Journal: Frontiers in Cellular Neuroscience

Article Title: Adipose-derived stem cell-conditioned medium mitigates ischemia-induced neuronal injury via the JAK1/STAT3 signaling pathway

doi: 10.3389/fncel.2026.1744887

Figure Lengend Snippet: ADSC-CM promotes neurite outgrowth in OGD-injured neurons through the JAK1-STAT3 signaling pathway. (A) On day 5 of cultivation, neuronal neurites extended and formed an extensive neural network. Immunofluorescence staining showed that the neurons were positive for the neuronal-specific marker Tuj1. After OGD injury, neuronal neurites were damaged, partially interrupted, and disappeared. (B) Western blot bands of JAK1, pJAK1, STAT3, pSTAT3, and GAPDH for each group. (C) Comparison of the relative ratio of pJAK1 to JAK1 across experimental groups ( n = 4). (D) Comparison of the relative ratio of pSTAT3 to STAT3 across experimental groups ( n = 4). (E) Immunofluorescence images of neurons from each group, showing the effects of ADSC-CM and GLPG0634 on neurite outgrowth. Neurons were stained with the Tuj1 antibody (red) to label the neuronal cytoskeleton, and nuclei were counterstained with DAPI (blue). (F) Diagram illustrating how to measure the length of the longest neurite and the number of primary neurites in neurons. The green line shows the trajectory of the longest neurite, and white arrows point to primary neurites. (G,H) Comparison of the length of the longest neurite and the number of primary neurites in neurons across experimental groups ( n = 4). Data are expressed as means ± SEM. The difference between the groups was assessed using a one-way ANOVA followed by Bonferroni post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm (A) , 20 μm (E,F) .

Article Snippet: After overnight incubation with rabbit anti-Tuj1 antibody (1:200, BM3881, Boster, Wuhan, China) at 4 °C, cells were washed and then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, AS007, ABclonal) for 1 h at room temperature in the dark.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Comparison

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Chemical structure of standard Cbls with their corresponding β-axial groups shown. b , Co-crystal structure of the env8 -OHCbl complex (PDB 4FRG 19 ). Global RNA architecture (left) is displayed as cartoon, nucleotides critical for recognition of the variable β-axial group are shown in cyan (A68), green (A20), and yellow (G19), and OHCbl is represented by magenta van der Waals spheres. The ligand binding pocket (right) consists of a π-stacking network between nucleotides G19-A20 and A20-A68 that is unimpeded by the small (-OH) β-axial group. c , Simplified chemical structures of previously characterized 20 β-axial-modified Cbls 4 - 7 tested against env8 . d , Reduction-free synthetic route 25 , 26 to make Cbl derivatives from CNCbl using a variable alkyne. e , Overview of our expanded β-axial-modified Cbl library.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Ligand Binding Assay, Modification

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Binding curve of the fluorescence induction of CNCbl-5×PEG-ATTO590 binding to env8 . b , Representative displacement binding curve for MeCbl and Cbl 4 . Binding curves are shown as mean and s.e.m. from independent experiments ( n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 3). c , Log-transformed K D values for 1 - 44 shown as mean and s.d. from independent experiments ( n = 4 except for data from 1 , 2 , 4 - 7 , 10 , 14 , 19 , 41 , and 42 where n = 3 and data from 21 , 36 , and 37 where n = 5.). d , Locations of the training and test set from the Q 2 -focused modeling in two-dimensional chemical space constructed from PC1 and PC2 of the whole data set. e , Measured ln [K D ] values plotted with the value predicted by the Q 2 -focused model. f , Overview of our lead compound screen from a 513-compound alkyne library using our binding-based models. g , Simplified chemical structures of representative derivatives that our binding-based models predict to be tight ( 45 - 48 ), moderate ( 49 and 50 ), and weak ( 51 - 53 ) binders. h , Measured ln [K D ] values plotted with the value predicted by our binding-based models. The experimental data are represented as mean and s.d. from independent experiments ( n = 3 except for data from 45 , 48 , and 53 where n = 4) and the predicted data are shown as the mean and s.d. from independent predictions ( n = 3) from our three models (baseline, R 2 -focused, and Q 2 -focused).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: RNA Binding Assay, Binding Assay, Fluorescence, Transformation Assay, Construct

