EMBO/SourceData huggingface dataset:
Figure 3(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. (A) SOX9, (B) Pan-CK, Representative images are shown.(A)-(D)Liver cell rosettes in PSC patients do not acquire other markers of bile duct cells or intermediate hepatobiliary cells. Immunofluoresence for different bile duct cell or intermediate hepatobiliary cell type marker (magenta), F-Actin (green) and nuclei (grey) in healthy parenchyma (left) and bile duct (middle) in control patients and liver cell rosettes (right) in PSC patients (N=3 control patients and N=4 PSC patients). Scale bar 10 µm. TROP2 CD133/PROM1. Representative images are shown.
Figure 43D reconstruction of the bile canaliculi network reveals fundamental changes in the network architecture of PSC patients. (A) Top panel: 3D reconstructed bile canaliculi network across full central vein to portal vein (CV-PV) axes in control (left) and PSC (right) patients. Bottom panel: Projection of the 3D reconstruction. Colours correspond to the mean bile canaliculi diameter in µm. Scale bar 100 µm. (B)-(I) Quantification of bile canaliculi network characteristics from the 3D reconstruction. Represented are mean values with standard deviation of control and PSC patients at different zones across the CV-PV axis. 0 represents the zone surrounding the CV and 10 the area surrounding the PV. N=3 with 1x axis/patient for control patients and N=4 with 3x axis/patient for PSC patients. Paired t-test *P < 0.05, **P < 0.01, ***P < 0.001. (B) bile canaliculi radius [µm], (C) volume fraction [%], (D) surface area/volume (µm2/mm3), (E) connectivity (1/# disjointed bile canaliculi), (F) branches crossing regions [#/mm2]. (G) junctions/length [#/µm
Figure 5(A) Liver cell rosettes occur in early- and late-stage PSC patients and in end-stage alcoholic liver disease (ALD) patients. Immunofluorescence of Fibronectin (magenta), F-actin (green) and nuclei (grey) in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients showing liver cell rosettes. Representative images are shown. Scale bar 50 µm. Examples of rosettes are labelled with a red asterisk and examples of sinusoids are labelled with a red arrowhead. (B) Similar numbers of liver cell rosettes in early-stage and end-stage PSC patients. Quantification of the total number of rosettes in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5) patients. Unpaired t-test *P < 0.05. (C) Similar mean diameter of analysed liver cell rosettes in different liver diseases. Quantification of the mean rosette diameter in early-stage PSC (N=3), late-stage PSC (N=5) and end-stage ALD (N=5). Unpaired t-test. Data information: Boxplot shows the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.
Figure 6(C) -(D) DCA treatment induces a cholestatic response in primary hepatocytes. Gene expression analysis of genes involved in bile acid metabolism and transport in primary hepatocytes treated with DMSO or 200 µM DCA for 16h. Represented are mean Cq values normalized to Gapdh expression (N=4 biological replicates). One sample t-test *P < 0.05, ***P < 0.001. (C) Abcb11/Bsep, (D) Cyp7a1/Cyp7. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.(I-J) DCA treatment in 3D hepatocyte organoids also induce canalicular dilation and rosette-like canaliculi and Sox9 expression. Hepatocyte organoids were treated with DMSO or 200 µM DCA for 24h (N=3 biological replicates). (I) Maximum projection of full organoids stained for F-actin (green). Scale bar 50 µm. Inset A and B. Maximum projection of zoomed-in parts of the bile canaliculi network in DMSO-treated (A) and DCA-treated (B) hepatocyte organoids stained with F-actin (green), nuclei (white) and the tight junction protein ZO-1 (yellow). Scale bar 10 µm. (J) High-resolution microscopy of individual bile canaliculi in DMSO-treated and DCA-treated hepatocyte organoids stained for F-actin (green). Red arrows indicate apical bulkheads. Scale bar 2 µm.(K) Maximum projection of F-actin (green) and nuclei (white), and the bile duct cell marker Sox9 (magenta) in DMSO-treated and DCA-treated hepatocyte organoids (N=3 biological replicates). Untreated bile duct cell organoids serve as positive control.Scale bar 50 µm.
