Journal: Current Research in Structural Biology

Article Title: Screening for novel chemical scaffolds targeting PCNA identifies the Hsp90alpha inhibitor SNX-2112

doi: 10.1016/j.crstbi.2026.100183

Figure Lengend Snippet: PCNA AI-CADD molecule thermal shift assay screening hits . AI-CADD screening: Left , plot of normalized melt curves of compounds + PCNA melt. Center , normalized derivative RFU of melt curve data. The DMSO control is plotted in black dashed lines. Right , . Calculated Δ Tm values of 19 AI-CADD hits. Thermal shift assays were performed using a Bio-Rad CFX Connect Real-Time PCR Detection System. and the final concentration of recombinant purified PCNA was 9 μM, and the final concentration of each compound was 1 mM. The reaction plates were heated from 25 °C to 95 °C with heating increments of 0.5 °C/min. Fluorescence intensity was measured within the excitation/emission ranges 470–505/540–700 nm. A negative control of 100% DMSO was included in each assay to obtain an apo- PCNA melting temperature (Tm apo ). The melting temperature of each reaction well was extrapolated from the midpoint of excitation of the produced melt curves by the CFX Maestro Software (Bio-Rad). The thermal shift values were calculated using the following equation: ΔTm = Tm + compound – Tm apo .

Article Snippet: Thermal shift assays were performed using a Bio-Rad CFX Connect Real-Time PCR Detection System. and the final concentration of recombinant purified PCNA was 9 μM, and the final concentration of each compound was 1 mM.

Techniques: Thermal Shift Assay, Control, Real-time Polymerase Chain Reaction, Concentration Assay, Recombinant, Purification, Fluorescence, Negative Control, Produced, Maestro Software

Journal: Current Research in Structural Biology

Article Title: Screening for novel chemical scaffolds targeting PCNA identifies the Hsp90alpha inhibitor SNX-2112

doi: 10.1016/j.crstbi.2026.100183

Figure Lengend Snippet: PCNA AI-CADD molecule thermal shift assay screening hits . AI-CADD screening: Left , plot of normalized melt curves of compounds + PCNA melt. Center , normalized derivative RFU of melt curve data. The DMSO control is plotted in black dashed lines. Right , . Calculated Δ Tm values of 19 AI-CADD hits. Thermal shift assays were performed using a Bio-Rad CFX Connect Real-Time PCR Detection System. and the final concentration of recombinant purified PCNA was 9 μM, and the final concentration of each compound was 1 mM. The reaction plates were heated from 25 °C to 95 °C with heating increments of 0.5 °C/min. Fluorescence intensity was measured within the excitation/emission ranges 470–505/540–700 nm. A negative control of 100% DMSO was included in each assay to obtain an apo- PCNA melting temperature (Tm apo ). The melting temperature of each reaction well was extrapolated from the midpoint of excitation of the produced melt curves by the CFX Maestro Software (Bio-Rad). The thermal shift values were calculated using the following equation: ΔTm = Tm + compound – Tm apo .

Article Snippet: Thermal shift assays were performed using a BioRad CFX Connect Real-Time PCR Detection System.

Techniques: Thermal Shift Assay, Control, Real-time Polymerase Chain Reaction, Concentration Assay, Recombinant, Purification, Fluorescence, Negative Control, Produced, Maestro Software

Journal: PLOS Genetics

Article Title: Genetic and functional characterization of AMH Signaling in Zebrafish - Evidence for Roles of Amh-Bmpr2a-Bmpr1bb Pathway in Controlling Gonadal Homeostasis

doi: 10.1371/journal.pgen.1011958

Figure Lengend Snippet: (A) Intrafollicular distribution of amh and BMP receptors ( bmpr2a, bmpr2b, bmpr1ba, bmpr1bb ) in the FG follicle. The housekeeping gene ef1a serves as a reference, while lhcgr and gdf9 are the marker genes expressed in the follicle layer and oocyte respectively. (B-C) Temporal expression profiles of amh and BMP receptors ( bmpr2a, bmpr2b, bmpr1ba, bmpr1bb ) during folliculogenesis. Quantitative assessment of mRNA levels was conducted using real-time qPCR, normalized to the expression of the housekeeping gene ef1a . The data are expressed as a fold change relative to the PG stage. Follicle staging was confirmed by examining the expression patterns of lhcgr and fshr as references. The values are mean ± SEM (n ≥ 3) from a representative experiment. Different letters indicate statistical significance (P < 0.05). PG, primary growth; PV, previtellogenic stage; EV, early vitellogenic stage; MV, mid-vitellogenic stage; LV, late vitellogenic stage; FG, full-grown. (D) Gene expression UMAP plots of amh, bmpr2a, bmpr2b, bmpr1ba, and bmpr1bb . Cells exhibiting expression of the specified gene are represented in orange, with varying shades indicating the levels of expression. The gradient of orange correlates with expression intensity, as detailed by the intensity scale on the right of each plot. The plots were all downloaded from the open-access Single Cell Portal ( https://singlecell.broadinstitute.org/single_cell ). (E) The colocalization of amh with BMP receptors ( bmpr2a, bmpr2b, bmpr1ba, bmpr1bb ) is observed in zebrafish granulosa cells, represented by histogram. The scRNA-seq raw data utilized for this analysis were sourced from the Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ), under accession number GSE191137 . The data was visualized using a custom R script, which was developed internally and incorporated Perl scripting.

Article Snippet: The HRMA was performed on a CFX Real-Time PCR System (Bio-Rad, Hercules, CA), using the following protocol: an initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec, a pre-determined optimal annealing temperature of 60°C for 15 sec, and 72°C for 20 sec. A final melting curve analysis, ranging from 70°C to 95°C with increments of 0.2°C at each step, was then performed to distinguish wild-type and mutant alleles based on their distinct melting profiles.

Techniques: Marker, Expressing, Gene Expression, Single Cell