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Box-and-whisker plot of the OD 600 -normalized relative fluorescence units (RFU) of E. coli cells with and without reporter plasmid ( env8 -GFPuv) and ligand (MeCbl and Cbl 4 ) added. Data are shown from independent experiments ( n = 4). b , Fold repression (defined as the ratio of “(−)-Cbl RFU” and “(+)-Cbl RFU” values) 33 for our env8 reporter system in the presence of Cbl 1 - 44 shown as mean and s.d. from biological replicates ( n = 4 except for data from 6 where n = 3). c , Plot comparing the log-transformed fold repression and K D data. d , Locations of the training and test set from the Q 2 -focused modeling in two-dimensional chemical space constructed from PC1 and PC2 of the whole data set. e , Measured fold repression values plotted with the value predicted by the Q 2 -focused model. f , Overview of our lead compound screen from a 513-compound alkyne library using our function-based models. g , Simplified chemical structures of representative derivatives that our function-based models predict to be strong ( 54 - 57 ), moderate ( 58 and 59 ), and weak ( 60 - 62 ) repressors. h , Measured fold repression values plotted with the value predicted by our function-based models. The experimental data are represented as mean and s.d. from biological replicates ( n = 4 except for data from 58 , 59 , 61 , and 62 where n = 3) and the predicted data are shown as the mean and s.d. from independent predictions ( n = 3) from our three models (i.e., baseline, R 2 -focused, and Q 2 -focused).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Activity Assay, Whisker Assay, Fluorescence, Plasmid Preparation, Transformation Assay, Construct

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Representative MD starting states of the env8 -CNCbl and env8 -Cbl 4 complexes from one (of three) independent 1 μs trajectory. The A20(C6)-G19(C6) and A20(C6)-A68(N7) distances are shown as double arrows. In all structural representations, the binding pocket nucleotides G19 (yellow), A20 (green), and A68 (cyan) are numbered in reference to full-length env8 and colored, the β-axial group is shown in magenta, and dashed lines represent proposed hydrogen bonds. b , The average A20(C6)-G19(C6) (left) and A20(C6)-A68(N7) (right) distances measured over the course of three independent MD trajectories for both env8 -CNCbl and env8 -Cbl 4 complexes. c , Co-crystal structure of env2 -CNCbl showing only the binding pocket nucleotides and the β-axial group. All mesh representations correspond to a simulated annealing 2F o -F c map where A20 and the ligand were omitted from the model and are shown at 1 σ contour. d , Same as in c but for env2 -Cbl 4 . e , Same as in c but for env2 -Cbl 32 (left) and showing a van der Waal sphere representation of the binding pocket (right). f , Same as in e but for env2 -Cbl 29 . g , Same as in c but for env2 -Cbl 42 . h , Schematic representation of design hypotheses that emerge from our 11 RNA-ligand co-crystal structures.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Binding Assay

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Modified click reaction 25 to make Cbl derivatives from 58 using a variable azide. b , Simplified chemical structures of Cbls 63 - 65 that were made to systematically test our design hypotheses. c , Fluorescence displacement binding curve for CNCbl and 63 - 65 shown as mean and s.e.m. from independent experiments ( n = 4 except for data from CNCbl where n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 4 except for data from CNCbl where n = 3). d , To verify the near-picomolar binding of 63 and 64 , we used ITC with the env8 aptamer domain. Given that this RNA shows weaker ligand binding than the full-length env8 , 19 it is in the perfect regime for ITC. Representative ITC binding isotherm for the env8 aptamer domain with CNCbl, 63 , and 64 . e , Co-crystal structure of env2 -Cbl 63 showing only the binding pocket nucleotides and the β-axial group. Proposed hydrogen bonds, which are weakly supported by the electron density, are shown as double arrows. All mesh representations correspond to a simulated annealing 2F o -F c map where A20 and the ligand were omitted from the model and are shown at 1 σ contour. f , Same as in e but for env2 -Cbl 64 . g , Fold repression for our reporter system in the presence of CNCbl and 63 - 65 and for cells with CNCbl and excess amounts of 63 - 65 added. Mean and s.d. from biological replicates ( n = 4) are shown. For comparison, the mean fold repression of 10 nM CNCbl is shown as a dashed line its s.d. is represented as a shaded box. h , Simplified chemical structure of the titratable pyridine Cbl 66 . i , Representative ITC binding isotherms for env8 aptamer domain with CNCbl, 29 , and 66 at pH 8 and pH 5. For all ITC data, K D values are reported as mean and s.d. from independent experiments ( n = 3).