Figure 7(A)Rosette-like canaliculi with reduced number of apical bulkheads can emerge from usual canaliculi with accumulated apical bulkheads. Timelapse imaging with brightfield phase-contrast of primary hepatocytes either untreated (top row), DMSO-treated (middle row) or DCA-treated (bottom row). An image was acquired every 15 minutes. Representative images are shown (N=3 biological replicates). Inserts show magnification of bile canaliculi with red arrows pointing towards apical bulkheads. Red stars mark areas in between apical bulkheads that markedly bulge out. Scale bar 10 µm.(B) Pharmacological inhibition of canalicular water influx can prevent canalicular dilation upon DCA treatment. Confocal microscopy of CD13 (magenta), F-actin (green) and nuclei (white) in primary hepatocytes treated with DMSO and 200 µM DCA with 200 µM phloretin and 1 µM ouabain for 16h. Scale bar 10 µm. Representative images are shown.(C) Quantification of the mean bile canaliculi volume in µm3of primary hepatocyte treated with DMSO and 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h (N=4 biological replicates). Unpaired t-test ***P < 0.001. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.(D)- (E) Pharmacological inhibitors do not affect key bile acid signalling pathways. Gene expression analysis of different genes involved in bile acid signalling in primary hepatocytes treated with DMSO or 200 µM DCA with 200 µM Phloretin and 1 µM Ouabain for 16h. Represented are mean Cq values normalized to Gapdh expression and whiskers show min and max values (N=5 biological replicates). Unpaired t-test. (D) Cyp7a1/Cyp7, (E) Nr0b2/Shp. Data information: Boxplots show the 25th and 75th percentiles and the central band shows the median value. Whiskers extend to the min and max values.
Figure 1(A-C) Extra-mitochondrial puncta formation of CHCHD2T61I in MEFs. Chchd2KO cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids. At the indicated times, cells were fixed and stained with anti-HA and anti-Tom20 antibodies, and observed by confocal microscopy. In (A), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown in Appendix Fig. S1D. (B, C) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In (B, C, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01(D, E) Extra-mitochondrial puncta formation of CHCHD2T61I in Neuro2a cells. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, then cultured with medium containing 2% FBS and 10 µM retinoic acid for neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-Tom20 antibodies. In (D), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown (E) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In E, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01(F) Neuro2a cells were treated as described in (D), and cell lysates were fractionated into cytosol, organelles (including mitochondria), nuclei, and cytoskeleton (including insoluble matter). The expression of each protein was analyzed by western blotting using an anti-HA antibody. Cox4 was used as a control. (G) A semiquantitative analysis of protein expression in (F) is shown. In G), data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01
Figure 2Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with an anti-HA antibody and ProteoStat protein aggregation dye (A, B), anti-HA and anti-Nefl antibodies (C, D), anti-HA and anti-p-Nefl473 antibodies (E, F), In (A, C, E, representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and aggresomes or the indicated proteins. Representative images at lower magnification are shown (B, D, F, The amount of aggresomes (B), Nefl (D), p-Nefl473 (F) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In (B, D, F, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-p-α-Synuclein129 (p-α-Syn129) antibodies (G, H). In G), representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated proteins. Representative images at lower magnification are shown H) The amount of p-α-Syn129 (H) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In H, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01In (I), cell lysates were harvested at 28 hr, and the expression of each protein was analyzed by western blotting. (J) A semiquantitative analysis of protein expression in (I) is shown. Data are shown as the mean ± SD (n = 3). In J), comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01
