EMBO/SourceData huggingface dataset:
Figure 5(A) The interaction of WASH with Beclin 1 was validated by yeast two‐hybrid assays. Yeast strain AH109 was co‐transfected with Gal4 DNA‐binding domain (BD) fused WASH and Gal4 activating domain (AD) fused Beclin 1. p53 and large T antigen or a known WASH interactor Bloc1s2 was introduced as positive controls.(B, C) WASH co‐immunoprecipitates Beclin 1 in mammalian cells. Flag‐tagged Beclin 1 and Myc‐tagged WASH were co‐transfected into HEK293T cells and immunoprecipitations were performed at 24 h post transfection. IP, immunoprecipitation.(D) Mapping the interacting regions between WASH and Beclin 1.(E) The indicated Flag‐tagged Beclin 1 constructs encoding different regions of Beclin 1 were co‐transfected with full‐length HA‐tagged WASH (HA-WASH) into HEK293T cells followed by immunoprecipitation.(F) WASH directly interacts with Beclin 1. Recombinant MBP‐tagged WASH (MBP-WASH) or Beclin 1 (rBeclin 1) was expressed in E. coli and then subjected to MBP pull‐down assay.(G) GST-Beclin 1 precipitates WASH from HeLa cell lysates. GST‐Beclin 1 or GST was bound to GST beads and incubated with HeLa cell lysates followed by immunoblotting.(H) Endogenous WASH colocalizes with Beclin 1 via confocal microscopy. HeLa cells cultured in CM or EBSS for 1 h were stained with antibodies against Beclin 1 and WASH. The colocalization rate (Pearson's correlation coefficient) between Beclin 1 and WASH was calculated and shown as means±s.d. in the lower panel. Scale bar, 10 μm.(I) HeLa cells were treated with CM or EBSS for 1 h, followed by immunoprecipitation with antibodies against WASH (left panel) or Beclin 1 (right panel), and the immunoprecipitates were detected with the indicated antibodies.***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.
Figure 6(A) Endogenous Beclin 1 is ubiquitinated through K63 linkage during the process of autophagy. EBSS cells starved in EBSS for the indicated times were lysed and immunoprecipitated with anti‐Beclin 1 antibody. Immunoprecipitates were dissociated with 1% SDS and re‐immunoprecipitated with anti‐Beclin 1 antibody, followed by immunoblotting with antibody specific for lysine 63‐linked poly‐ubiquitin chains (anti‐K63‐Ub). ((B) The autophagy‐specific Beclin 1 is ubiquitinated through K63 linkage. After starved in EBSS for 1 h, HeLa cells were lysed and HeLa with anti‐Atg14L antibody before dissociated with 1% SDS. Immunoprecipitates were further precipitated with anti‐Beclin 1 antibody, followed by immunoblotting with anti‐K63‐Ub antibody. The left panel denotes the scheme for separation of autophagy‐specific Beclin 1. The right panel indicates ubiquitination state of autophagy‐specific Beclin 1.(C) Lysine 437 mutation to arginine (K437R) abrogates ubiquitination of Beclin 1. Different constructs of Flag‐tagged Beclin 1 mutants were co‐transfected with HA‐tagged ubiquitin (HA-Ub) into HEK293T cells.(D) Beclin 1 (WT) or K437R‐Beclin 1 (K437R) mutant was stably expressed in Beclin 1‐silenced HeLa cells. Beclin 1‐knockdown HeLa cells were infected with lentivirus encoding 3XFlag‐tagged WT Beclin 1 (WT) or K437R‐Beclin 1 (K437R).(E) K437R‐Beclin 1 mutant abrogates the enhanced interaction of Beclin 1 with Vps34 during autophagy. HeLa cells expressing 3XFlag-Beclin 1 or 3XFlag-K437R‐Beclin 1 were treated with EBSS for 1 h, and harvested for immunoprecipitation.(F) Vps34 kinase activity is impaired by K437R‐Beclin 1 mutant. HeLa cells were transfected with the indicated vectors for 24 h and immunoprecipitated with anti‐HA antibody followed by a Vps34 kinase assay. Immunoprecipitates were separated into two equal parts, one for kinase assays and the other for input detection.(G) K437R‐Beclin 1 reduces the activation of Vps34. Beclin 1‐silenced HeLa cells stably expressing GFP‐HeLa were rescued with WT‐ or K437R‐Beclin 1, followed by stimulation with 1 h for 1 h. GFP‐WIPI1 dots were visualized by confocal microscopy.(H) K437R‐Beclin 1 mutant fails to induce autophagy after starvation. Beclin 1‐knockdown HeLa cells expressing WT or K437R mutant of Beclin 1 (K437R) were starved with EBSS for the indicated times.(I) K437R‐Beclin 1 mutant suppresses autophagosome formation. Beclin 1‐knockdown HeLa cells expressing WT‐Beclin 1 or K437R‐Beclin 1 were infected with lentivirus encoding GFP‐LC3, and stimulated with EBSS for 1 h in the presence or absence of 20 nM BafA1. GFP‐LC3 dots were visualized by confocal microscopy. Scale bar, 10 μm. Data are shown as means±s.d. **P0.01 and ***P0.001. All the above experiments were repeated for at least three times with similar results.Source data for this figure is available on the online supplementary information page.
