inner viral membrane leaflet of a virion inside an endosome and a purified virion before infection.
Figure 2(G-J) Slices of a tomogram showing a filamentous EBOV VLP composed of VP40 and GP after incubation at low pH (n=18). White arrows in (H) and (J) highlight areas adjacent to the VLP membrane devoid of protein densities in contrast to corresponding slices of VLPs at neutral pH.(K) Slices of tomograms showing filamentous VLPs composed of VP40 after incubation at neutral (n=22) and low pH (n=8), respectively. (L) Line density profiles determined adjacent to the inner membrane monolayer of VLPs incubated at neutral (blue) and low pH (magenta). At neutral pH, the VP40 matrix detectable as regular densities in (D) have a 5-6 nm pitch.
Figure 3(E) Free energy profiles of VP40-lipid interactions at pH 7.4 and pH 4.5 determined from MD simulations. The plot shows free energy (in kBT) at increasing membrane-VP40 distances (nm) with indicated three states shown in (A).(F) EBOV VLP lipid composition showing highly abundant lipids determined by mass spectrometry in mol%. Lipid abbreviations: phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lyso-phosphatidylcholine (LPC), sphingomyelin (SM). Prefix "a" indicates acyl-linked glycerophospholipids, prefix "e" indicates ether-linked (plasmanyl) or the presence of one odd and one even chain fatty acyl. Bars represent mean and error bars represent standard error of the mean (red); n=3 biological replicates.
Figure 4(B) Overview confocal fluorescence microscopy image showing pleomorphic pHluorin-labelled VLPs composed of VP40, pHluorin-VP40 (ratio 10:1) and GP. (C) Magnified images of representative VLPs acquired during time-lapse microscopy at neutral pH and after acidification to approximately pH 5. Frames are exemplarily shown at 0, 1, 3, 5 and 10 min after lowering the external pH.(D) Plot showing the mean relative fluorescence intensities and standard deviation of VLPs imaged at neutral pH, low pH and in the presence of T-X100 at low pH over time. Data was obtained from 3 independent VLP preparations. Number of technical replicates: n=42 (pH 7.4); n=19 (pH 4.5 and pH 5 + T-X100).(E) Plot showing the drop of pH inside VLPs over time after lowering the pH of the surrounding buffer to 5. The dots represent the mean values, and the dashed lines are the theoretical fit to Eq. 3.
Figure 5(B) Stalk formation energy as a function of VP40-membrane interaction energy. The stalk energy for non-interacting VP40 matrix (u0 = 0) is 65 kBT. VP40 matrix layer Young's modulus legend - red 5.6 MPa, blue 11.2 MPa, black 16.8 MPa, and orange is infinitely rigid. The bending rigidity ratio between the VP40 matrix layer and lipid monolayer are 1, 2, 3, and infinity, respectively. The line represents an infinitely rigid layer.(D) Fusion pore formation energy as a function of VP40-membrane interaction energy. The discontinuity in the energy is located at the phase line between configurations (see C). The change in pore formation energy, ΔEpore is defined as the difference between fusion-pore formation energy at interaction energy density 0.2 kBT/nm2 (the value found using MD simulations) to the matrix-free case (no interaction energy). A-D) Dotted lines serve as a guide to the eye.(F) Quantification of FACS data showing EBOV VLP entry as measured by a fluorescence shift of infected cells from emission at 510 nm (no entry) to 450 nm (entry). VLPs were treated prior to infection as indicated on the x-axis, with control: uninfected control cells, - : no treatment, T: thermolysin-treatment at neutral pH, Lp: low pH treatment. Target cells were treated with media or ammonium chloride (NH4Cl), n= 3 with 10'000 cells measured per sample.
Figure 1E. Survival curves of mice challenged with SARS-CoV-2 Wuhan-D614G at 104 PFU, after a primo-infection by BANAL-236 or Wuhan-372 viruses at 103 or 104 PFU. Survival of K18-hACE2 naive mice infected by SARS-CoV-2 Wuhan-D614G at 104 PFU is presented as a reference (yellow).F. Histopathological analysis, 3 days post-inoculation, of the lung of K18-hACE2 mice infected with 104 PFU of BANAL-236 (a-d) or SARS-CoV-2 Wuhan-372 (e,f) viruses. Interstitial pneumonia characterized by interstitial inflammation, often centered on blood vessels (perivasculitis, black arrows) or bronchi/bronchioles (black arrowheads), and endothelial cell injury and inflammation (endothelitis). Lesions were globally more severe after Wuhan-372 infection (n=4; all of moderate severity) than after BANAL-236 infection (n=4; 3/4 of minimal severity (a-c) and 1/4 of moderate severity (d)). Scale bar: 100 µ. Low magnification images are provided in Figure S3.
