EMBO/SourceData huggingface dataset:
Figure 3(D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 but not Vasa released the cleaved target RNA from Ago3-piRISC. (F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.
Figure 4(C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain.(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA".
Figure 1(A) Masson's trichrome representative images of pulmonary fibrosis and quantification.(B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH.(E) Masson's trichrome representative images of vascular wall thickening and quantification.(F) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients).(G) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph.
Figure 2(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001).Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients.Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients.(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.(E) Relative CD68 mRNA expression normalized to GAPDH(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.
Figure 3(A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target.(C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular.Relative expression of Prolactin-induced protein (PIP) mRNA (D) normalized to GAPDH in PF and PF-PH patients.Relative expression of Prolactin-induced protein (PIP) expression normalized to GAPDH in PF and PF-PH patients.(F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification.Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.Proliferation assay on healthy pulmonary arterial SMC J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.
Figure 4(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA.(F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol.(H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model.Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH.(L) Masson's trichrome representative images and quantification of vascular remodeling.(M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph.
Figure 5(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model.Relative expression of Slug mRNA (B) normalized to GAPDH.Relative expression of Slug protein (C) normalized to GAPDH.(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.
Figure 6Relative expression of PIP mRNA (A) normalized to GAPDH.Relative expression of PIP protein (B) normalized to GAPDH.(C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph.
Figure 7Relative expression of Slug protein (B) normalized to GAPDH.Relative expression of Slug mRNA (C) normalized to GAPDH.(D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm.Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH.(I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness.(K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung.(L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.
Figure 8(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green).(B) Relative expression of CD68 mRNA normalized to GAPDH.(C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung.Relative expression of PIP mRNA (D) normalized to GAPDH.Relative expression of PIP protein (E) normalized to GAPDH.(F) Representative images and quantification of PIP immunohistochemistry.
Figure 1E) H2AQ105me and H3K56ac levels in deletions of genes important for H3K56ac depositionG-J) ChIP-qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points. Samples were either normalized to input or to the core histone. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, relative to t=0, based on unpaired, 2-tailed t-test
Figure 2B) Volcano plot of quantitative SILAC experiments. Left upper quadrant indicates significantly, 2-fold enriched proteins bound specifically to H2AQ105me. Members of the FACT complex (SPT16/POB3) are highlighted in red.
Figure 3C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were slot blotted and detected using streptavidine-HRP.E) ChIP-seq profile of Nhp2 (orange) and its respective input (black) across the entire chromosome XII of yeast. H2A-normalised H2AQ105me (grey) was directly taken as comparison. Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks. Scale bar is for the magnification.
Figure 4D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoffG) Peptide pulldowns from human cells (HEK293T) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me. Dyskering (Cbf5 homolog) was probed to test for the presence of members of the H/ACA complex. Pulldown efficiency was analyzed by western blotting using the indicated antibodies.I) Quantification of Northern Blot results. Normalised to GAPDH, n=3, error bars are standard error of the mean. Asterix indicates p-value (*, p<0.05, based on unpaired, 2-tailed t-test).
Figure 1E. Normalized ThT kinetics of 10μM Biot-Aβ1-42 with timepoints of samples that incubated with Aβ1-42 membranes.I. ThT kinetics of Biot-Aβ1-42 seeding. 10μM of Biot-Aβ1-42 incubated with 0.5 or 1 μM of Biot-Aβ1-42 seeds.
Figure 2D. Binding of Biot-Aβ1-42 to homologue peptides derived from ~520 randomly selected proteins.E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.
Figure 3A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.
Figure 4A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.G. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.
Figure 5A. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)D. No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions (α-synuclein aggregates). (statistics: hypergeometric test with Bonferroni correction)
Figure 6D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).E. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm
Figure 1(A) Chromatin immunoprecipitation (ChIP) analysis of receptor binding to the ER8 element (region 1) of the nrf2 promoter. Immunoprecipitation with nonspecific IgG and PCR amplification of an adjacent region (2) served as controls. ChIP assays were also carried out to analyse binding to ER8s in the il10, ddit3 and tyrobp genes. RAR, retinoic acid receptor; VDR, vitamin D receptor.(B) Reverse transcription-PCR analysis of 1,25‐dihydroxyvitamin D3 (1,25D3)‐ or retinoic acid (RA)‐regulated expression of ddit3, il10, and tyrobp in U937, Calu‐3 and MCF‐7 cells.