Figure 1(C) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype (left). Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting (right). Cropped blots are shown
Figure 3(F) Representative PCR amplification of MYC, TP53R249S, KRASG12D, NRASG12D, CTNNB1S45F, BRAFV600E, AXIN1G652S, IL6, PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC-PHHs (Tumor) and their corresponding overexpression vector plasmids as positive controls (OCs)(I) Expression of MYC, p53, and KRASG12D was detected in the iHCC samples (n=3, biological replicates) by Western blotting. Cropped blots are shown
Figure 4(B) Genomic DNA of colonies that were derived from single cells of iHCC1-1 and iHCC2-1 tumor samples were extracted for PCR amplification of MYC, TP53R249S, and KRASG12D
Figure 5(B) Volcano plot of RNA-seq data showing genes with differential expression (1,591 upregulated and 2,243 downregulated genes, fold change≥2 and P value≤0.05) in iHCC cells compared to PHHs. TP53, KRAS, MYC, MUC1, and AFP were highly expressed in iHCC cells, whereas hepatocyte signature markers, including FOXA2, FOXA3, ATF5, HNF4A, and ALB were highly expressed in PHHs(D) Heat map analysis of the log2(RPKM) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells. These genes were classified into three categories based on their functions
Figure 6(A) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs. Within these 20 genes, seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA-HCC (TCGA-LIHC) analysis(B) Representative cytotoxicity CARFAP-T and CARMUC1-T cells assessed by coincubation with iHCC2-1 cells. P=0.0038 (CARFAP-T vs. mock T), and P<0.0478 (CARMUC1-T vs. mock-T). P values were determined by paired t-test, **P≤0.05. Cells were seeded at E: T ratios ranging from 4:1 to 1:4; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3)(D) Clonogenic assay of iHCC1-1 cells treated with a MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates(F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of iHCC in xenografts (n=5) treated with PBS or XL413 at 50 mm3 tumor volume. P=0.0486 (XL413 vs. PBS group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.
Figure 1(D) ACE2 levels were characterized using RNAscope. A representative 3D view of a 232 x 232 μm2 field of view of the AT layer of an uninfected control at 3 days at air-liquid interface (3C) is shown. The nuclear labelling is indicated in electric indigo and ACE2 mRNA is labelled in spring green LUT. (G) Same as in (D) with infected LoC at 3 dpi (G) shown.(I, J) A plot of the fold change in expression of host proteins required for viral entry in the AT and endothelial layer of LoCs at 1 day-post infection (n=4 biological replicates) and 3 days-post infection (n=3 biological replicates) vs. uninfected control LoCs (n=3 biological replicates). Data information: The bars represent the mean value, the solid line represents the median value, and the error bars represent the standard deviation. P-values are calculated using a Kruskal-Wallis one-way ANOVA test, * represents p<=0.05, ** represents p<=0.01, ** represents p<=0.001. Scale bar = 20 μm.
