Journal: International Journal of Biological Sciences

Article Title: ARID1A deficiency activates OSM-STAT3 axis in endometrial cancer, creating vulnerability to JAK/STAT3 inhibition

doi: 10.7150/ijbs.129142

Figure Lengend Snippet: Identification of synthetic lethality of ARID1A and JAK/STAT3 in endometrial cancer through drug library screening. A. Schematic illustration of the synthetic lethality screenings with the Epigenetics Drug Library and the Kinase Inhibitor Library. B. Immunoblot analysis showing ARID1A knockout in A1 and B7 single clone. C. Drugs with selectivity index (SI)≥2 were selected as synthetic lethality candidates. SI=IC50 ARID1A+/+ /IC50 ARID1A-/- . D. Synthetic lethality in HEC1B-ARID1A -/- cell treated with stattic (D, F) and gandotinib (E, G) for 72h. The cell images were taken with IncuCyte ZOOM. Scale bar, 300 μm. H. Immunoblot analysis validated the ARID1A overexpression in HEC1B-ARID1A -/- cell. I. Overexpressed ARID1A rescued the synthetic lethality mediated by stattic. Data are mean±sd. * P <0.05, ** P <0.01. J-K. HEC1B ARID1A isogenic cell pair were transfected with STAT3-siRNA for 72 h. J. The quantitative analysis of synthetic lethality induced by STAT3-siRNA. Data are mean±sd. *** P <0.001. K. The growth curve of HEC1B ARID1A isogenic cell pair treated with STAT3-siRNA. **** P <0.0001. Two-way ANOVA test. L- N. IC50 test treated with stattic and gandotinib in endometrial cancer cell line. Data are mean±sd. * P <0.05.

Article Snippet: Epigenetics Drug Library and the Kinase Inhibitor Library screening were purchased from Selleck Chemicals.

Techniques: Drug discovery, Western Blot, Knock-Out, Over Expression, Transfection

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

Journal: iScience

Article Title: A dual SHOX2:GFP; MYH6:mCherry knockin hESC reporter line for derivation of human SAN-like cells

doi: 10.1016/j.isci.2022.104153

Figure Lengend Snippet: Generation of human SAN-like cells from hESCs (A) Schematic representation of the differentiation protocols 1–8. The protocol involves three stages (S1, S2, and S3) during the first 9 days of directed differentiation. (B–D) Quantitative PCR (B), live fluorescence images (C), and FACS plots (D) of the day 30 cells derived from the SHOX2:GFP;MYH6:mCherry H9 line using the corresponding differentiation protocols (n = at least 3 biological replicates for each condition). For quantitative PCR data, fold changes were normalized to protocol #1. p values calculated using ordinary one-way ANOVA with Fisher’s test (∗∗p < 0.01, ∗∗∗p < 0.001 ). Data are represented as mean ± SEM. The x axis in panel D is side scatter. (E) Quantitative PCR analysis for SHOX2 transcriptional expression of GFP + cells purified after sorting. n = 3 biological replicates. p value calculated by unpaired two-tailed Student’s t-test ∗∗∗p < 0.001. Data are represented as mean ± SEM, and normalized to GFP-negative population. (F) Schematic and results of the epigenetic library chemical screen. (G) Schematic of the final differentiation protocol. (H) Immunofluorescence image and FACS plot of day 30 cells derived using the protocol described in (G), Scale bar = 100 μm. AA: Activin A, B: BMP4, Ch: CHIR99021, Wi: Wnt inhibitor, RA: Retinoic Acid, Fi: FGF inhibitor, Cu: cucurbitacin, Tyr490: tyrphostin AG 490. See also Figures S1 and .

Article Snippet: Cayman Epigenetic Chemical library , Cayman , #11076.

Techniques: Real-time Polymerase Chain Reaction, Fluorescence, Derivative Assay, Expressing, Purification, Two Tailed Test, Immunofluorescence

Journal: iScience

Article Title: A dual SHOX2:GFP; MYH6:mCherry knockin hESC reporter line for derivation of human SAN-like cells

doi: 10.1016/j.isci.2022.104153

Figure Lengend Snippet:

Article Snippet: Cayman Epigenetic Chemical library , Cayman , #11076.

Techniques: Recombinant, Software

Journal: Frontiers in Pharmacology

Article Title: Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids

doi: 10.3389/fphar.2024.1335246

Figure Lengend Snippet: Schematic representation of the ADM screen. Seeded organoids originating from wildtype (WT) or p48 Cre/+ (Cre) mice were treated with the Cayman small-molecule epigenetic modulator library (ESL) at final concentration of 1 µM using an automated dispensing system and left to incubate for 72 h. Following incubation, organoids were stained using the calcein AM viability dye and imaged using a high throughput imaging system at ×20 magnification to obtain high resolution images for further image analysis.

Article Snippet: We screened the Cayman’s small-molecule epigenetic modulator library (ESL) which contains 144 compounds with the goal to identify important regulators of ADM with therapeutic potential.

