EMBO/SourceData huggingface dataset:
Figure 2Top-left - tSNE plot of the Seurat clusters, boxes demarcate compared clusters. Dashed boxes and labels indicate the cell clusters that are compared in panels A-C. (A) Volcano plot of differential gene expression (DGE) between liver vascular endothelial cells in the tumor and non-tumor samples. (B) Volcano plot of DGE between mononuclear phagocytes in the malignant and non-malignant samples. (C) Volcano plot of DGE between T cells in the tumor and non-tumor samples. (D) DGE analysis between tumor mononuclear phagocytes classified by cancer type (cholangiocarcinoma in dark purple and metastasis in light purple). Wilcoxon rank-sum tests were used to generate p-values, Benjamini-Hochberg multiple hypotheses correction was used to compute q-values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two clusters.
Figure 3Key marker genes for the four mesenchymal clusters (RBP1, COL1A1, RGS5, and MYH11). Light gray dots denote cells originating from non-tumor samples. Dark gray dots denote cells originating from the tumor samples. Left - Representative smFISH image of patient p1 stained for RGS5 and COL1A1 showing distinct spatial localization of CAFs and Pericytes. Scale bar 10µm. Dashed lines mark the shortest distance of cells (2a) and (2b) from the cell (1). Middle - zoom-in of (1) from left panel, showing a blood vessel like structure formed by endothelial cells marked by PDGFB (magenta) wrapped by pericytes marked by RGS5 (green). Dashed lines are two consecutive cell layers of endothelial cells and pericytes. Scale bar 2.5µm. Right - zoom-in of (2a and b) from the left panel, showing two distant CAFs expressing high COL1A1 signal but not RGS5. DAPI used for nuclei staining. Scale bar 2.5µm.Violin plot of the distance from blood vessels of low/high RGS5 expressing cells (n=358 and n= 360 respectively) and low/high COL1A1 expressing cells (n=359 and n= 359 respectively). 'p' is the p-value determined by Wilcoxon rank-sum test. Empty circles are the medians over all repeats.
Figure 4Dot-plot of selected genes highlight shared interaction motifs between different clusters colored by max normalized expression for each genes across all clusters in (A). For each gene, dot size represents the fraction of positive cells for each cluster.
Figure 5H&E staining of malignant and non-malignant sites from patient p4, separated by the white dashed line. Scale bar is 100µm. Non-malignant human liver lobule from patient p4 used for LCM stained with HistoGene Staining Solution (Methods). Top - sequential zones from the central vein (cv) to the portal node (pn) before microdissection. LCM captured zones marked by the black dashed line. White dashed line marks the boundaries of NPC (non-parenchymal cells) located in the pn and not included in the analysis. Bottom - Tissue after microdissection. Scale bar is 100µm.Zonation of human hepatocytes. Left - Max normalized expression over the reconstructed six lobule layers (L1 to L6), sorted by center of mass from the central vein (cv) to the portal node (pn). Right - selected pericentrally (top), and periportally (bottom) zonated genes. Blue patches are standard errors of the mean.Zonation profiles of selected genes from human (blue) and mouse (green). Each profile is normalized by the maximal expression across zones. Patches are standard errors of the mean.
Figure 6tissue sections from patients p1 and p3 stained with HistoGene Staining Solution. Laser captured zones are marked with dashed black line including: tumor core (tc), the border between the tumor and the fibrotic regions (ftb), tumor islets within the fibrotic regions (ti) and fibrotic zones (fz) in panel (A), and tumor border (tb) in panel (B). Small images in (A) are magnifications of the corresponding zones in the large image for the left. White dashed line in (B) marking the non-malignant liver (nml) border. Scale bars in all panels are 100µm.
Figure 1(A) siRNA knockdown of Tollip in SH-SY5Y cells was performed over 72 hours, with cells reverse transfected with siRNA oligos specific for Tollip (or mock) for 24 hours before a second transfection was performed for a further 48 hours. Knockdown was confirmed by western blotting.(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.(C, D) The number of TOM20+ve/PDH-ve MDVs (C) or PDH+ve/TOM20-ve MDVs (D) per cell were quantified from immunofluorescence images captured(E) SH-SY5Y cells were transfected with GFP-labelled Tollip for 24 hours then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 hours prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP-Tollip was observed on TOM20+ve/PDH-ve MDVs (arrowheads). Zoom insets represent 7x magnification.(F) Images captured using super-resolution radial fluctuations (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH-SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm.
Figure 2(A) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).(H) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).