Figure 2(A The kinetics of release of SARS-CoV-2 progeny was assessed from samples obtained from a wash of the apical face of the chip and the vascular effluent (A) collected at each day-post infection. (A) The number of viral genomes in each sample was quantified using a one-step qRT-PCR assay for the SARS-CoV-2 N gene. The starting inoculum introduced to the apical side (labelled 'Inn') corresponded to 200-300 PFU. Lines join datapoints from the same LoC, reconstituted with (green squares) and without (dark blue circles) macrophages. Viral genome numbers within the apical wash showed a declining trend over 3 days of infection. Viral genomes are not detected ('nd') in the vascular effluent for all the chips shown. Data information: In all plots, the bars represent the mean value, and the error bars represent the standard deviation, the solid black line in (A) represents the median.
Figure 3(A-D) 3D views of representative 232 x 232 μm2 (A, B) and 155 x 155 μm2 (C, D) fields of view from confocal imaging of an uninfected control (A), and infected chips without macrophages at days 2 (B) and 3 (C, D) post-infection, respectively. Actin and nuclear labelling are shown in azure and indigo, respectively. (B) At 2 dpi, adjacent areas of endothelial cell clusters identified by a stronger nuclear stain and brighter actin staining (yellow arrows) and cells with increased permeability and loss of confluence (white arrows) are indicated. (C) At 3 dpi, a significant loss of barrier integrity is observed in the endothelial layer, whereas the AT layer directly above this field of view (D) is relatively intact. A small area of missing cells in the AT layer is indicated with a white arrow in (D).(G-J) The fields of view in (C, D) are shown also in (G, H) respectively, SARS-CoV-2 spike (S) proteins labelled via immunofluorescence are false-colored in amber. Zooms in (I) and (J) show clear evidence of intracellular spike protein within both endothelial and ATs, respectively.
Figure 4(A-D) Maximum intensity projects of 232 x 232 μm2 fields of view from the AT (A, B) and endothelial layers of an uninfected control LoC. ATs show strong actin (A) and TJP1 expression (B, identified via antibody staining) at the cell junction boundaries. Endothelial cells also show high levels of TJP-1 expression (C) together with CD31 expression at cell junctions (D, identified via antibody labelling). Actin, TJP-1, CD31, and nuclear labelling are indicated with the azure, bright pink, spring green and electric indigo LUTs, respectively.(E-H) 3D views of one 232 x 232 μm2 field of view of the AT layer (E) and (F-H) three 232 x 232 μm2 fields of view of the endothelial layer of an infected LoC at 1 dpi. Areas with lower TJP1 expression in the AT layer in (E) are indicated by white arrows. (F-H) Areas with loss of tight junctions between cells on the endothelial surface is indicated by white arrows. The proportion of the surface area covered by confluent cells decreases sequentially from (F) to (H). Endothelial cell clusters with low CD31 expression are clearly visible, and the proportion of the surface area covered by these clusters increases sequentially from (F) to (H). In (F-H), spike protein is identified via an anti-SARS-spike antibody and shown in amber LUT, and von Willebrand Factor identified by antibody labelling is shown in bring pink LUT.(I-L) Zooms of two endothelial cell clusters (I, J and K, L) in the endothelial layer of another infected LoC at 1 dpi confirm that little or no CD31 expression is detected (I, K) despite high levels of actin and TJP-1 protein expression in these cells. (J, L).(M) Plot of the fold change in expression of TJP1 (AT and endothelial layers) and CDH5, CD31, and CD146 (endothelial layers only) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs, uninfected controls (n=3 biological replicates). (N) Plot of the fold change in gene expression of endothelial cell specific coagulation markers - Tissue Factor (F3), Tissue Factor Pathway Inhibitor (TFPI), Thrombomodulin (THBD), Plasminogen Activator Inhibitor (SERPINE1) and von Willebrand Factor (VWF) in infected LoCs at 1 and 3 dpi (n=3 biological replicates in each case) vs. uninfected controls (n=3 biological replicates). Data information: In all plots, bars represent the mean value and error bars represent the standard deviation. Scale bar = 20 μm. P-values are calculated using a one-way Kruskal-Wallis ANOVA test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.
