Journal: EMBO Molecular Medicine

Article Title: Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl‐tRNA synthetase 1

doi: 10.15252/emmm.202216940

Figure Lengend Snippet: A WI‐26 VA4 cells were made to stably express Flag‐EPRS1 in the presence of doxycycline (WI‐26 VA4 Flag‐EPRS1 stable cells). The cells were transfected with negative control or untranslated region (UTR) of EPRS1‐targeting siRNAs for 72 h. The cells were also incubated in the presence of doxycycline for 68 h. After starved of serum for 6 h, the cells were treated with TGF‐β for 15 h. The level of COL1A1 was determined by immunoblot assay. The level of β‐actin was used as loading control. B, C WI‐26 VA4 cells were transfected with the indicated siRNAs for 72 h and incubated with TGF‐β for 15 h. The level of COL1A1 was determined by immunoblot assay. D The proportion of proline and the half‐lives of proteins detected in (B) and (C) are listed in the table. The proportion of proline in the polypeptide sequence was calculated based on UniProtKB. The half‐lives of proteins were cited from a study that measured half‐lives of whole proteome in NIH3T3 cells via mass spectrometry (Schwanhausser et al , ). Source data are available online for this figure.

Article Snippet: WI‐26 VA4 cells (human) were obtained from Korean Cell Line Bank (KCLB) (10095.1) and authenticated by STR profiling.

Techniques: Stable Transfection, Transfection, Negative Control, Incubation, Western Blot, Control, Sequencing, Mass Spectrometry

Journal: EMBO Molecular Medicine

Article Title: Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl‐tRNA synthetase 1

doi: 10.15252/emmm.202216940

Figure Lengend Snippet: A A total 523 HF derivatives were tested for in vitro efficacy, intracellular efficacy, cytotoxicity, in vivo efficacy, and in vivo toxicity as described in the Methods section. DWN12088 was selected as the final candidate. At each step, the compounds that met each screening criteria are shown in pink lines. The detailed screening strategy is described in Fig . B Dose–response curve of HF and DWN12088 in WI‐26 VA4 cells. The IC 50 value for collagen level was measured as a representative of chemical efficacy. The cells were starved of serum for 6 h and then incubated with the indicated concentrations of compounds in the presence of TGF‐β (2 ng/ml) for 72 h. The secreted pro‐collagen I levels were determined by ELISA. The CC 50 values were determined by CellTiter‐Glo assay. Dose–response curves were determined by combining at least two biologically independent experiments. The IC 50 values were calculated using GraphPad Prism 7.0 (Collagen level, n = 5 from three independent experiments (singlicate for one experiment and duplicate for two experiments; mean ± SEM); Celltiter‐Glo assay, n = 4 from two independent experiments (duplicate for each experiment; mean ± SEM). C The TI of HF and DWN12088 was determined by dividing the CC 50 with the IC 50 for collagen level (Welch's t test; *** P < 0.001; mean ± SEM). D Schedules of two different in vivo efficacy tests. E–I In vivo efficacy of HF and DWN12088 was determined in a bleomycin‐induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for 2 weeks from a week after intratracheal injection of bleomycin (D, model 1). The saturation of percutaneous oxygen (SpO 2 ) (E) was determined as a measurement of lung function. The number of cells in bronchoalveolar lavage fluid (BALF) was counted as a measure of inflammation response (F). The collagen level was determined by hydroxyproline assay (G) and Masson's trichrome staining (H and I) of lung tissues. The remaining Masson's trichrome staining images shown in Fig are presented in Appendix Fig ( n = 5; Mann–Whitney test after Kruskal–Wallis test; * P < 0.05, ** P < 0.01; mean ± SEM). scale bar = 200 μm. BLM, bleomycin; PO, per oral; QD, once a day; 12088, DWN12088; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg; b, bronchiole; v, blood vessel. Source data are available online for this figure.

Article Snippet: WI‐26 VA4 cells (human) were obtained from Korean Cell Line Bank (KCLB) (10095.1) and authenticated by STR profiling.

