Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blot analysis: 53BP1 (Bethyl Laboratories, A300-272A, 1:3000), LIN37 (Santa Cruz Biotechnology, sc-515686, 1:200), BLM (Bethyl Laboratories, A300-572A, 1:2000), BRCA1 for mouse (R and D Systems, gift from Dr. Andre Nussenzweig, NCI, 1:1000) , BRCA1 for human (Millipore Sigma, 07-434, 1:1000), RAD51 (Millipore Sigma, ABE257, 1:2000), BARD1 (Thermo Fisher Scientific, PA5-85707, 1:1000), CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), RIF1 (Abcam, ab13422, 1:500), SHLD1/C20orf196 (Thermo Fisher Scientific, PA5-559280, 1:200), GAPDH (Sigma, G8795, 1:10,000), KAP1 (Genetex, GTX102226, 1:2000), FANCD2 (R and D Systems, MAB93691, 1:1000), BRCA2 for human (Proteintech, 19791-1-AP, 1:500), Rb1 (Thermo Fisher Scientific, LF-MA0173, 1:1000), Phospho-Rb (Ser780) (Cell Signaling Technology, 8180T, 1:1000), Phospho-Rb (Ser807/811) (Cell Signaling Technology, 8516T, 1:1000), PCNA (Bethyl Laboratories, A300-276A, 1:3000), CDK4: (Novus Biologicals, NBP1-31308, 1:1000), CDK4 (phosphor Thr 172) (GeneTex, GTX00778, 1:1000), and RPA (Cell Signaling Technology, 2208S, 1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy

Journal: medRxiv

Article Title: BLM overexpression as a predictive biomarker for CHK1 inhibitor response in PARP inhibitor–resistant BRCA-mutant ovarian cancer

doi: 10.1101/2022.12.02.22283037

Figure Lengend Snippet: ( A ) Basal levels of BLM in parental ( BRCA -mutant PEO1 and UWB), acquired PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and UWB/OlaR), and de novo PARPi-resistant (PEO4) cell lines were analyzed by immunoblotting. Densitometric values of BLM relative to GAPDH (top) and representative colony formation images of cells with or without prexasertib treatment are shown (bottom). ( B-C ) Cells transfected with BLM overexpression plasmids for 48 hours were treated with or without prexasertib for another 48 hours. Cell viability was measured by XTT assay (B). IC50 values (C) from (B) were calculated using GraphPad Prism v7.1. Experiments were repeated at least in triplicate and data are shown as mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Abbreviations: CHK1i, CHK1 inhibitor; PARPi, PARP inhibitor; UWB, UWB1.289.

Article Snippet: BLM (#2742), CHK1 (#2360), pCHK1-S296 (#2349), ECL goat anti-mouse IgG HRP (#7076), and ECL goat anti-rabbit IgG HRP (#7074) antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

Techniques: Mutagenesis, Western Blot, Transfection, Over Expression, XTT Assay

Journal: Cell Death & Disease

Article Title: Cellular adaptations impact the biological activity of naphthalene diimide G-quadruplex ligands in ALT-positive osteosarcoma cells

