EMBO/SourceData huggingface dataset:
Figure 3(A) Real-Time RT-PCR analysis for IL-1β mRNA levels in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(B) ELISA assay for IL-1β protein level in the culture medium of PC3 cells with control or CTH knockdown. Data information All data shown represent the means ± SD (n=3 biological replicates). Student's t-test (B) was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(C) PC3 cells transfected with control or CTH knockdown were incubated for invasion (24h) assay with recombinant IL-1β (10ng/ml) added to the medium of the lower chamber. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).(D) PC3 cells transfected with control or CTH siRNA were exposed to IL-1β (20ng/ml) for 1hr. Subcellular localization of p65 was detected by immunocytochemistry. Nuclei were counterstained with DAPI. The representative images are shown. Scale bars: 25μm.(E) The percent of nuclear translocation for p65 was scored by counting the number of nuclear positive stained cells of p65 to the total number of cells in random microscopic fields. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).Real-Time RT-PCR analysis for VEGF (F) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).Real-Time RT-PCR analysis for MMP-13 (G) mRNA level in PC3 cells with control or CTH knockdown. Recombinant IL-1β (10ng/ml) was added for 24h in serum-free medium. Data information: All data shown represent the means ± SD (n=3 biological replicates). ANOVA followed by Tukey's post-hoc test was used for the statistical analysis. (*P<0.05; **P<0.01; ***P<0.001).
Figure 5(A) Western blot analysis of the CTH expression in PC3 cells stably expressing control or CTH shRNA.1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. The tumor weight (B), and tumor volume (C) were compared. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. Incidences of paraaortic lymph nodes metastasis (D) and bone metastasis (E) are shown. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).(F) Real-Time RT-PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group. Data shown represent normalized means ± SEM (n=10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using CD31 (G) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using LYVE-1 (H) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).
Figure 1Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH. Shown are maximum projections of 50 µm z-stacks covering an entire CV-PV axis (CV, left; PV, right). Indicated regions of the CV (orange) and PV (blue) areas are shown as magnifications on the left and right, respectively.Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) in livers from untreated animals or mice at indicated timepoints post PH. The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=3-6 mice per timepoint. BC diameter of untreated mice vs. 0.8 d, p=1.62*10-11; untreated mice vs. 1.5 d, p=1.18*10-12; untreated mice vs. 2.0 d, p=5.69*10-11; untreated mice vs. 3.0 d, p=6.92*10-12; untreated mice vs. 5.0 d, p=1.65*10-7.EM images of BC on liver tissue sections of untreated mice (left) and 1.8 d post sham OP (middle) or PH (right).Quantification of BC perimeter (d) and total BC membrane length (e) from EM images as representatively shown in (c). Box-whisker plot with median, 25-75 quartiles and minimum/maximum error bars, n=5-6 mice per condition. In (d), BC perimeter of untreated vs. sham condition, p=0.94 (n.s.); Untreated vs. PH condition, p=0.01. In (e), BC membrane length of untreated vs. sham condition, p=0.86 (n.s.); Untreated vs. PH condition, p=0.03.IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL. Shown are maximum projections of 50 µm z-stacks in the CV (left, orange) and PV (right, blue) region. Images in (a) and (f) are background-subtracted. Scale bars, 50 µm (a), 1 µm (c), 5 µm (f).
Figure 2Fluorescence stainings for F-actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of apical F-actin intensity from images as representatively shown in (a, as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) mice per timepoint.Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=2-5 (d) mice per timepoint.
Figure 3Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.Quantification of the mean nuclear YAP (green) and PCNA (magenta) intensity from images of liver tissue sections at indicated timepoints post PH (solid line) and sham OP (dashed line) as representatively shown in (a) and Appendix Fig. S4A. Data was normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of PH vs. sham time course, p=2.97*10-5. Nuclear PCNA intensity of PH vs. sham time course, p=7.51*10-6.Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in (a) and Appendix Fig. S4A, at indicated time points post PH (solid line) or sham OP (dashed line) in 10 zones within the CV-PV axis (zone 1, CV area; zone 10, PV area). Data is normalized to untreated animals (not shown). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of sham vs. PH mice at 0.8d, p=5.35*10-5; 1.5d, p=3.95*10-5; 2d, p=5.99*10-5; 3d, p=2.16*10-5; 5d, p=0.01.Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.Fractionation of liver tissue at 2d post PH or sham (Sh) OP and untreated (UT) mice for nuclear (Nuc), cytoplasmic (Cyto) and membrane (Mem) fraction. TRF2, transferrin receptor2 (membrane marker); Histone H3 (nuclear marker); GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (cytoplasmic marker). Quantification of YAP in the nuclear, cytoplasmic and membrane fraction from immunoblots as representatively shown in (f). YAP signal is normalized to the fraction marker (GAPDH, cytoplasm; Histon H3, nuclei; TRF2, membrane) and to the untreated condition (not shown). n = 3 (sham) or 4 (PH); data is shown as mean +/- s.e.m. Differences between sham and PH conditions for cytoplasmic, nuclear and membrane fractions are not significant.
Figure 4EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.
Figure 5Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compoundsFluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).
Figure 6Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area. Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.
Figure 1b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).
Figure 2a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise).
Figure 4c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).
Figure 5b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.
Figure 4A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.
Figure 5A. B-factor putty representation of the intertwined loop structure. Shades of blue and a narrower tube indicate regions with lower B-factors, whereas orange to red and a wider tube indicate regions with higher B-factors. The figure was prepared using PyMOL (http://pymol.org/).B. The 2Fo-Fc electron density map for the intertwined loop (yellow) "spike" region residues contoured at 1.0 sigma is represented in blue.C. Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop. W115 forms a hydrogen-bonding interaction with E74 from α2 helix. W118 inserts into the hydrophobic interspace between α1 and α2 helix, formed by P36, L42, V71, L75 and I78, and W118 also interact with P36 and D37 by hydrogen-bonding interactions.
f1(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg-1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.(d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively.(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.(f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments.(g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).
f2(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate.(b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm.(c) Mitochondrial content of H-Ras 12 V expressing WT, p53−/− and p21−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. Results are shown from four independent experiments performed in duplicate.(d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate.(e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown.(f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate.(g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heartmitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).
f3(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies.(b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody.(c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies.(d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53.(f) Schematic representation of the GST-p53 fusion proteins. (g) GST pull-down assays using GST-p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81-160 of p53 were sufficient to bind Parkin.