Figure 5(A-H) 3D views of 232 x 232 μm2 fields of view of the AT (A, G) and endothelial layer (B, H) from infected LoCs reconstituted without macrophages at 1 (A, B) and 3 dpi (G, H). Orf1ab antisense RNA, S RNA, ACE2 mRNA, and nuclear staining with DAPI are false-colored pink, amber, spring green, and electric indigo, respectively. (C, D) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (C) An example of infection and intracellular replication in a cell with no detectable ACE2 expression (white arrow) as well as nuclear localization of viral RNA (white arrowhead). (D) An example of an uninfected cell with ACE2 mRNA expression (yellow arrow). (E, F) Zooms corresponding to the regions in (B) representing increased nucleic acid staining (hyperplasic) and normal levels of nucleic acid staining highlighted with white and yellow boxes, respectively. (E) Examples of infection of hyperplasic endothelial cells both with and without ACE2 expression (F) Examples of endothelial cell infection with no ACE2 expression, nuclear localization of viral RNA in an infected endothelial cell is indicated (white arrow).
Figure 6(A) Expression relative to GAPDH of pro-inflammatory cytokines (TNFA, IL1B, IL6), interferon genes (IFNB, IFNL1, IFNL3) and interferon stimulating genes (IP10, ISG15) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages (n=3 biological replicates for IFNB, IFNL1 and IFNL3 in the AT layer, n=4 biological replicates for all other data). (B) Fold-change in the markers in (A) relative to uninfected controls (n=3 biological replicates) (data in Appendix Fig. S12) at the same timepoint. The bar represents the mean, and the error bars the standard deviation. ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.(D) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi (n=3 biological replicates for the AT layer and n=4 biological replicates for endothelial layer). (E) Fold-change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls (n=3 biological replicates for both layers). ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001.(F-I) 3D views of representative 232 x 232 μm2 fields of view of the AT (F, H) and endothelial layer (G, I) from infected LoCs reconstituted with macrophages at 1 (F, G) and 3 dpi (H, I). IL6 mRNA, and nuclear staining are indicated with the chartreuse and electric indigo LUTs, respectively.
Figure 7(A-F) Maximum intensity projections of representative 232 x 232 μm2 fields of view of the endothelial layer from an infected LoC reconstituted without macrophages at 2 dpi with (A) and without Tocilizumab treatment (D). Actin and nuclear staining are indicated with the azure and electric indigo LUTs respectively, the actin staining is saturated for the endothelial cell clusters to highlight cells with lower levels of actin. (B, C) Zooms corresponding to the region in (A) highlighted with white and yellow boxes, respectively. (B) A region with normal vascular profile, (C) A region of endothelial cell cluster formation with increased actin staining. (E, F) Zooms corresponding to the regions in (D) representing regions highlighted with white and yellow boxes, respectively. (E) An example of reduced tight junctions between endothelial cells; bare patches are indicated with arrows. (F) A region of endothelial cell clusters. Scale bar = 20 μm.(G, H) 3D views from two 232 x 232 μm2 field of views of the endothelial layer of a Tocilizumab treated infected LoC at 2 dpi, immunostaining for the tight junction protein CD31 is shown in the Spring Green LUT. Endothelial cell clusters with low CD31 expression and altered actin staining are also visible and indicated by yellow arrows.
Figure 1B. IHLs were isolated from the liver of Tie2-GFP mice intrasplenically injected with NaCl (n=3) or with 5x103 CT26 at the specified time points (day 25 post-injection n=6, day 35 post-injection n=5). The number of GFP+ TEMs was estimated by flow cytometry as 7AAD-/CD45+/CD11b+/CD11c-/GFP+ cells per liver; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.C. Representative images (top panels) and corresponding H&amp;amp;E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&amp;amp;E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.
Figure 2A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher's exact test.C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test.D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test.F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test).G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).
Figure 3A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2-GFP or Tie2-IFNαmice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFPmice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).B. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).