Figure 3(E, F) Interaction between CHCHD2T61I and Csnk1e/d. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids together with pmax-GFP (to detect transfected cells), and were differentiated into neuronal cells at 4 hr after transfection. At 28 hr after transfection, cells were fixed and the PLA was performed using anti-HA and anti-Csnk1e/d antibodies and Duolink PLA reagents. In (E), PLA signals were clearly observed in cells expressing CHCHD2T61I-HA, but were faint and localized in the cytosol in cells expressing CHCHD2WT-HA. Representative images at lower magnification are shown In (F), the number of PLA signals were counted (n  = 50 cells in each experiment). Red bars indicate mean values. In F), comparisons were performed using the unpaired two-tailed Student t-test and one-way ANOVA followed by the Tukey post-hoc test. *p < 0.05. **p < 0.01. NS: not significant
Figure 4(D-I) Neuro2a cells were transfected with the CHCHD2T61I-HA plasmid together with the indicated siRNAs, and were differentiated into neuronal cells after 4 hr. At 28 hr after transfection, cells were fixed and stained with anti-HA, anti-p-Nefl473, and anti-p-α-Syn129 antibodies, or ProteoStat aggresome detection dye. Similar experiments were performed by the addition of PF-670462 (10 µM) after 4 hr of transfection instead of gene silencing. In (D, F, H), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated phosphorylated proteins or aggresomes. In (E, G, I), the amount of p-Nefl473 (E), p-α-Syn129 (G), and aggresomes (I) was measured as the fluorescence intensity per cell (n = 30 cells in each experiment). Red bars indicate mean values. In E, G, I), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests or unpaired two-tailed Student t-test. *p < 0.05; **p < 0.01. NS: not significant
Figure 5Reduction in TH signals in the SNpc of knock-in mice. In (D), brain cryosections were immunostained with the dopaminergic cell marker TH. Representative images of the SNpc and ventral tegmental area (VTA) are shown. Dashed lines indicate the SNpc and VTA regions. Bars = 200 µm. In (E), TH signals in the SNpc (average fluorescence intensity per region) are shown as the mean ± SD (n = 3). In E), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests. *p < 0.05; **p < 0.01. NS: not significantReduction in TH signals in the SNpc of knock-in mice. In (F), cryosections of the SNpc were immunostained with anti-Chchd2 and anti-TH antibodies. Arrowheads indicate Chchd2T61I puncta in TH-positive cells. Dashed lines indicate cell shapes. (G-K) Extra-mitochondrial aggresome formation by p-Nefl473, p-α-Syn129, and Csnk1e/d in the SNpc of knock-in mice. Cryosections of the SNpc were immunostained with anti-Chchd2 and anti-Ant1/2 (G), anti-p-Nefl473 (H), anti-p-α-Syn129 (I), and anti-Csnk1e/d antibodies (J), and with ProteoStat dye (K). Ant1/2 are mitochondrial membrane proteins and arrowheads indicate extra-mitochondrial Chchd2T61I (G). In (H-K), arrowheads indicate the colocalization of puncta with Chchd2T61I and the indicated proteins or aggresomes. Dashed lines indicate cell shapes.(L) Isolated midbrain and diencephalon lysates were subjected to western blotting.
Figure 6(F-J) The same experiments as Fig. 5G-K were performed using PF-670462-infused and vehicle-infused Chchd2T61I knock-in mice.
