Journal: bioRxiv

Article Title: MicroRNA-195-3p as a potential regulator of hypoxic injury in HUVECs

doi: 10.1101/2023.02.23.529661

Figure Lengend Snippet: miR-195-3P inhibitor regains hypoxia-induced HUVECs proliferation rate. ( A, B ) EdU results shows the proliferation rate of each group. The cells were stained with Hoechst 33342 (5 μg/mL), and the proliferation population was analyzed (n=3). Scale bar, 50 μm. ( C ) The viabilities of cells in the different treatment groups was measured using the CCK-8 assay (n=5). ( D, E ) Representative images of cell colonies. Colony formation assays determined cell proliferation in HUVECs. **p < 0.01, ***p < 0.001 vs. the control group. ##p < 0.01, ###p < 0.001 vs. hypoxia induction for 6 h group. HUVECs, human umbilical vein endothelial cells; EdU, 5-ethynyl-2’-deoxyuridine; CCK-8, Cell Counting kit-8.

Article Snippet: HUVECS viability was assessed by Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay as described previously.

Techniques: Staining, CCK-8 Assay, Control, Cell Counting

Journal: Protein Science : A Publication of the Protein Society

Article Title: Single domain antibodies for the knockdown of cytosolic and nuclear proteins

doi: 10.1002/pro.3154

Figure Lengend Snippet: Reports of Cytosolic/nuclear sdAb Mediated Knockdown

Article Snippet: Tanaka et al. 57 Endothelial and epithelial kinase (Etk) Human single domain VL library (Domantis) derived from a single human framework of a ĸ light chain variable region.

Techniques: Knockdown, Recombinant, Plasmid Preparation, Immunofluorescence, Expressing, Selection, Transfection, Virus, Enzyme-linked Immunosorbent Assay, Inhibition, Infection, Purification, Flow Cytometry, In Vivo, Transgenic Assay, Retroviral, Injection, Irradiation, Activation Assay, Modification, Binding Assay, Clone Assay, Activity Assay, Construct, Cotransfection, Blocking Assay, Western Blot, Transformation Assay, Cell Culture, Isolation, Generated, Derivative Assay, Translocation Assay, Membrane, Migration, Luciferase, Sequencing, Incubation, Phospho-proteomics, Clinical Proteomics, Variant Assay, In Vitro, Cytotoxicity Assay, Mutagenesis, Solubility, Bioassay, Starch, Knock-In, Ubiquitin Proteomics, Knock-Out

Journal: eLife

Article Title: Control of endothelial cell polarity and sprouting angiogenesis by non-centrosomal microtubules

doi: 10.7554/eLife.33864

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , AMAXA huvecs nucleofector kit , Lonza , Lonza:VPB-1002 , .

Techniques: Cell Culture, Recombinant, Sequencing, Amplification, Software

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a) UMAP embedding of 63,581 single cells from hPCLS color coded by meta cell type identity. (b) Stacked bar plots show relative cell type frequency changes with regards to treatment. (c-e) UMAP embedding of hPCLS data color coded by cell state (c), tissue donor (d) and tissue donor/treatment (e), respectively. (f) UMAP feature plots show relative expression of lineage markers EPCAM+ (epithelial cells), CLDN5 + (endothelial cells), COL1A2 + (stromal cells), and PTPRC + (CD45, immune cells) cells in the ex vivo hPCLS data. (g) Clinical metadata of hPCLS tissue donors. (h) UMAP embedding of 481,788 single cells from the integrated multi-cohort pulmonary fibrosis cell atlas, color coded by meta cell type identity. (i) Stacked bar plot of relative cell type frequency changes. (j,k) UMAP embedding of the integrated pulmonary fibrosis cell atlas color coded by cell state (j) and cohort (k), respectively. (l) UMAP feature plots UMAP feature plots showing relative expression of lineage markers EPCAM+ (epithelial cells), CLDN5+ (endothelial cells), COL1A2+ (stromal cells), and PTPRC+ (CD45, immune cells) in the in vivo integrated pulmonary fibrosis cell atlas (m) In vivo marker genes signatures of meta-cell type identities. (n,o) matchSCore comparison of ex vivo against in vivo marker gene signatures from Control Cocktail (CC) treated hPCLS (d6) and control lungs (n) as well as Fibrotic Cocktail (FC) treated hPCLS (d6) and pulmonary fibrosis lungs (o).