Figure 2(A) Electrophoretic mobility shift assay (EMSA) of binding of vitamin D receptors (VDRs)/retinoid X receptors (RXRs) to thep19ink4dER8, with binding to the mouseosteopontin (mop) DR3vitamin D responsive element as a control, along with a comparison of binding to ER6 and ER8 motifs (right‐hand panel).(B) EMSA of retinoic acid receptor (RAR)/RXR binding to the p19ink4d ER8, and the rarβ DR5 retinoic acid response element, as a control.(C) Chromatin immunoprecipitation (ChIP) analysis of binding of VDRs and RARs to the p19ink4d ER8 (region 1) and RNA polymerase II (POLII) binding to the transcription start site.(D) ReChIP analysis confirms the presence of VDR/RXRs and RAR/RXRs but not of VDR/RARs on the p19ink4d ER8.(E) Cloning of p19ink4d promoter containing or lacking ER8 upstream of a luciferase reporter. 3 (1,25D3)‐ (F) and retinoic acid (RA)‐regulated (G) luciferase expression is dependent on the ER8 element in the p19ink4d promoter. Luciferase expression driven by a thymidine kinase (tk) control promoter is shown.
Figure 33 and retinoic acid induce p19ink4d expression. Expression of p19ink4d messenger RNA by reverse transcription-PCR (A) or protein by western blotting (B) was carried out on extracts of cells treated with 3 (1,25D3) or retinoic acid (RA), as indicated.(C) Treatment with 1,25D3 or RA transiently enhances co‐immunoprecipitation (IP) of p19INK4D with cyclin‐dependent kinase CDK4 in U937 cells. Similar results were obtained in SCC25 cells (not shown). A control western blot (W) of CDK4 levels in ligand‐treated cells is shown below.(D) Treatment with 1,25D3 enhances co‐immunoprecipitation of p27KIP1 with CDK4 in U937 cells. Extracts of U937 cells were probed for p27KIP1 and CDK4, and immunoprecipitated with an anti‐CDK antibody and probed for p27KIP1 co‐immunoprecipitation.
Figure 4(A) Short interfering RNAs (siRNA) knockdown of p19ink4d messenger RNA and protein in SCC25 cells. SiRNAs were directed against p19ink4d, scrambled p19ink4d (Scr.) or cyclophilin (cyc.) controls.(B) Combined knockdown of p27KIP1 and p19INK4D in SCC25 cells analysed by western blotting for p27KIP1 (top) and p19INK4D (bottom).(C,D) Fluorescence‐activated cell sorting analysis of cell‐cycle distribution of control SCC25 cells, or cells transfected with scrambled (Scr), p19ink4d‐ or p27kip1‐directed siRNAs, individually or together (Dbl). (C) Cells were treated with vehicle or 1,25‐dihydroxyvitamin D3 (1,25D3), as indicated. (D) Cells were treated with DMSO vehicle, retinoic acid (RA) or 1,25D3, as indicated.(E) Loss of p19 enhances lysosomal β‐galactosidase activity in SCC25 cells. (Left) Histogram of numbers of cells staining for β‐galactosidase activity in the absence (−) or presence of 1,25D3 in control cells (−) or in cells transfected with scrambled (Scr) or p19 siRNAs. (Right) Bright field images of untransfected cells (−/−) or p19INK4D‐depleted cells treated with 1,25D3 (p19/D3).(F) Loss of p19INK4D induces lysosomal fusion. Analysis of lysosomal fusion (autophagosomes) in SCC25 cells treated as in (E), stained for incorporation of lysotracker red. Nuclei were counterstained with Hoechst (blue).