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Modification, Fluorescence, Binding Assay, Ligand Binding Assay, Comparison

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Chemical structure of the eight hits that emerged from our structure-informed docking, with their biphenyl-like scaffold highlighted in gray. b , TO-based displacement assay to identify env8 binding compounds that induce fluorescence attenuation. This was used as an orthogonal binding experiment because we observed ligand-induced fluorescence induction of CNCbl-5×PEG-ATTO590. c , MST trace of fluorescently labeled env8 alone and in the presence of CNCbl, 68 , and 71 . All ligands induced an observable change to the MST trace of env8 suggestive of binding. d , TO-displacement binding curve for CNCbl, 68 , and 71 shown as mean and s.d. from independent experiments ( n = 3). K D values are reported as mean and s.d. from independent experiments ( n = 3). e , Native polyacrylamide gel showing a gradual shift from a slower migrating, extended conformation to a faster migrating, kissing-loop conformation of env8 upon titration of CNCbl (0.01–10 μM). Data are shown from an individual experiment. f , Same as in e but after addition of CNCbl, 68 , and/or 71 to demonstrate competitive binding of 68 and 71 . Similar results were obtained from repeated experiments ( n = 8 for CNCbl, n = 3 for 68, and n = 2 for 71). g , MST trace from a competition experiment where env8 -CNCbl was monitored with and without the addition of excess amounts of 68 or 71 . Both ligands induced an observable change to the MST trace of env8 -CNCbl suggestive of competitive binding. All MST data are shown as representative traces from multiple independent experiments ( n = 4). h , Top-ranked docking poses of 68 and 71 to env2 in the A20-out and A20-in state.

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Binding Assay, Fluorescence, Labeling, Titration

Journal: Nature chemical biology

Article Title: Designing small molecules targeting a cryptic RNA binding site via base displacement

doi: 10.1038/s41589-025-02018-8

Figure Lengend Snippet: a , Schematic representation of the secondary structures of full-length env8 , env8 aptamer domain, and the env8 and env2 RNA crystallography constructs. The binding pocket nucleotides G19 (yellow), A20 (green), and A68 (cyan) are colored and the sites of variation between the env8 and env2 RNA sequences are indicated in magenta. Other changes to the env8 wild-type RNA sequence are the insertion of a gGAAAc tetraloop at L5, the variable lengths of helices P1 and P5 (either four or six base pairs, bp), and a 3′-end adenosine, which were all incorporated to aid crystallization. b , Binding curve of the fluorescence induction of CNCbl-5×PEG-ATTO590 binding to env8 aptamer domain and env2 crystal construct shown as mean and s.e.m. from independent experiments ( n = 3). The K D values are reported as mean and s.d. from independent experiments ( n = 3). c , Fold repression values for our env8 and env2 reporter systems in the presence of 10 μM CNCbl, reported as mean and s.d. from biological replicates ( n = 4). d , Co-crystal structure of env2 -CNCbl aligned to the env8 -OHCbl complex (PDB 4FRG 19 ). Global RNA architecture (left) is displayed as cartoon, with sites of variation between the env8 and env2 shown in magenta. The ligand binding pocket (right) is shown in sticks and shows very strong agreement between the two RNA-ligand complexes (RMSD = 0.45 Å). Given that env2 shows near-identical ligand binding, regulatory activity, and local and global RNA architecture, all structural information from env2 can be translated to env8 .

Article Snippet: Plasmids harboring env8 -GFPuv (Addgene #99831) 33 or pBR322 empty vector control were transformed into E. coli cells (Keio collection, JW3805).

Techniques: Biomarker Discovery, Construct, Binding Assay, Sequencing, Crystallization Assay, Fluorescence, Ligand Binding Assay, Activity Assay