Figure 7(A) Photos of the midbrain of a control subject (left) and PD patient harboring the CHCHD2T61I mutation (right). The dashed lines indicate the SNpc. A portion of the midbrain of the control subject was lost when slicing.(B-G) Colocalization of extra-mitochondrial CHCHD2T61I with CSNK1E/D, p-NEFL472, and p-α-SYN129 in the brain of a PD patient harboring the CHCHD2T61I mutation. Each brain section was deparaffinized and immunostained with anti-CHCHD2 and anti-ANT1/2 (B), anti-CSNK1E/D (C), anti-p-NEFL472 (D, E), or anti-p-α-SYN129 antibodies (F, G). Arrowheads indicate extra-mitochondrial CHCHD2T61I puncta (B), and puncta showing colocalization of CHCHD2T61I with CSNK1E/D (C), p-NEFL472 (D), and p-α-SYN129 (F). Arrows indicate weaker colocalization between CHCHD2T61I and the indicated proteins in the control brain (C, D, F). Dashed lines indicate cell shapes. In (E, G), the amount of p-NEFL472 (E) and p-α-SYN129 (G) was measured by the average fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. Comparisons were performed using the unpaired two-tailed Student t-tests. **p < 0.01
Figure 8iPSCs were prepared from a healthy control and a PD patient harboring the CHCHD2T61I mutation. An isogenic control iPSC line was generated by the correction of the gene mutation Then, these iPSC cells were differentiated into dopaminergic (DA) neurons, and analyzed by immunofluorescence using anti-CHCHD2 (A-E), anti-ANT1/2 (A), anti-CSNK1E/D (B), anti-p-NEFL472 (C), and anti-p-α-SYN129 antibodies (D) Arrowheads indicate extra-mitochondrial CHCHD2T61I (A), puncta showing colocalization of CHCHD2T61I with CSNK1E/D (B), p-NEFL472 (C, , and p-α-SYN129 (D, Dashed lines indicate cell shapes.CHCHD2T61I iPSC-derived DA neurons were treated with PF-670462 (10 µM) for 20 hr and analyzed by immunofluorescence using anti-CHCHD2 and anti-p-α-SYN129 antibodies (G), and ProteoStat aggresome dye (H). Arrowheads indicate puncta showing colocalization of CHCHD2T61I with p-α-SYN129 G) or aggresomes H). Dashed lines indicate cell shapes.
Figure 1(B) Representative images of β-Gal staining (left panel) and quantification of β-Gal positive cells (right panel) of BMSCs isolated from 2‑month-old and 24-month-old mice. Scale bar: 100 μm. β-Gal, beta‑galactosidase.(C) Representative images of Alizarin Red staining at 21 days of osteogenic induction (left panel) and quantification of calcification (right panel) of BMSCs isolated from 2-month-old and 24-month-old mice by detecting the amount of Alizarin Red extracted from the matrix.
Figure 2(A-B) Representative images (A) of immunofluorescence staining and quantification (B) of YBX1 (green) in primary BMSCs.(C-D) Representative immunohistochemical staining images of YBX1 (red) and LEPR (green) (C) and quantification of YBX1+ LEPR + cell numbers (D) in femoral bone marrow. MP, metaphysis; T Ar, Tissue area. Arrows point to YBX1+ LEPR + cells.(E) Age‑associated changes in YBX1 levels in human BMSCs from 30 males (left panel) and 30 females (right panel).(F and G) Representative images of Alizarin Red staining (F) and quantification of calcification (G) by detecting the amount of Alizarin Red extracted from the matrix in BMSCs transfected with adenovirus driven control and Ybx1 shRNA at 21 days of osteogenic induction.(H and I) Representative images of Oil Red O staining (H) and quantification of lipid formation by detecting the amount of Oil Red O extracted from the matrix (I) in BMSCs after 10 days of adipogenic induction.(J and K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells among BMSCs.(L-M) Relative mRNA levels of osteogenic differentiation related genes (L), adipogenic differentiation and senescence related genes (M) between BMSCs transfected with adenovirus driven control and Ybx1 shRNA.
Figure 3(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative μCT analysis of trabecular bone microarchitecture (C-F) in distal femora from 3- and 12-month-old male Ybx1Prx1-CKO mice and Ybx1flox/flox mice (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).(G) Representative images of H&E staining (Hematoxylin-Eosin Staining) in distal femora. Scale bar: 100μm. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin (OCN) immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. Scale bar: 50 μ (J) Quantification of osteocalcin positive cells on the bone surface. Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. Scale bar: 100 μm. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm).(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50 μm. (N) Quantification of the bone formation rate (BFR) based on calcein double labeling. (O) Quantification of the mineral apposition rate (MAR) based on calcein double labeling.
