Journal: PLoS ONE

Article Title: Ectopic Otoconin 90 expression in triple negative breast cancer cell lines is associated with metastasis functions

doi: 10.1371/journal.pone.0211737

Figure Lengend Snippet: A) Kaplan Meier estimation of overall survival probability with OC90 co-expressed genes, HMGA2 (left panel), POLE2 (middle panel), TRIB3 (right panel). B) qPCR demonstrating co-expression with OC90 in TNBC. Linear regression of BT20, BT549 and HCC38 under basal, knockdown and overexpression conditions (left panel OC90 vs. HMGA2, middle panel OC90 vs. POLE2, right panel OC90 vs. TRIB3).

Article Snippet: TaqMan Gene Expression Assay targets were predesigned; Hs00903174_m1 (OC90), Hs00971724_m1 (HMGA2), Assay Id Hs01123904_m1 (POLE2), Assay Id Hs00221754_m1 (TRIB3).

Techniques: Expressing, Knockdown, Over Expression

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

doi: 10.1093/toxsci/kfac131

Figure Lengend Snippet: Total cellular RNAs were extracted from human Sertoli cells after a 24 h treatment with DMSO (0 μM) or 7–10 μM CBD. Gene expression profiles (n=4/group) were analyzed by mRNA-seq. (A) A heatmap shows concentration-dependent changes of representative DNA replication-related genes. Relative expression of E2F1 (B), E2F7 (C), MCM3 (D), PRIM1 (E), POLE2 (F), and POLD1 (G) was determined using real-time PCR. The bar graphs represent means ± standard deviation (SD) (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

Article Snippet: To measure gene expression levels, real-time PCR assays were performed using FastStart Universal Probe Master (Rox, MilliporeSigma) and the following probes (Thermo Fisher Scientific): E2F1 (Hs00153451_m1), E2F7 (Hs00987777_m1), MCM3 (Hs00172459_m1), PRIM1 (Hs00265388_m1), POLE2 (Hs00160277_m1), POLD1 (Hs01100821_m1), CDC25A (Hs00947994_m1), CDKN1A (Hs00355782_m1), EXO1 (Hs01116190_m1), MRE11 (Hs00967437_m1), NEIL3 (Hs00217387_m1), FANCB (Hs00537483_m1), CDKN2A (Hs00923894_m1), IL-1A (Hs00174092_m1), IL-6 (Hs00174131_m1), and GAPDH (Hs02758991_g1).

Techniques: Gene Expression, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Control

Journal: Nature Communications

Article Title: The non-catalytic DNA polymerase ε subunit is an NPF motif recognition protein

doi: 10.1038/s41467-025-67284-5

Figure Lengend Snippet: A NPF tripeptides were described to interact with multiple types of NPF motif receptors, making prediction of partners of putative motifs highly ambiguous. B The native holdup (nHU) affinity interactomic approach is ideal to capture motif-mediated interactions because of high ligand concentration allowing abundant complex formation of weak interactions and no washing procedures that would eliminate transient interactions. C – H Six different NPF motif peptides were assayed with nHU coupled with mass spectrometry. Interaction partners belonging to known or suspected NPF motif receptors are highlighted. Experiments were carried out with 2 biological and 3 technical replicates (total N = 6). Two-tailed unpaired T-test, assuming equal variance was used during analysis. POLE/POLE2 (in red) and detected EH proteins (in violet) are shown on the volcano plots even if they were not identified as partners to show specificity of NPF motifs. I Fluorescence polarization was measured by titrating recombinant avi-His 6 -MBP-POLE2 to a fluorescent NPF motif peptide, confirming binary complex formation. J Competitive titration experiments show that the non-fluorescent NPF motif peptide can compete with the binding of the labeled one with similar affinity. In vitro binding experiments were carried out in triplicates. K Ex vivo measured affinity of HA-POLE2, measured by nHU in cell extract, shows higher affinity than the affinity measured in vitro with purified recombinant protein. L The affinity of HA-MBP-POLE2 was found to be weaker than of HA-POLE2 with a clear partial binding activity. Titration nHU experiments were carried out with at least two technical replicates and the obtained standard deviations are displayed for each measurement points to indicate range of measurements. Source data can be found in Supplementary Data , , Supplementary Figs. , , , , and in the Source Data File.

Article Snippet: In direct FP measurements, a dilution series of MBP-POLE2 was prepared in 96 well plates (96 well skirted PCR plate, 4ti-0740, 4titude, Wotton, UK) in a 20 mM Hepes pH 7.5 buffer that contained 150 mM NaCl, 0.5 mM TCEP, 0.01% Tween 20 and 50 nM fluoDONSON peptide.

Techniques: Concentration Assay, Mass Spectrometry, Two Tailed Test, Fluorescence, Recombinant, Titration, Binding Assay, Labeling, In Vitro, Ex Vivo, Purification, Activity Assay

Journal: Nature Communications

Article Title: The non-catalytic DNA polymerase ε subunit is an NPF motif recognition protein

doi: 10.1038/s41467-025-67284-5

Figure Lengend Snippet: A Recombinant avi-His 6 -MBP-POLE2 was used as a bait in a nHU experiment coupled with mass spectrometry revealing numerous binding partners. Experiments were carried out with 2 biological and 3 technical replicates (total N = 6). Two-tailed unpaired T-test, assuming equal variance was used during analysis. Those significantly depleted partners that contain putative NPF motifs, as well as PYGO2 that is not significant but the known partner of the WNT enhanceosome, are highlighted with green or blue circles. B List of partners with putative NPF motifs. C Schematic pipeline of our de novo motif discovery approach. Based on the sequences of the identified partners, NPF motifs are the most evolutionary conserved ones among all enriched motifs. D Superposition of all 7 identified POLE2-bound motifs in AF3 predictions reveal that although most partners bind via a common NPF motifs with a close to identical core motif conformation, the same binding interface is also compatible with a putative NPY motif of SEC23IP. Source data can be found in Supplementary Data , Supplementary Figs. , , , , and in the Source Data File.

Article Snippet: In direct FP measurements, a dilution series of MBP-POLE2 was prepared in 96 well plates (96 well skirted PCR plate, 4ti-0740, 4titude, Wotton, UK) in a 20 mM Hepes pH 7.5 buffer that contained 150 mM NaCl, 0.5 mM TCEP, 0.01% Tween 20 and 50 nM fluoDONSON peptide.

Techniques: Recombinant, Mass Spectrometry, Binding Assay, Two Tailed Test