Figure 3(A) HeLa cells expressing mycBioID-Tollip and HA-Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 hours in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole cell extracts and pulldowns from each condition were then separated by SDS-PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID-Tollip were used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions.(B) HeLa cells expressing mycBioID alone and HA-Parkin were used to confirm the specificity of the Tollip-Parkin interaction.(C) Hela cells co-expressing GFP-Tollip and HA-Parkin were either left untreated or treated with AO for 2 hours prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP and western blot analysis was performed using antibodies against Parkin and GFP.(D) HeLa cells expressing mycBioID-Tollip containing the CUE domain mutation (CUEmut) and HA-Parkin were subjected to biotinylation and streptavidin pulldowns, followed by western blot analysis.(E) HeLa wild-type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID-Tollip and HA-Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins.
Figure 4(A) Cells were reverse transfected with Parkin or Tom1 siRNA oligos for 24 hours before a second transfection was performed for a further 48 hours, then fixed and stained with antibodies specific to TOM20 (green) and PDH E2/E3bp (red). Nuclei are labelled with Hoechst (blue). Zoom insets represent 5x magnification and arrowheads indicate TOM20-positive MDVs.(B) Knockdowns were confirmed for both Parkin and Tom1 by western blotting.(C, D) Quantification of MDVs, defined as TOM20+ve/PDH-ve, (C) or PDH+ve/TOM20-ve (D) per cell under steady-state conditions were performed (n = 32-52 cells per condition from 3 independent experiments).(E) Western blot analysis of lysates harvested from SH-SY5Y wild-type (empty vector) and CRISPR generated Parkin KO cells. Immunoblot analysis was performed using antibodies against Parkin and GAPDH.(F) Immunofluorescence microscopy was performed on SH-SY5Y wild-type and Parkin KO cells either untreated or treated with AO for 2 hours. Cells were immunostained for TOM20 (red), PDH E2/E3bp (blue), and LAMP1 (green). Zoom insets represent 5x magnification. Circles indicate TOM20 MDVs positive for LAMP1 and arrowheads indicate TOM20 MDVs negative for LAMP1. Scale bar represents 10 µm.(G) Quantitation of TOM20-positive MDV colocalisation with LAMP1 were performed across all treatment groups and represented as the % of TOM20-positive MDVs colocalising with LAMP1. Results represent 3 independent experiments (28-53 cells/condition) and statistical significance was determined using a Two-way ANOVA (Sidak).(H) HeLa cells transiently transfected with HA-Parkin were subjected to mitochondrial stress by treatment with 25 μM antimycin A (AA) for 2 hours, then fixed and stained with antibodies specific to HA, PDH and TOM20. TOM20+ve/PDH-ve MDVs were quantified in cells that were either expressing or not expressing HA-Parkin (n = 12-14 cells per condition from 3 independent experiments).(I) Hela cells stably coexpressing BioID-Tollip and either HA-Parkin WT or Δ1-76 (ΔUBL) were incubated with and without biotin in the presence of AO for 6 hours prior to being subjected to cell lysis and streptavidin pulldowns. Western blot analysis was performed on lysate inputs and streptavidin pulldowns (Streptavidin PD) using antibodies against the indicated proteins or epitope tags.
Figure 5(A-B) siRNA knockdown of Tollip or Parkin in SH-SY5Y cells was performed over a 72 hour period, then 18 hours prior to treatment cells were transfected with GFP-2xFYVE to label endocytic membranes. Cells were treated with 25 μM antimycin A (AA) for 2 hours then fixed and immunostained with antibodies specific to PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs that colocalise with GFP-2xFYVE and arrows denote TOM20+ve/PDH-ve MDVs that do not.(C) Quantification of TOM20+ve/PDH-ve MDVs that are positive for GFP-2xFYVE are shown as a % of TOM20 MDVs positive for GFP, with each data point indicating 1 cell. MDV colocalisation were quantified by eye from images captured (n = 20-22 cells per condition from 3 independent experiments).(D) SH-SY5Y cells were left untreated or treated with 25 μM AA for 2 hours prior to fixation and immunostaining for EEA1 (green), PDH E2/E3bp (red), and TOM20 (blue). Arrowheads indicate TOM20+ve/PDH-ve MDVs positive for the early endosome marker EEA1.(E) siRNA knockdown of Parkin was performed in SH-SY5Y cells followed by 25 μM AA for 2 hours prior to fixation and immunostaining with antibodies specific to TOM20 (green), Tollip (red) and Cytochrome c (blue). We observed Tollip colocalisation with TOM20+ve/PDH-ve MDVs (denoted by arrowheads), which was still maintained following Parkin siRNA knockdown.(F) SH-SY5Y cells were transfected with GFP-Tollip and treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A (BfnA1) for 2 hours prior to fixation and immunostaining with antibodies specific to GFP (green), Rab7a (red) and PDH E2/E3bp (blue). (G) Quantification of GFP-Tollip/Rab7a colocalisation using Pearson's correlation.