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Expressing, Ex Vivo, In Vivo, Marker, Comparison, Control

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a,b) UMAP embedding of 2,741 single epithelial cells from FC- and CC-treated hPCLS, color coded by cell type identity (a) and treatment (b) . (c) Ex vivo marker gene signatures of epithelial cells. The heatmap shows the scaled average expression in each cell type. (d) Cell type proportion analysis of epithelial cells in FC-vs CC-treated hPCLS ( ex vivo , left) and PF vs control patients ( in vivo , right): Stacked bar plot shows the relative frequency of each cell type with regards to treatment and tissue donor. (e) Boxplot of in vivo basaloid gene signature (derived from the in vivo reference PF cell atlas) score after treatment with FC or CC per tissue donor. Mann-Whitney-Wilcoxon test, ****p ≤ 0.0001. (f) Boxplot of in vivo AT2 cell gene signature (derived from the in vivo reference PF cell atlas) score after treatment with FC or CC per tissue donor. Mann-Whitney-Wilcoxon test, ****p ≤ 0.0001. (g) The Venn diagram illustrates the intersection of genes in alveolar epithelial cells that are uniformly upregulated both in vivo and ex vivo . (h) Qualitative analysis of conserved responses on the gene, pathway and Transcription Factor (TF) regulon level in alveolar epithelial cells. The bar plots illustrate the uniform behavior of conserved genes (log2FC), PF- and health-specific TF regulons (Regulon Specificity Score - RSS), and IPA pathways (IPA pathway activity score). (i) Staining and analysis pipeline assessing the presence of KRT17+/KRT5-basaloid cells by immunofluorescence (IF), high-throughput imaging and machine-learning segmentation of FFPE sections derived from histopathological and microCT staged lung tissues from IPF and control patients (scale bars = 1 mm). (j) Representative IF images of SFTPC (light gray), KRT17 (green) and KRT5 (red) stained lung sections demonstrating the presence of SFTPC-/KRT17+/KRT5-basaloid cells (red arrowheads) already in early-stage IPF (IPF1, scale bars = 50 μm). (k) Quantification of the percentage of SFTPC-/KRT17+/KRT5-basaloid cells by single-cell segmentations of 4 randomly selected regions of interest (ROIs) per patient (n = 5 controls, and n = 3 - 4 per IPF stage). In total, 249.700 single cells were analyzed. Statistics: Unpaired t-test. Bar plot depicts the Mean ± standard deviation. (l) ROI1 and ROI2 from IPF1 shown in panel (j) demonstrate distinct epithelial cell states: SFTPC+/KRT17-/KRT5-(green dashed contour), SFTPC+/KRT17+/KRT5-(yellow dashed contour), SFTPC-/KRT17+/KRT5-(red dashed contour) and SFTPC-/KRT17+/KRT5+ (blue dashed contour). Scale bars = 20 μm. (m) Stacked bar plots depicting the relative frequencies (Mean ± standard deviation) of the four distinct epithelial states across the different IPF stages. Statistics: Mixed effect model for two factors “stage” and “cell state”. p(stage x cell state) = 0.016. The same ROIs as in panel k and 25,466 single cells were quantified.

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Ex Vivo, Marker, Expressing, Control, In Vivo, Derivative Assay, MANN-WHITNEY, Activity Assay, Staining, Immunofluorescence, High Throughput Screening Assay, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a-c) UMAP embedding of 2,741 single EPCAM+ epithelial cells from FC and CC treated hPCLS, color coded by cell type (a) , tissue donor (b) , and treatment / sample (c) . (d-n) UMAP feature plots illustrating relative expression of KRT17+/KRT5-basaloid marker gene signature. (o) DC embeddings of trajectory inference analysis on AT2 cells, basaloid cells, and basal cells color coded (from left to right) cell type overlaid by RNA velocity, diffusion pseudotime, condition, relative expression of AT2 marker SFTPC, relative expression of shared basal and basaloid cell marker KRT17 and relative expression of basaloid marker MMP7. (p-r) UMAP embeddings of 181,453 single EPCAM+ epithelial cells from the in vivo pulmonary fibrosis cell atlas, color coded by cell type (p) , disease status (q) , and cohort (r) . (q) In vivo marker gene signatures. The heatmap shows the scaled average expression of markers in each epithelial cell type. (t,u) Qualitative analysis of non-conserved responses to FC on the gene (t) and pathway (u) level in alveolar epithelial cells. The bar plots illustrate the diverging behavior of non-conserved genes (log2FC) (t) , and IPA pathways (IPA pathway activity score) (u) in alveolar epithelial cells.

