Journal: eLife

Article Title: Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes

doi: 10.7554/eLife.47970

Figure Lengend Snippet: ( A ) Bar graph shows expression profile of MYH isoforms in hiPSC-1-derived myotubes. Data are shown as mean ± S.E.M.; n = 3, ***p<0.001. ( B ) Bar graph shows the ratio of % MyHC-stained area to % DAPI area in myotubes resulting from treatment with five candidates identified by the small molecule screening. Data show significant increase (***p<0.001) compared to DMSO in all three PS cell lines analyzed (hESC-1, hiPSC-1 and hiPSC-2). Data from three independent replicates are shown, normalized to DMSO, as mean ± S.E.M. ( C ) Bar graph shows the ratio of % MyHC-stained area to % DAPI area in iPS cell-derived myotubes that had been differentiated in the presence of all candidates combined, or with individual candidates excluded from the overall combination. Data from three independent replicates are shown normalized to DMSO. Values are shown as mean ± S.E.M. ***p<0.001. ( D ) Representative images show immunostaining for MyHC (in red) in hiPSC-1 myotubes differentiated with combinatory treatments of small molecules or DMSO. DAPI stains nuclei (in blue). Scale bar is 100 μm. ( E ) Bar graph shows fusion index analysis of myotubes that were differentiated with small molecule combinations or DMSO. Data are shown as mean of three independent replicates ± S.E.M. ***p<0.001. ( F ) Stacked bar graph shows the frequency of number of nuclei per myotube upon differentiation with combinatory treatments or DMSO. Data are shown as mean of three independent replicates ± S.E.M. Statistical analysis compares each combination to DMSO. *p<0.05 **p<0.01 ***p<0.001. 10.7554/eLife.47970.006 Figure 1—source data 1. Tocriscreen Stem Cell Toolbox compounds tested during myogenic terminal differentiation of PS cell lines.

Article Snippet: To determine whether small molecule compounds may enhance the maturation of PS cell-derived myotubes, we performed a small molecule library screening using the Tocriscreen stem cell toolbox kit (Tocris).

Techniques: Expressing, Derivative Assay, Staining, Immunostaining

Journal: eLife

Article Title: Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes

doi: 10.7554/eLife.47970

Figure Lengend Snippet: ( A ) Schematic representation of the small molecule screening procedure. Each well from a 96-well plate contained an individual compound from the Tocriscreen Stem cell toolbox added in the differentiation medium. ( B ) Representative images of MyHC (red) immunostaining in myotubes differentiated with selected candidates upon small molecule screening ( A ). DAPI stains blue. Scale bar is 100 μm. ( C ) Bar graph shows ratio of % MyHC-stained area to % DAPI area from hiPSC-1 myotubes differentiated with compounds candidates at 5, 10 or 20 μm relative to DMSO. Data are shown as mean of three independent replicates ± S.E.M. Statistical analyses showed no significant differences among concentrations for each compound. ( D ) Bar graphs show gene expression analysis of MYOG and MYH isoforms relative to GAPDH of hiPSC-1 myotubes differentiated with compound candidates. Data are shown as mean of three independent replicates ± S.E.M. *p<0.05 **p<0.01 ***p<0.001.

Article Snippet: To determine whether small molecule compounds may enhance the maturation of PS cell-derived myotubes, we performed a small molecule library screening using the Tocriscreen stem cell toolbox kit (Tocris).

Techniques: Immunostaining, Staining, Expressing

Journal: eLife

Article Title: Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes

doi: 10.7554/eLife.47970

Figure Lengend Snippet:

Article Snippet: To determine whether small molecule compounds may enhance the maturation of PS cell-derived myotubes, we performed a small molecule library screening using the Tocriscreen stem cell toolbox kit (Tocris).