Techniques: In Vitro, In Vivo, Incubation, Enzyme-linked Immunosorbent Assay, Glo Assay, Injection, Hydroxyproline Assay, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl‐tRNA synthetase 1

doi: 10.15252/emmm.202216940

Figure Lengend Snippet: WI‐26 VA4 cells were incubated in the presence of the indicated concentrations of HF and DWN12088 for 72 h. The chemical concentrations were determined based on the IC 50 for collagen level (HF, 15 nM; DWN12088, 2 μM). The levels of proteins were determined by immunoblot assay. The proline content of the proteins based on Uniprot amino acid sequence is shown on the right side of the immunoblot images. The plot of “proline percentage in the protein” versus “chemical concentration relative to IC 50 for collagen” is displayed based on (A). The lowest concentration at which protein levels started to decrease was normalized by IC 50 values for collagen level and used as the y‐axis value. Proteins with no change in level, such as RagC in the DWN12088‐treated group, were excluded from the plot. The slope and R 2 value were calculated using GraphPad Prism 7.0. ULK1, unc‐51 like autophagy activating kinase 1; AIMP2, aminoacyl tRNA synthetase complex interacting multifunctional protein 2; AMPKα, protein kinase AMP‐activated catalytic subunit alpha; RagC, Ras‐related GTP binding C. WI‐26 VA4 cells were incubated with the indicated concentrations of HF and DWN12088 in the presence of 2 ng/ml TGF‐β for 15 h. The chemical concentrations were determined based on the IC 50 values for collagen levels (HF, 15 nM; DWN12088, 2 μM). The activation of the TGF‐β pathway was determined by monitoring the phosphorylation of SMAD2. Immunoblot images are representative of three biologically independent experiments. The band intensities of the immunoblot images in (C) were quantified using ImageJ ( n = 3; One‐way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001; mean ± SEM). Source data are available online for this figure.

Article Snippet: WI‐26 VA4 cells (human) were obtained from Korean Cell Line Bank (KCLB) (10095.1) and authenticated by STR profiling.

Techniques: Incubation, Western Blot, Sequencing, Concentration Assay, Binding Assay, Activation Assay, Phospho-proteomics

Journal: EMBO Molecular Medicine

Article Title: Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl‐tRNA synthetase 1

doi: 10.15252/emmm.202216940

Figure Lengend Snippet: A The role of F1097, E1123 and R1152 in the binding to the compounds, ATP and proline in complex structures of PARS1‐HF‐ANP (PDB: 4HVC), PARS1‐DWN12088‐ATP and PARS1‐proline (PDB: 4K87). The color codes of the structures are the same as those shown in Fig . B The binding of HF and DWN12088 to in vitro purified PARS1 WT and F1097A/E1123A/R1152L (FA/EA/RL) mutant was determined via microscale thermophoresis (MST). K d values are listed in the top left part of the graph (technical replicates n = 3; mean ± SD). C Catalytic activities of PARS1 WT and FA/EA/RL mutant were determined by prolylation assay in the presence of [ 3 H]proline. To monitor the effect of PARS1 RA/EA/RL mutant on the activity of PARS1 WT, the same concentrations of the proteins were pre‐incubated for 10 min and then used for the assay. The formation of Pro‐tRNA Pro was measured using liquid scintillation counter (technical replicates n = 3; mean ± SEM). D The kinetic values of PARS1 with homodimer (WT/WT) and heterodimer (WT/MT) settings were determined by prolylation assay in the presence of [ 3 H]proline. MT, PARS1 FA/EA/RL (biological replicates n ≥ 3; mean ± SEM). E 293 T cells were transfected with the indicated plasmid DNAs for 24 h. The cell lysates were prepared and pulled down with Strep bead for 2 h. Co‐precipitated proteins were determined by immunoblot assay. WT, PARS1 wild type; MT, PARS1 FA/EA/RL. F Schematic representation of the strategy for generating a monomer‐like EPRS1. Endogenous EPRS1 dimer would function as a monomer‐like EPRS1 by forming a dimer with exogenous PARS1 mutant that cannot bind to the compounds and does not show the catalytic activity. G, H WI‐26 VA4 cells stably expressing each of EV (empty vector) or PARS1 FA/EA/RL mutant were incubated with the indicated compounds for 72 h. Dose–response curves of the compounds for collagen levels and cytotoxicity were determined and relative ratio of CC 50 to the IC 50 of collagen for HF (G) and DWN12088 (H) were calculated. The IC 50 values were calculated using GraphPad Prism 7.0 (collagen ELISA, n = 12 from four independent experiments (triplicate for each experiment); cytotoxicity assay, n = 8 from four independent experiments (duplicate for each experiment); Welch's t test; *** P < 0.001; mean ± SEM). I, J Relative slopes of dose–response curves of HF (I) and DWN12088 (J) for collagen levels were determined in cells stably expressing each of EV or PARS1 FA/EA/RL mutant. The slopes were determined by using GraphPad Prism 7.0 ( n = 3 in triplicates for collagen ELISA, n = 4 in duplicates for cytotoxicity assay; mean ± SEM; Welch's t test; *** P < 0.001; mean ± SEM). K WI‐26 VA4 cells stably expressing each of EV or PARS1 FA/EA/RL mutant were incubated with the indicated compounds in the presence of TGF‐β for 72 h. The levels of the indicated proteins were determined by immunoblotting. The percentage of proline in polypeptide sequences is shown on the right side of immunoblot images. Source data are available online for this figure.