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Expressing, Marker, Diffusion-based Assay, In Vivo, Activity Assay

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a) Additional representative indirect iterative IF image (4i) of eight different epithelial (KRT17, KRT5, KRT8, SFTPC, PDPN) and stromal (TNC, aSMA, DES) cell markers demonstrating the interaction of elongated basaloid (SFTPC-/KRT17+/KRT8+/KRT5-) cells (white dashed contour) and myofibroblasts (TNC+/aSMA+/DES-) in fibroblastic foci of early-stage IPF (IPF1). Scale bars = 100 µm (overview image) and 20 µm (enlarged view). (b) Basaloid cells and myofibroblasts are absent in control lungs. (c) The interaction of SFTPC-/KRT17+ cells (white arrowheads) and TNC+/aSMA-/DES-(myo-)fibroblasts is also recapitulated ex vivo in FC-treated hPCLS at d6. In addition, an interaction of KRT17+/SFTPC-cells with TNC+/aSMA+/DES+ cells in the thickened alveolar septum has been observed. Scale bars = 100 µm (overview image) and 50 µm (enlarged view). (d) No interaction of KRT17+ cells and (myo-)fibroblasts are found in CC-treated hPCLS (d6). Scale bars = 100 µm (overview image) and 20 µm (enlarged view).

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Control, Ex Vivo

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a-c) UMAP embedding of 10,418 single CLDN5+ epithelial cells from FC and CC treated hPCLS, color coded by cell type (a) , tissue donor (b) , and treatment / sample (c) . (d-h) UMAP feature plots illustrating relative expression of VWA1+/PLVAP+ ectopic EC marker gene signature. (j-m) UMAP embeddings of trajectory inference analysis on endothelial cells, color coded by cell type and overlaid by estimated cell type connectivity as measure by partition-based graph abstraction (PAGA) (j) , CellRank initial states probability (k) , CellRank terminal states probability (l) , and CellRank latent time (m) . (n-p) UMAP embeddings of 29,534 single CLDN5+ immune cells from the in vivo pulmonary fibrosis cell atlas, color coded by cell type (n) , disease status (o) , and cohort (p) . (q) In vivo marker gene signatures. The heatmap shows the average scaled expression of markers in each endothelial cell type. (r,s) Qualitative analysis of non-conserved responses to FC on the gene (r) and pathway (s) level in vascular endothelial cells. The bar plots illustrate the diverging behavior of non-conserved genes (log2FC) (r) , and IPA pathways (IPA pathway activity score) (s) in vascular endothelial cells.

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Expressing, Marker, In Vivo, Activity Assay

Journal: bioRxiv

Article Title: Ex vivo tissue perturbations coupled to single cell RNA-seq reveal multi-lineage cell circuit dynamics in human lung fibrogenesis

doi: 10.1101/2023.01.16.524219

Figure Lengend Snippet: (a) Ex vivo cell-cell communication routes in hPCLS after treatment with FC. Circle plot shows contributions of ligands upregulated after FC treatment (vs CC) in sender cell types to gene expression changes after treatment with FC (vs CC) in receiver cell types. The edge weight (thickness) represents the sum of the ligand scores, which is the product of the ligands expression in the sender cell and the NicheNet target gene prediction score. The color of the edge reflects the origin (sender cell) of the cell-cell communication route. The node size is proportional to the sum of the strength of outgoing cell-cell communication routes. (b) In vivo cell-cell communication routes in IPF. Circle plot shows contributions of ligands upregulated in IPF (vs healthy) in sender cell types to gene expression changes in IPF (vs healthy) in receiver cell types. (c) Number of conserved ligands ( in vivo and ex vivo ) from sender cells (rows) to receiver cells (columns) at meta-cell type level. The heatmap is clustered hierarchically and highlights a conserved cell-cell circuit between alveolar epithelial cells, vascular endothelial cells, fibroblasts, MCs/pericytes, and macrophages. (d) Variation of gene expression of the conserved ligands ( in vivo and ex vivo ) across IPF progression. Heatmap shows the log2FC of the identified conserved ligands IPF stages 1, 2 and 3 (vs healthy control tissue) as derived from publicly available bulkRNA-seq data (GEO GSE124685). (e) Overview of the cell-cell communication routes between five FC-induced cell states as predicted by NicheNet. The left panel shows the average expression of ligands predicted by NicheNet across sender cells. The right panel visualizes the corresponding predictive score of all ligands to induce their downstream target gene signature in the respective receiver cell types as measured by the Pearson correlation target gene prediction ability. Ligands in bold letters are conserved compared to in vivo . (f) Schematic of the pro-fibrotic cell-cell circuit. Ligands are derived from panel (e) and coloured according to the sender cell type they originate from.