doi: 10.1038/s41419-025-07908-2

Figure Lengend Snippet: A Representative western immunoblotting showing the amount of the indicated proteins in U-2 OS ( top panel ) and Saos-2 ( bottom panel ) cells either untreated (UNT) and after a 2-day exposure to an equitoxic amount (IC 50 ) of NMe2 or QN-302. Vinculin or β-tubulin were used to ensure equal protein loading. Cropped images of selected proteins are shown. The graphs on the side of each panel report the quantification of the protein amounts in treated vs . untreated cells following normalization with respect to vinculin or β-tubulin and represents mean values ± s.d. (N = 3); * p < 0.05; ** p < 0.01; **** p < 0.0001 (two-tailed unpaired t -test); B Representative Western immunoblotting showing BLM protein amounts in the cytoplasmic (c) and nuclear (n) fractions of untreated U-2 OS cells and after a 2-day exposure to NMe2 (IC 50 ). Lamin B1 and vinculin were used to ensure equal protein loading and proper nucleus/cytoplasm fractionation. Cropped images of selected proteins are shown. The graph on the bottom reports the quantification of the protein amounts. Data have been reported as relative protein amounts with respect to vinculin (cytoplasmic fraction) or lamin B1 (nuclear fraction) and represents mean values ± s.d. ( N = 3); ** p < 0.01 (two tailed unpaired t -test); C Representative photomicrographs showing the co-localization between BLM (green) and TRF1 (red) assessed by immunofluorescence in untreated U-2 OS cells and after a 2-day exposure to NMe2 (IC 50 ). Nuclei were counterstained with DAPI. Arrows indicate the fluorescence foci (yellow) arising from the co-localization between the two proteins. Merged images are shown; scale bars: 10 μm; magnification: × 60; D Representative western immunoblotting showing the amount of BLM protein in non-transfected (UNT), siCTR- and siBLM-transfected as well as DMSO-exposed and ML216-treated U-2 OS cells. β-actin was used to ensure equal protein loading. Cropped images of selected proteins are shown. The graphs on the bottom reports the quantification of the protein amounts. Data have been reported as relative protein amounts with respect to β-actin and represents mean values ± s.d. ( N = 3); **** p < 0.0001 (two tailed unpaired t -test); n.s.: not statistically significant. E Quantification of telomeric C-circle DNA levels in untreated (UNT), siCTR- and siBLM-transfected U-2 OS cells and after a 2-day exposure to 30 μM ML216. Data have been reported as relative CCA score in the indicated samples with respect to UNT cells and represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann–Whitney test); F Assessment of cell growth kinetics in siCTR (•)- and siBLM (▲)-transfected U-2 OS cells either untreated (blue) or after a 2-h exposure (pulse) to subtoxic amounts of NMe2 (green) or QN-302 (red). Data have been reported as the percentage of phase image confluency (determined by Incucyte® SX5 Live-Cell Imaging and Analysis System) normalized to the first time point (T 0 ) using the normalization function in GraphPad. Data represent mean values ± s.d. ( N = 4); * p < 0.05; ** p < 0.01; *** p < 0.001 (2-way ANOVA). The panel on the right reports a representative western immunoblotting showing BLM protein amounts in siCTR- and siBLM-transfected U-2- OS cells at the indicated time points. The graphs report the quantification of BLM protein amounts. Data have been reported as relative protein amounts with respect to β-actin and represents mean values ± s.d. ( N = 3). G Quantification of cell growth inhibition in U-2 OS cells exposed for 2 days to NMe2 (0.25 μM) and ML216 (30 μM) alone or in combination. Data have been reported as the extent of cell growth inhibition (fraction affected) and represent mean values ± s.d. ( N = 4); * p < 0.05 (two-tailed Mann–Whitney test); H Synergistic pharmacological interaction observed in U-2 OS cells after a 2-day exposure to the combination NMe2:ML216 (1:120) as indicated by the values of the combination index (C.I.) as a function of the fraction affected (Fa) calculated as previously reported .

Article Snippet: Target-specific siRNAs designed to silence the expression of TP53 (sip53; sc-29435); CDKN1A (sip21; sc-44214), BLM (siBLM; sc-29808) and NFE2L2 (siNrf2; sc-44332) as well as a control siRNA (siCTR; sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX). siRNAs were dissolved in RNase-free water to make a 10 μM stock solution, stored at −20 °C and diluted to obtain a final concentration of 50 nM immediately before use.

Techniques: Western Blot, Two Tailed Test, Fractionation, Immunofluorescence, Fluorescence, Transfection, MANN-WHITNEY, Live Cell Imaging, Inhibition

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Action of MMAF and cetuximab on the proliferation of ovarian cancer cell lines. A. Comparative dose-response studies in five ovarian cancer cell lines (A2780, OVCAR3, IGROV1, SKOV3, OVCAR8) treated with the free payload (MMAF) and the corresponding ADCs. Cells were incubated for 5 days with the indicated doses. Results, relativized to the untreated controls, are represented as the mean ± SD of triplicates of a representative experiment that was repeated at least twice. B. Action of naked cetuximab on the different ovarian cancer cell lines. Cells were treated with 1 or 10 nM of cetuximab for 5 days, counted, and data, relativized to untreated controls, represented as the mean ± SD of triplicates of a representative experiment that was repeated at least twice.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Incubation