Figure 2Neutralizing antibody titer (expressed in ED50) of mice primo-infected at 103 (left) or 104 (right) PFU by BANAL-236 (red) or SARS-CoV-2 Wuhan-372 (blue) viruses. The antibody response against the Wuhan-D614G challenge strain of mice primo-infected by BANAL-236 is presented as a dashed line. PNT means pseudo-neutralization.
Figure 3B. Effects of infection by BANAL-236 virus in non-human primates. Animal #MF1 is presented in green and animal #MF2 in purple. Clinical score, body weight variation (in %), CT scores (compared to Wuhan-372) and neutralizing antibodies are measured from 10 days before infection to 43 days post-infection.C. Viral load expressed in log10 copies of genomic RNA (solid line) or sub-genomic RNA (dashed line) per mL of tracheal, nasopharyngeal, BAL and rectal fluids. Lower limit of quantification is presented in red for both genomic (solid line) and sub-genomic (dashed line) RNA, and limits of detection are presented in green for both genomic (solid line) and sub-genomic (dashed line) RNA. Results were compared to historical data of SARS-CoV-2 Wuhan-372 virus inoculated at 106 or 105 PFU (presented as the mean virus load +/- 95% CI expressed in log10 copies of genomic RNA/mL).
Figure 5(A-C) Results of Pseudoneutralisation (PNT), Luciferase immunoprecipitation (LIPS) and LuLISA tests are shown. Pre-pandemic French sera (black) were used as negative controls and Laotian sera samples from confirmed SARS-CoV-2 infection (green) were used as cross-reacting positive controls. Laotian sera samples collected in the general population before 2019 (blue, n=100) or late 2020 (gray, n=100) or in people exposed to bats (purple, n=74) were tested for BANAL-236 (A), BANAL-52 (B) and BANAL-103 (C) antibody responses. The ANOVA non-parametric Kruskal-Wallis test was conducted to compare each sub-population to the reference French population (* P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001). The red bars represent the median.
Figure 1(A)- (D) Hepatocytes accumulate apical bulkheads upon bile duct ligation (BDL) and MDR2 KO. (A)-(B) Overview images of (A) murine liver tissue after control surgery or bile duct ligation (BDL) for 24h (N=3 biological replicates), or (B) control and MDR2 KO mice after 12 weeks (N=3 biological replicates). Immunofluorescence for the apical membrane marker CD13 (magenta), F-Actin (green) and nuclei (grey). Representative images are shown. Scale bar 10 µm. (C)-(D) High-resolution imaging of individual bile canaliculi in murine liver tissue (C) after control and 24h BDL surgery, or (D) in control and MDR2 KO mice, stained for F-Actin (green). Representative images are shown. Scale bar 2 µm. Apical bulkheads are highlighted with red arrowheads.Apical bulkheads in the adult liver resemble those described in the embryonic liver. (E) Transmission electron microscopy (TEM) longitudinal 80 nm-thin serial section on liver tissue with BDL for 24h shows the ultrastructure characteristic of apical bulkheads. Representative images are shown. Scale bar 1 µm. I-III represent consecutive sections with 460 nm distance showing four apical bulkheads (B1-B4) sealed by electron-dense tight junctions. Scale bar 1 µm.Hepatocytes form aberrantly dilated canaliculi formed by >3 cells after BDL. (I) Transmission electron microscopy (TEM) in liver tissue after 24h BDL. Scale bar 10 µm. Bile canaliculi (BC) can be severely dilated similar to the size of sinusoids (S) and are formed by multiple hepatocytes (see inset A, scale bar 1 µm). Apical bulkheads (marked with red arrowheads) are visible. Individual hepatocytes are part of a liver cell rosette with one part of their apical membrane while they form normal canaliculi with apical bulkheads at another pole (see inset A, cell 3 and 4).
Figure 2(A)-(B) Hepatocytes accumulate apical bulkheads in the liver tissue of primary sclerosing cholangitis (PSC) patients (N=3 control patients and N=4 PSC patients). (A) Overview images in liver tissue of control and PSC patients. Immunofluoresence for the apical membrane marker BSEP (magenta), F-Actin (green) and nuclei (grey). Scale bar 10 µm. (B) High-resolution imaging of F-Actin (green) showing individual bile canaliculi in control or PSC liver tissue. Red arrowheads mark apical bulkheads. Scale bar 2 µm.(C) Different lumen morphologies of a typical bile canaliculus (left column) and bile duct (middle column) in control patients and rosette (right column) in PSC patients. Immunofluorescence for BSEP (magenta) and F-Actin (green). Shows also differences dependent on the imaging plane i.e. transversal (top row) or longitudinal (bottom row) cut of the lumen. White dashed lines mark the lumen. Scale bar 2 µm.(F) Liver cell rosettes in PSC patients are formed by hepatocyte-like cells (N=3 control patients and N=4 PSC patients). Immunofluoresence for BSEP (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes in PSC patients (right). Scale bar 10 µm.