Techniques: Control, Recombinant, Imaging, Proliferation Assay, Sequencing

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A MST traces of the DNMT2 enzyme reaction (250 nM DNMT2, 5 µM tRNA, 0.9 µM SAM) incubated for variable durations (0–120 min) and yielded significant thermophoresis shifts. B Substrate conversion plots (F norm vs incubation time) reveal steady-state DNMT2 kinetics. C Dose-response curves of aptaSAH1&2 (both 50 nM) in the presence of SHO108/SH112 inhibitors (0–100 µM) indicated these SAH-analogs are not binding to the aptamer at relevant concentrations. D – F Inhibitor characterization of SHO108 from left to right: MST traces at variable inhibitor concentrations; MST-derived dose-response curves; orthogonal 3 H-incorporation assay-derived dose-response curves from detected counts per minute (CPMA). G – I Inhibitor characterization of SH112 analogous to subplot ( D – F ). All inhibitor characterizations were performed in triplicates (mean ± SD, n = 3).

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Incubation, Binding Assay, Derivative Assay

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A Screening a library including 160 drug-like compounds resulted in the identification of ten potential hit compounds (green) with an F norm > 1000‰ (equals >80% inhibition). Initial hits were confirmed by triplicate validation (mean ± SD, n = 3). Positive reaction control: aptamer spiked with 1 µM SAH; negative control: aptamer mock-treated with DMSO. B Exemplary MST traces of the library’s first 80 compounds. C Orthogonal screening of the library by FP displacement experiments using FTAD as the fluorescent tracer. FP experiments reveal that only alexidine ( Cpd 3 , green) is binding to the SAH-binding site. D An inhibition selectivity panel was established by MST aptamer assays using the MTase assays described in this study and 500 µM of the respective hit compound. Only DNMT2 was inhibited significantly. E Alexidine’s ( Cpd 3 ) dose-response curves and K D determination by DNMT2 FP assays (mean ± SD, n = 3). F FP assays to confirm the reversibility of alexidine binding to DNMT2. The polarization of the DNMT2-FTAD complex (96 mP) is displaced in the presence of alexidine (5 mP) and can be effectively restored by analytical size-exclusion chromatography (92 mP). A second treatment with alexidine leads to a repeated FP displacement (4 mP). G Chemical structures of the identified DNMT2 hits.

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Inhibition, Biomarker Discovery, Control, Negative Control, Binding Assay, Size-exclusion Chromatography

Journal: Communications Chemistry

Article Title: A microscale thermophoresis-based enzymatic RNA methyltransferase assay enables the discovery of DNMT2 inhibitors

doi: 10.1038/s42004-025-01439-9

Figure Lengend Snippet: A Screening a library including 80 cysteine-focused covalent compounds resulted in the identification of one single hit compound (green) with an F norm > 1000‰ (equals >90% inhibition). Initial hits were confirmed by triplicate validation (mean ± SD, n = 3). Positive reaction control: aptamer spiked with 1 µM SAH; negative control: aptamer mock treated with DMSO. B Orthogonal screening of the library by fluorescence polarization displacement experiments using FTAD as the fluorescent tracer. FP experiments reveal that only adamantanyl-acryloylurea ( Cpd 11 , green) is binding to the SAH-binding site. C MST-derived dose-response curve for adamantanyl-acryloylurea ( Cpd 11 ) including the chemical structure. All inhibitor characterizations were performed in triplicates (mean ± SD, n = 3). D FP assay with Cpd 11 showing time-dependent DNMT2 inhibition with hyperbolic FP displacement plots. E Covalent dose-response analysis of subfigure D: k obs vs. [I] for the determination of covalent inhibition constants ( K I , k inact ) , .

Article Snippet: A compound library for the DNMT2 pilot screening (including Cpd 1–10 ) was commercially obtained (Tocriscreen Epigenetics 3.0, Cat. No. 7578).

Techniques: Inhibition, Biomarker Discovery, Control, Negative Control, Fluorescence, Binding Assay, Derivative Assay, FP Assay