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Internalization of cetuximab-vc-MMAF. A. OVCAR8 cells were treated with 10 nM cetuximab-vc-MMAF (red) at 37°C for the indicated times. For time 0 h, cells were incubated on ice for 30 minutes to visualize membrane staining. Nuclei were stained with Hoechst 33342 in all cases. Scale bar: 10 μm. B. Colocalization of cetuximab-vc-MMAF (red) with endocytic markers (green). Cells were treated as above, and colocalization (yellow) with markers for early endosomes (EEA1), late endosomes (RAB7), and lysosomes (LAMP1) analysed at the indicated times. Scale bar: 5 μm. C. Quantitation of colocalization was performed using ImageJ and expressed as a percentage. Data are presented as the mean ± SD of 10 fields per time point, for each vesicle type.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Incubation, Membrane, Staining, Quantitation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Action of cetuximab-vc-MMAF on the cell cycle. A. Morphological changes of OVCAR8 and SKOV3 cells treated with cetuximab-vc-MMAF (10 nM). Phase-contrast images were taken with a 20X objective. Scale bars: OVCAR8, 50 µm; SKOV3, 80 µm. B. Cell cycle profile analysis of OVCAR8 and SKOV3 cell lines treated with cetuximab-vc-MMAF (10 nM) for 24 h and 48 h. Different cell cycle phases are indicated in the graphs. C. Quantitative representation of the percentage of cells in each cell cycle phase. Data are plotted as mean ± SD of three independent experiments. D. Western blot analyses of various cell cycle proteins (50 µg of total protein per lane) following treatment with 10 nM cetuximab-vc-MMAF for the indicated times. Molecular weight markers are shown at the right. Tubulin was used as a loading control. E. Immunofluorescence analyses of aberrant mitotic spindles (tubulin, green) during treatment with 10 nM cetuximab-vc-MMAF. DNA was stained with Hoechst 33342 (blue). Scale bar: 5 µm. F. Quantitation of aberrant mitotic spindles, represented as the number of aberrant versus normal spindles out of 100 mitotic cells.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Western Blot, Molecular Weight, Control, Immunofluorescence, Staining, Quantitation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Action of cetuximab-vc-MMAF on cell viability. A. Flow cytometry-based apoptosis assay of cells treated with the cetuximab-vc-MMAF (10 nM) for the indicated times. Live cells are shown in the lower left quadrant (Annexin V-/PI-), while the other three quadrants represent different stages of non-viable cells. B. Graphical representation of non-viable cells for each condition. Data are plotted as mean ± SD of three independent experiments. C. Western blot analysis of apoptosis-related proteins at the indicated time points following treatment with 10 nM cetuximab-vc-MMAF. Actin was used as a loading control, and molecular weight markers are shown at the right. D. Analysis of caspase-3 activity in cells treated with cetuximab-vc-MMAF (10 nM) for the indicated times. Data represent mean ± SD of fluorescence intensity resulting from substrate cleavage (Ac-DEVD-AFC) by activated caspase-3 in two independent experiments.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Flow Cytometry, Apoptosis Assay, Western Blot, Control, Molecular Weight, Activity Assay, Fluorescence