Figure 6(A) Quantitation of Tollip vesicular clusters in SH-SY5Ys treated with 5 μM antimycin A/10 μM oligomycin B (AO) for 2 hours. The percentage of cells with vesicular clusters were quantified from 7-23 cells for each condition from 4 independent experiments.(B) Quantitation of the prevalence of Tollip clusters in HEK293 WT or Tom1 KO cells treated with AO for 2 hours. The percentage of cells with vesicular clusters were quantified from an average of 50 cells for each condition from 5 independent experiments.(C) Endogenous Tollip and LAMP1 colocalise in SH-SY5Y cells treated with AO for 2 hours. Antibodies specific to LAMP1 (green) and Tollip (red) were used.(D-E) SH-SY5Y cells were transfected with either wild-type GFP-Tollip (WT), GFP-Tollip lacking its N-terminus (∆Nterm), or GFP-Tollip containing a CUE domain mutation (CUEmut) then treated with AO for 2 hours. Cells were fixed and immunostained with antibodies specific to GFP (green) and LAMP1 (red).(F) The extent of colocalisation between GFP-Tollip and LAMP1 from untreated and AO treated SH-SY5Y cells were quantitated from immunofluorescence images and represented as a Pearson's correlation (2-3 cells per experiment, from 2 independent experiments). (G-I) The extent of colocalisation between GFP-Tollip and LAMP1 in Tom1 KO (G), ATG5 KO (H), and VPS35 KO (I) compared to wild-type (WT) HeLa cells was quantified from immunofluorescence images immunostained for GFP and LAMP1 and represented as a Pearson's correlation (2-3 cells per experiment, from 3 independent experiments). Western blot analysis of lysates harvested from wild-type (WT) alongside either Tom1 KO (G) or VPS35 KO (I) Hela cells was performed using antibodies specific to the indicated proteins.
Figure 7(A) A subset of TOM20+ve/PDH-ve MDVs are trafficked to the lysosome. siRNA knockdown of Tollip was conducted in SH-SY5Y cells over a 72 hours period prior to treatment with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM antimycin A (AA) for 2 hours. Cells were then fixed and stained with antibodies specific to LAMP1 (green), PDH E2/E3bp (red) and TOM20 (blue). Arrows denote TOM20+ve/PDH-ve MDVs colocalising with LAMP1, whereas arrowheads denote MDVs that do not colocalise with LAMP1. (B) From immunofluorescence images, the total number of TOM20+ve/PDH-ve MDVs were counted as well as the number of TOM20 MDVs that colocalised with LAMP1 to calculate the % of TOM20 positive MDVs colocalising with LAMP1 per cell (n = 25-32 cells per condition from 3 independent experiments).(C) siRNA knockdown was performed in SH-SY5Y cells, then 18 hours prior to fixation cells were transfected with GFP-Tollip wild-type (WT), ∆Nterm, CUEmut, or R78A. Cells were fixed and immunostained for PDH E2/E3bp (red) and TOM20 (blue). Arrowheads denote TOM20+ve/PDH-ve MDVs. (D) Quantification of TOM20 MDVs per cell was performed by eye (n = 11-14 cells per mutant from 2 independent experiments). Cells expressing a GFP-Tollip construct were identified using the GFP channel. Surrounding cells not expressing GFP were counted as the Tollip siRNA only population.
Figure 1(A) GSCs contain the higher levels of stemness markers (Nestin, SOX2, Musashi-1, NANOG, and OCT4) compared with their DFCs. Total RNAs from GSCs and DFCs were subjected to real-time PCR. The average relative amounts of GSCs were normalized with those of DFCs. Shown are means +S.D. of the normalized mRNA levels of GSCs obtained from the three independent experiments.(B) Immunoblotting analysis of markers for stemness (SOX2, Nestin and Musashi-1) and differentiation (GFAP and TUJ1 (Neuron-specific class III beta-tubulin)) in GSCs in comparison with GSC-derived cells at different passage numbers (P1-P7).(C) Measurement of the cell doubling time. Approximately 3×104 cells for each of XO8 GSCs and XO8 DFCs were seeded in a 60 mm culture dish and counted every day. The cells were treated with trypsin-EDTA, and the number of viable cells was counted with a hemocytometer. The doubling time was calculated from the cell growth curve over 5 days. Shown are means ±S.D. obtained from three independent experiments.(D) FACS analysis of CD133 and GFAP using X08 GSCs and DFCs. Data are from representative experiments repeated at least three times.(E) Immunoblotting analysis of CD133 and GFAP in GSCs in comparison with DFCs.(F) Quantitation of (G). (G) Equal numbers of XO8 GSCs and DFCs were injected orthotropically into mouse brains. After six weeks, brains were harvested and tumorigenic potential was assessed by evaluating tumor formation. Representative microphotographs of the brain sections of mice injected with XO8 GSCs and XO8 DFC controls.