Article Snippet: WI‐26 VA4 cells (human) were obtained from Korean Cell Line Bank (KCLB) (10095.1) and authenticated by STR profiling.

Techniques: Binding Assay, In Vitro, Purification, Mutagenesis, Microscale Thermophoresis, Activity Assay, Incubation, Transfection, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay

Journal:

Article Title: HtrA Homologue of Legionella pneumophila : an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

doi: 10.1128/IAI.69.4.2569-2579.2001

Figure Lengend Snippet: Intracellular growth kinetics of GT20 and the parental strain AA100 in (a) U937 macrophage-like cells, (b) WI-26 alveolar epithelial cells, and (c) A. polyphaga. Strain GT20(pLP102) is a plasmid-complemented clone of GT20. These data are representative of at least three independent experiments performed in triplicate. The absence of error bars indicates very small standard deviations that could not be displayed.

Article Snippet: Macrophage-like U937 cells and human type I alveolar epithelial cells (WI-26 VA4; ATCC CCL-95.1) and axenic A. polyphaga were maintained and used for infections as described previously ( 26 , 27 ).

Techniques: Plasmid Preparation

Journal:

Article Title: HtrA Homologue of Legionella pneumophila : an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

doi: 10.1128/IAI.69.4.2569-2579.2001

Figure Lengend Snippet: Cytopathogenic defect of GT20 is due to the defect in intracellular replication. (a) Cytopathogenicity of GT20 and the parental strain AA100 to U937 macrophage-like cells as determined by Alamar Blue dye reduction at various time points following infection at an MOI of 0.5. Strain GT20(pLP102) is a plasmid-complemented clone of GT20. Percent killing was normalized to uninfected cells, which were considered 100% viable. These data are representative of at least three independent experiments performed in triplicate. (b) Induction of apoptosis as evidenced by DNA fragmentation in U937 macrophages following 3 h of incubation after 1 h of infection at an MOI of 50. The φχ size standard is shown on the left. (c) Contact-dependent hemolysis of sheep red blood cells following 1 h of incubation at a bacterium-blood cell ratio of 25:1. Hemolysis was measured spectrophotometrically at A415. RBC represents red blood cells incubated in the absence of bacteria. These data are representative of at least three independent experiments performed in triplicate. The absence of error bars indicates very small standard deviations that could not be displayed.

Article Snippet: Macrophage-like U937 cells and human type I alveolar epithelial cells (WI-26 VA4; ATCC CCL-95.1) and axenic A. polyphaga were maintained and used for infections as described previously ( 26 , 27 ).

Techniques: Infection, Plasmid Preparation, Incubation