Figure 1A. ROCK2 immunohistochemistry stained sections of normal and stages I, II and III cases of human pancreas adenocarcinoma. Scale bar = 50 µm (left). Histoscores of ROCK2 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma stage I (n=22), stage II (n=46) and stage III/IV (n=4). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.B. Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and/or ROCK2 gene amplification, significantly increased mRNA or truncation mutation from TCGA research network. Survival p value determined by Log-Rank test.C, D. Log2 median-centered ROCK1 or ROCK2 RNA expression in normal (n=39) vs PDAC (n=39), normal (n=5) vs pancreatic adenocarcinoma (PAC) (n=12), or normal (n=6) vs pancreatic carcinoma (PC) (n=11) samples from indicated studies. Exact p values determined by Mann-Whitney test.E. ROCK1 and ROCK2 mRNA expression in human PDAC (n=146) from the TCGA research network. Significance (p value) of slope deviation from 0 determined by Deming regression.F. Representative hematoxylin and eosin (H&amp;amp;E) and ROCK2 immunohistochemistry stained sections of normal mouse pancreas, acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1-3, and PDAC from indicated genotype mice. Scale bar = 50 µm.G. Quantification of ROCK2 staining in epithelial cells of normal, ADM/PanIN1 (KC), PanIN2 (KPC), PanIN3 (KPC) and PDAC (KPC) pancreatic tissue (n=5 mice per group). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.
Figure 2A. H&amp;amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test.B. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally-activated human ROCK1, human ROCK2 and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody.C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.D. H&amp;amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=30), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.G. Ki67 positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n=30; n=12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).
Figure 3A. Immunofluorescence of collagen matrix sections co-stained for cleaved collagen neo-epitope Col1 3/4C and DAPI (left panels). Second harmonic generation (SHG) images of collagen fibers within 3-dimensional collagen matrices at 15 µm and 45 µm below the cell surface (middle panels). Scale bar = 20 µm. Graphs indicate the mean ± SEM fibrillary collagen areas at 2.5 µm increments below the cell/collagen interface (n=5). SHG fibrillar collagen (red); cells (green).B, C. Grey level correlation matrix (GLCM) texture analysis of SHG correlation (n=6) and contrast (n=5). Mean ± SEM at each distance.D. Transmission electron microscopy of a collagen matrix section. Scale bar = 1 µm. Positions of magnified areas indicated by corresponding colored squares.E. Transmission electron microscopy of collagen matrix sections with collagen stained by Tannic acid. Yellow arrows indicate long collagen bundles, Red arrows indicate short collagen bundles. Scale bar = 0.5 µm.
Figure 4A. Volcano plots of RNA sequencing data of genes that pass a threshold false discovery rate (FDR) < 5%, with log10 fold-change (FC) in expression in 4HT treated ROCK1:ER expressing cells relative to GFP:ER expressing cells (left) or 4HT treated ROCK2:ER expressing cells relative to GFP:ER expressing cells (right) versus log10 adjusted p value (n=3). ROCK1:ER+4HT vs GFP:ER+4HT; 3828 genes. ROCK2:ER+4HT vs GFP:ER+4HT; 4481 genes.C. Heatmap of 33 differentially expressed genes in the MetaCoreTM process network Cell adhesion: Cell-matrix interaction. High relative expression in red and low relative expression in blue.D. MMP average RNA sequence reads. Mean ± SEM (n=3) * adjusted p-value <0.05.E. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp10 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.F. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-) or 1 µM 4HT for 24 h (left). Means ± SEM (n=4), p value by unpaired t test. Mmp13 mRNA levels relative to Gapdh determined by qPCR following treatment with EtOH vehicle (-), 1 µM 4HT or 4HT + 1 µM H1152 ROCK inhibitor for 24 h (right). Means ± SEM (n=3), one-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.G. MMP10 immunohistochemistry stained sections of human pancreas adenocarcinoma. Scale bar = 100 µm (left). Histoscores of MMP10 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma grade 1 (n=23), grade 2 (n=27) and grade 3 (n=13). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.