Figure 2Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO.(I) XO8 GSCs and DFCs were treated with 50 nM MG132 for 24 hrs. Cell viability was measured using trypan blue exclusion assay. Error bars represent the mean ± SD from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. (*P < 0.05).(J) XO8 GSCs were treated with MG132 were cultured in 0.3% soft-agar as described in Materials and Methods. The colonies bigger than 100 µm in diameter in four randomly chosen fields were subjected to blind counting. Bars, means +S.D. from three independent experiments.
Figure 3(A) XO8 GSCs and DFCs were differentially labeled with PKH67 (green) and PKH26 (red), respectively, and mixed before plated on laminine-coated plates. The cells were treated with 50 nM MG132 and subjected to fluorescent microscopy. Scale bar, 100 μM. (B) Quantitation of (A). Cell numbers from three independent experiments were normalized to those of control cells treated with DMSO and presented as means +S.D. (*p=0.0035, n=3). Student t-test (two-sided, one type) were used for statistical analysis.(C) The harvested cells from (A) were subjected to FACS analysis.(D) XO8 GSCs were treated with various PIs (salinosporamide A, epoxomicin, and PS341), followed by cell viability assays. Bars, means ±S.D. from three independent experiments.(E) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay. Bars, means ±S.D. from three independent experiments. (F) XO6, XO8, and XO10 GSCs were treated with MG132 and subjected to cell viability assay in serum-containing media. Bars, means ±S.D. from three independent experiments.
Figure 4(A) Immunoblotting of XO8 GSCs and DFCs.(B) Immunoblotting analysis of Ub in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (C) Similar to (B) except that XO6 GSCs were used.(D) Immunoblotting analysis of ubiquitin ligases and proteasomal subunits in XO8 GSCs and XO8 GSC-derived cells in the course of differentiation (P1-P7). (E) Similar to (E) except that XO6 GSCs were used.(F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039.(F-I) In vitro proteasome activity assays were performed as described in Materials and Methods. Bars represent means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: (F), 0.0039; (G), 0.0052; (H), 0.0009, (I) DFC, 0.00032; (I)-HEK293, 0.0039; (I)-HeLa: 0.00051.(J) XO8 GSCs and XO8 DFCs treated with MG132 as described in Materials and Methods were labeled with annexin V and visualized using fluorescent microscopy. Scale bar, 100 μM.(K) Cells from (J) were subjected to flow cytometry analysis. Annexin V-positive cell numbers were obtained from three independent experiments and normalized to those of controls cells treated with DMSO. Bars, means +S.D. from three independent experiments. For statistical analysis, student t-test (two-sided, one type) were used. * P value: 6 hrs, 0.0012; 17 hrs, 0.0041.(L) Cells treated with 50 nM MG132 were subjected to immunoblotting analysis of apoptotic markers.(M and N) Similar to (L) except that XO6 and XO10 cells were used.
Figure 5(A) Real-time PCR analysis of the proteasomal subunits PSMB5, PSMB6, and PSMB7 in XO8 GSCs and XO8 DFCs. Shown are means +S.D. values from the three independent experiments following normalization to those of GSCs. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.053, PSMB6: 0.034, PSMB7: 0.067. (B) Similar to (A) except that the cells were treated with 50 nM MG132 for 17 hrs. Shown are means +S.D. values from the three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value=PSMB5: 0.022, PSMB6: 0.08, PSMB7: 0.603.(C) Immunoblotting analysis of proteasomal subunits in XO8 GSCs in comparison with XO8 DFCs at passages 11 and 12. Note that part of Figure 5C is also shown as the left panel of Figure 4A. (D) Same as (C) except that the cells were treated with MG132 for 17 hrs.(E) Immunoblotting analysis of the NF-κB pathway in XO8 GSCs and XO8 DFCs.(F) Fractionation analysis of NF-κB in XO8 GSCs and XO8 DFCs treated with 50 nM MG132 for 17 hrs. PARP1 and tubulin were used as markers for the nucleus and cytoplasm, respectively.(G) Immunoblotting analysis of JNK and p38.(H) Cell viability assay was performed as in Fig. 2 with cells treated with MG132 alone or in combination with SB203580. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. ** P value = 0.724, 0.502, 0.439 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.1 μM MG132, and 0.760, 0.683, 0.498 for 0.5, 1.0 and 5.0 μM SB203580, respectively at 0.5 μM MG132.(I) Experimental conditions were similar to (H) except that SP600129 was used. Shown are means +S.D. values from three independent experiments following normalization to those of control cells treated with DMSO. For statistical analysis, student t-test (two-sided, one type) were used. * P value = 0.009, 0.002, 0.07 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.1 μM MG132, and 0.018, 0.025, 0.09 for 0.5, 1.0 and 5.0 μM SP600129, respectively at 0.5 μM MG132.(J) Immunoblotting analysis of XO8 GSCs treated with 50 nM MG132 alone or in combination with the JNK inhibitor SP600129.