Techniques: Concentration Assay, Incubation, Staining, High Throughput Screening Assay, Imaging

Journal: Frontiers in Pharmacology

Article Title: Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids

doi: 10.3389/fphar.2024.1335246

Figure Lengend Snippet: ADM inhibition from epigenetic compound library screen (compounds 1–68 grouped by target). The ADM inhibition assay screen was performed in duplicate using the Cayman ESL on wildtype mouse organoids. The mean percentage of viable ducts and acinar clusters (± SD) 72 h post treatment. BET = bromodomain and extraterminal domain inhibitors; DMT = DNA methyltransferase inhibitors; HDM = histone demethylase inhibitors; HMT = histone methyltransferase inhibitors; HAT = histone acetyltransferase inhibitors; HDAC = histone deacetylase inhibitors (NAD + dependent); HCl = hydrochloride; TFA salt = trifluoroacetic acid salt. p values were calculated using two-tailed Student’s t -test with unequal variances. Significance was accepted at p ≤ 0.05 only when averages of clusters are higher than the respective vehicle control. * p -value = 0.05–0.01; ** p -value = 0.01–0.001; *** p -value < 0.001. Red asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects but had overall less than 50% viability of total objects (false positive). Black asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects and more than 50% of total objects were viable by calcein AM staining (true positive). Single biological replicate of quadruplicate technical replicates, mean ± SD.

Article Snippet: We screened the Cayman’s small-molecule epigenetic modulator library (ESL) which contains 144 compounds with the goal to identify important regulators of ADM with therapeutic potential.

Techniques: Inhibition, Drug discovery, Histone Deacetylase Assay, Two Tailed Test, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids

doi: 10.3389/fphar.2024.1335246

Figure Lengend Snippet: ADM inhibition from epigenetic compound library screen (compounds 69–144 grouped by target). The ADM inhibition assay screen was performed in duplicate using the Cayman ESL on wildtype mouse organoids. The mean percentage of viable ducts and acinar clusters (± SD) 72 h post treatment. HDAC = histone deacetylase inhibitors (Zn 2+ dependent); PARP = poly-ADP ribose polymerase inhibitors; HCl = hydrochloride; TFA salt = trifluoroacetic acid salt. p values were calculated using two-tailed Student’s t-test with unequal variances. Significance was accepted at p ≤ 0.05 only when averages of clusters are higher than the respective vehicle control. * p -value = 0.05–0.01; ** p -value = 0.01–0.001; *** p -value < 0.001. Red asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects but had overall less than 50% viability of total objects (false positive). Black asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects and more than 50% of total objects were viable by calcein AM staining (true positive). Single biological replicate of quadruplicate technical replicates, mean ± SD.

Article Snippet: We screened the Cayman’s small-molecule epigenetic modulator library (ESL) which contains 144 compounds with the goal to identify important regulators of ADM with therapeutic potential.

Techniques: Inhibition, Drug discovery, Histone Deacetylase Assay, Two Tailed Test, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids

doi: 10.3389/fphar.2024.1335246

Figure Lengend Snippet: ADM reversal from epigenetic compound library screen (compounds 1–68 grouped by target). The ADM reversal assay screen was performed in duplicate using the Cayman ESL on Cre mouse organoids. The mean percentage of viable ducts and acinar clusters (± SD) 72 h post treatment. BET = bromodomain and extraterminal domain inhibitors; DMT = DNA methyltransferase inhibitors; HDM = histone demethylase inhibitors; HMT = histone methyltransferase inhibitors; HAT = histone acetyltransferase inhibitors; HDAC = histone deacetylase inhibitors (NAD + dependent); HCl = hydrochloride; TFA salt = trifluoroacetic acid salt. p values were calculated using two-tailed Student’s t-test with unequal variances. Significance was accepted at p ≤ 0.05 only when averages of clusters are higher than the respective vehicle control. * p -value = 0.05–0.01; ** p -value = 0.01–0.001; *** p -value < 0.001. Black asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects and more than 50% of total objects were viable by calcein AM staining (true positive). Single biological replicate of quadruplicate technical replicates, mean ± SD.

Article Snippet: We screened the Cayman’s small-molecule epigenetic modulator library (ESL) which contains 144 compounds with the goal to identify important regulators of ADM with therapeutic potential.

Techniques: Drug discovery, Histone Deacetylase Assay, Two Tailed Test, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids

doi: 10.3389/fphar.2024.1335246

Figure Lengend Snippet: ADM reversal from epigenetic compound library screen (compounds 69–144 grouped by target). The ADM reversal assay screen was performed in duplicate using the Cayman ESL on Cre mouse organoids. The mean percentage of viable ducts and acinar clusters (± SD) 72 h post treatment. HDAC = histone deacetylase inhibitors (Zn 2+ dependent); PARP = poly-ADP ribose polymerase inhibitors; HCl = hydrochloride; TFA salt = trifluoroacetic acid salt. p values were calculated using two-tailed Student’s t-test with unequal variances. Significance was accepted at p ≤ 0.05 only when averages of clusters are higher than the respective vehicle control. * p -value = 0.05–0.01; ** p -value = 0.01–0.001; *** p -value < 0.001. Red asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects but had overall less than 50% viability of total objects (false positive). Black asterisks denote compounds that showed significantly higher cluster/duct ratios of live objects and more than 50% of total objects were viable by calcein AM staining (true positive). Single biological replicate of quadruplicate technical replicates, mean ± SD.

Article Snippet: We screened the Cayman’s small-molecule epigenetic modulator library (ESL) which contains 144 compounds with the goal to identify important regulators of ADM with therapeutic potential.

Techniques: Drug discovery, Histone Deacetylase Assay, Two Tailed Test, Control, Staining