Article Snippet: Percentages of epithelial cell types and states were quantified in four randomly selected ROIs from each patient analogously to the ex vivo hPCLS data using IMARIS 9.6.0 software (see above).

Techniques: Ex Vivo, Gene Expression, Expressing, In Vivo, Control, Derivative Assay

Journal: Biochemical and biophysical research communications

Article Title: Particulate matter 2.5 exposure induces epithelial-mesenchymal transition via PI3K/AKT/mTOR pathway in human retinal pigment epithelial ARPE-19 cells.

doi: 10.1016/j.bbrc.2022.05.072

Figure Lengend Snippet: Fig. 3. PM2.5 regulates EMT through PI3K/AKT/mTOR signaling. ARPE-19 cells were exposed to PM2.5 (0, 12.5, 25, and 50 mg/ml) for 48 h. (A) The protein expression of a-SMA, vimentin, ZO-1, E-cadherin, and GAPDH were determined by western blotting. (B) The protein expression of PI3K, p-mTOR, p-AKT, and GAPDH were determined by western blotting. Cells without PM2.5 stimulation were used as the control group (mock). Experiments were performed in triplicates and data were shown as mean ± SD. Bars with the same letter indicate no significant difference between the groups. Bars with different letters represent a statistically significant difference (P < 0.05) between the groups.

Article Snippet: Antibodies against matrix metalloproteinase (MMP)-2, MMP-9, phospho-serine/threonine kinase 1 (p-AKT), phosphoinositide 3- kinases (PI3K), E-cadherin, and vimentin were purchased from Santa Cruz Biotechnology Inc. Antibodies against tissue inhibitor of metalloprotease 1 (TIMP1), TIMP2, and phospho-mammalian target of rapamycin (p-mTOR) were purchased from Spring Bioscience.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

doi: 10.1074/jbc.m110.174037

Figure Lengend Snippet: FIGURE 9. Secreted Klotho inhibits EMT in A549 cancer cells. A, Klotho inhibits TGF-1-induced phosphorylation of Smad3 in A549 cells. A549 cells were incubated with secreted Klotho protein for 15 min and then stimulated with TGF-1 for 15 min at the indicated doses. Cell lysates were subjected to immunoblot analyses using antibody against phosphorylated Smad3 (pSmad3) or antibody that recognized Smad3 regardless of its phosphoryla- tion state (Smad3). B, Klotho inhibits TGF-1-induced activation of the Smad- responsive reporter. A549 cells were transfected with a luciferase reporter containing Smad response elements (pGTCT2 2-Luc) and a lacZ expression vector for normalization. These cells were incubated with TGF-1 or Klotho at the indicated doses for 6 h and subjected to a standard luciferase assay. The luciferase activity was normalized with that of non-treated cells. Data indicate means S.E. of three independent experiments. p 0.02 by one-way ANOVA. C, Klotho inhibits TGF-1 binding to A549 cells. TGF-1 binding assays were performed in A549 cells in the absence or presence of Klotho protein (0.1 or 0.3 nM). The amount of bound TGF-1 was normalized with that without Klotho. Data indicate means S.E. of four independent experi- ments. p 0.006 by one-way ANOVA. D, Klotho protein suppresses TGF-1- induced decrease in E-cadherin and increase in N-cadherin. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to immunoblot analyses 48 h later. E, Klotho protein suppresses TGF-1-induced cell migration. A549 cells were treated with TGF-1 and/or Klotho at the indicated doses for 6 h and then subjected to a standard Tran- swell migration assay. Data indicate means S.E. of three independent experiments. *, p 0.001 versus TGF-1 treatment alone by two-tailed t test.

Article Snippet: Primary antibodies used in this study were Klotho (KM2119)(30), SMA, Vimentin (Santa Cruz Biotechnology), E-Cadherin (BD Biosciences, San Jose, CA), N-cadherin (Santa Cruz Biotechnology), phosphorylated Smad2 (Cell Signaling Technology, Beverley, CA), phosphorylated Smad3 (Cell Signaling), Smad2 (Cell Signaling), Smad3 (Cell Signaling), GAPDH (Abcam).

Techniques: Phospho-proteomics, Incubation, Western Blot, Activation Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Binding Assay, Migration, Two Tailed Test