Figure 4(E-F) Representative μCT images, Scale bar: 1mm. (E) and quantitative μCT analysis of trabecular bone microarchitecture (F) in distal femora from 24-month-old mice with rAAV8-GFP-Ybx1 or rAAV8-GFP transfection (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).(G) Representative images of H&E staining in distal femora. (H) Quantification of the number of adipocytes related to the tissue area (N. adipocytes/T.Ar). (I) Representative images of osteocalcin immunohistochemical staining in distal femora. Arrows point to osteocalcin positive cells. (J) Quantification of osteocalcin positive cells in bone surface . Number of OCN+ cells per bone perimeter (N. Ocn+/B.Pm). (K) Representative images of TRAP staining in distal femora. (L) Quantification of TRAP positive cells on the bone surface. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). (M) Representative images of calcein double labeling of trabecular bone. (N) Quantification of the bone formation rate (BFR) and mineral apposition rate (MAR) based on calcein double labeling.
Figure 5(C) Semi-quantitative PCR showing the different isoforms of Fn1, Nrp2, Sp7, and Sirt2 from BMSCs of Ybx1Prx1-CKO mice and Ybx1flox/flox mice.(D-G) Representative images of Alizarin Red staining (D and F) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (E and G) in BMSCs transfected with different isoforms of Sp7 or different isoforms of Spp1.(H-I) Representative images (H) and quantification of Oil Red O staining (I) in BMSCs transfected with different isoforms of Spp1 with 10 days of adipogenic induction.(J-K) Representative images of β-Gal staining (J) and quantification (K) of β-Gal positive cells in BMSCs transfected with different isoforms of Sirt2.(L-T) BMSCs were isolated from Ybx1Prx1-CKO mice and Ybx1flox/flox mice, among them, the BMSCs from Ybx1flox/flox mice were transfected with the blank control, BMSCs from Ybx1Prx1-CKO mice were transfected with the blank control or different isoforms of the target genes. (L-P) Representative images of Alizarin Red staining (L and O) and quantification of calcification by detecting the amount of Alizarin Red extracted from the matrix (M and P). (Q-R) Representative images (Q) and quantification of Oil Red O staining (R) in BMSCs after 10 days of adipogenic induction. (S-T) Representative images of β-Gal staining (S) and quantification (T) of β-Gal positive cells among BMSCs.
Figure 6(D) Representative images of Alizarin Red staining (up panel), Oil Red O staining (middle panel, scale bar: 100 μm) and β-Gal staining (bottom panel, scale bar: 100 μm) in BMSCs treated with different compounds. (E-G) Quantification of calcium mineralization based on Alizarin Red staining (E), quantification of Oil Red O based on Oil Red O staining (F), and quantification of β-Gal positive cells based on β‑Gal staining in BMSCs treated with different compounds (G).(K) Co-IP of His-FBXO33 with HA-YBX1 and a series of mutant HA-YBX1 proteins following transfection into BMSCs.(M) Western blotting analysis of FBXO33 and YBX1 levels in BMSCs treated with different concentrations of sciadopitysin.(N) Semi-quantitative PCR showed the isoforms of Fn1, Nrp2 and Sirt2 in cultured BMSCs isolated from 2-month-old or 24-month-old mice then treated with or without sciadopitysin.
Figure 7(B-F) Representative μCT images, Scale bar: 1mm. (B) and quantitative analysis of trabecular bone microarchitecture (C-F) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle (BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation).(G, H) Representative images (G) and quantification (H) of osteocalcin (OCN) positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of Ocn+ cells per bone perimeter (N. Ocn+/B.Pm). Arrows point to osteocalcin positive cells. Scale bar: 50 μ(I, J) Representative images (I) of H&E staining in distal femora and quantification (J) of the number of adipocytes related to the tissue area (right panel, N. adipocytes/T.Ar) in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Scale bar: 100 μm.(K, L) Representative images (K) and quantification (L) of TRAP positive cells in distal femora of 25-month-old mice administered with sciadopitysin or vehicle. Number of TRAP+ cells per bone perimeter (N. Trap+/B.Pm). Scale bar: 50 μm.(M) Representative images of calcein double labeling of trabecular bone. Scale bar: 50μm. (N, O) Quantification of the bone formation rate (BFR, N) and mineral apposition rate (MAR, O) based on calcein double labeling.