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: In vivo antitumoral effect of cetuximab-vc-MMAF. A. Schematic representation of the in vivo experimental design for xenografts generated with OVCAR8 and SKOV3 cells. The overall duration of each experiment is indicated. Cetuximab-vc-MMAF was administrated once weekly by intraperitoneal injection (indicated by the syringe icon). SKOV3-bearing mice were treated using a weekly dose escalation (3 mg/kg during weeks 1 and 2, followed by 5 mg/kg in week 3 and 10 mg/kg in week 4), while OVCAR8 injected mice received a constant weekly dose of 5 mg/kg throughout the experiment . Blood sampling time points from the submandibular vein are indicated by a blood drop symbol. At the end of each experiment, tumors (generated from OVCAR8 and SKOV3), and organs (from SKOV3-injected animals) were collected for analysis. B-C. Mean tumor volume (mm³) for SKOV3 ( B ) and OVCAR8 ( C ) xenografts in vehicle-treated (PBS) and cetuximab-vc-MMAF-treated groups. Global p -values < 0.05 are considered significant. d -E. Body weight monitoring for SKOV3 ( D ) and OVCAR8 ( E ) xenografts. F-G. Kaplan-Meier survival curves for SKOV3 ( F ) and OVCAR8 ( G ) xenografts comparing vehicle and cetuximab-vc-MMAF treated groups. In the OVCAR8 experiment, all animals were euthanized at the experimental endpoint according to study design. H-I. Tumor growth change (%) for SKOV3 ( H ) and OVCAR8 ( I ), calculated for each tumor using the formula: TGC = ((Vf – Vi)/Vi) × 100, where Vi is the tumor volume at treatment initiation (Day 0) and Vf the final tumor volume at experiment endpoint. Values below zero indicate tumor regression.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: In Vivo, Generated, Injection, Sampling

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Detection of cetuximab-vc-MMAF in mouse tissues. A-B. Pharmacokinetic analyses of cetuximab-vc-MMAF in blood samples. Total human IgG (left panels), representing the cetuximab antibody irrespective of conjugation status, and MMAF bound to the antibody (right panels) were evaluated at different time points (−1, 1, 2, 5, 9, 14, and 19 days) by ELISA, as described in the Materials and Methods section. Treatment administration days are indicated. The 10 nM concentration used in most in vitro studies is also indicated for reference. C. SKOV3 tumoral tissues from vehicle-treated and cetuximab-vc-MMAF-treated animals were dissected at the end of the experiment, and total human IgG (left panel) or antibody-bound MMAF (right panel) were measured by ELISA. Data are plotted as mean ± SD of three independent tumors. D. Detection of human antibody heavy chain by western. Tumoral tissues were homogenized and 50 μg of protein loaded in SDS-PAGE gels. After transfer to PVDF membranes, human Ig heavy chain was detected by western. Actin was used as a loading control. E. Immunohistochemical analyses of tumor tissues and blood vessels using an anti-human IgG antibody. Scale bar: 40 µm. F. Western blot detection of the antibody heavy chain in different organs. After tissue homogenization, IgH was detected as described in D. G. Quantitative representation of IgH protein levels in tissues from four different animals . Data are plotted as mean ± SD. The experiments carried out in panels C-G were performed using tumors and organs derived from SKOV3 xenografts.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Conjugation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vitro, Western Blot, SDS Page, Control, Immunohistochemical staining, Tissue Homogenization, Derivative Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

doi: 10.1016/j.neo.2026.101295

Figure Lengend Snippet: Analyses of mechanistic markers in tumor tissues. A. Western blot analyses of EGFR, Ki67, and phospho-Histone H3 in SKOV3-derived tumors from vehicle and cetuximab-vc-MMAF treated mice. Tumoral tissues were homogenized and 50 μg of protein loaded in SDS-PAGE gels. After transfer to PVDF membranes, proteins were detected by western. Tubulin was used as a loading control. The molecular weights of the analyzed proteins are indicated. B. Quantitation of the western blot results shown in panel A. Data are plotted as mean ± SD. Each point represents an individual tumor. P -values were calculated using a two-tailed t -test. C. Immunohistochemical analyses of different markers in tumors from vehicle and cetuximab-vc-MMAF treated groups. The graphs on the right show the proportion of cells positive for each marker in 10 randomly selected fields per tumor, from tumors of three different animals (30 fields in total). Each point represents the mean for one tumor, boxes show the overall mean and error bars the SD.

Article Snippet: To generate the standard curve, we used a commercial antibody conjugated to MMAF (Belantamab Mafodotin, MedChem Express), allowing detection within a concentration range of 1.3-100 ng/ml.

Techniques: Western Blot, Derivative Assay, SDS Page, Control, Quantitation Assay, Two Tailed Test, Immunohistochemical staining, Marker