Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Ctrl-KO and SNX9-KO MCF10A cells were treated with etoposide at 25 and 50 μM (indicated by triangles) and analyzed by IB for total and phosphorylated (p53 ser15 ) p53, and the p53 target genes MDM2 and CDKN1A. Vinculin (VCL), loading control. B . RT-qPCR analysis of MDM2 and CDKN1A expression in the samples described in “A”. Data are from three independent experiments and expressed as mean ± SD. *, p<0.05; **, p<0.01. C. 3D reconstruction of MCF10A-p53-KO recipient cells treated for 8 h with EVs purified from the conditioned medium of HEK-293 cells transfected with p53-HA. Recipient cells were treated with etoposide (50 μM for 8 h) and analyzed by IF using an anti-p53 antibody (green) and DAPI (blue). Bar, 20 μm. D. MCF10A-p53-KO recipient cells were treated for 8 h with etoposide (50 μM) and EVs purified from HEK-293 WT (EV) or HEK-293 p53-HA (EV p53-HA) cells as indicated. After treatment cells were harvested and analyzed by RT-qPCR for CDKN1A levels. Data are from three independent experiments and expressed as mean ± SD. * p<0.05, ** p<0.01 and *** p<0.001 vs . same condition in cells not treated with EVs (only the most relevant statistical comparisons are shown). E. Scheme of the co-culture experiment shown in in panel F. a) MCF10A-p53-KO-H2B-Cherry cells were plated onto coverslips. b) After 24 h, coverslips were harvested and seeded (c) in plates in which the indicated cell lines had been previously seeded. Cells were then treated with etoposide (50 μM) or mock-treatment for 8 h. d) Coverslips were harvested and analyzed for purity of H2B-Cherry labeled cells (Fig. S10D) and for the levels of CDKN1A mRNA (panel F). Details are in Materials and Methods. F . RT-qPCR analysis of CDKN1A levels in harvested H2B-Cherry labeled MCF10A-p53-KO cells, co-cultured as described in panel “ E ”. Data are from three independent experiments and expressed as mean ± SD. **, p<0.01; n.s., not significant (only the most relevant statistical comparisons are shown). G . SAOS2 cells were treated with EVs purified from the indicated MCF10A cell lines (see experimental scheme in Fig. S10F), and cell viability/growth was assessed indirectly by quantifying intracellular ATP levels using a luminescence-based assay. Data are from ten samples/condition from two independent experiments and expressed as mean ± SD. One-way ANOVA test: *, and ***, p < 0.05 and < 0.001, respectively, vs . SAOS2 treated with EVs derived from MCF10A-p53-KO cells.

Article Snippet: Chemicals were: FLAG peptide, cat. F3290 (Merck Life Science); HA peptide, cat. 11666975001 (Merck Life Science); NUMB peptide corresponding to amino acids 537-551 of hNUMB (Genscript); Etoposide, cat. E1383 (Merck Life Science); Trehalose Dihydrate, cat. T9531 (Merck Life Science); Chloroquine, cat. C6628 (Merck Life Science); Ionomycin, cat. I0634 (Merck Life Science); Proteinase K, P4850 (Merck Life Science); MG132, cat. 474790 (Merck Life Science); U73122, cat. 6756 (Merck Life Science); GW4869, cat. S7609 (Selleck Chemicals); brain PI(4,5)P2, cat. 840046P; brain PI(4)P, cat. 840045P; brain PS, cat. 840032C; brain PC, cat. 840053C (all from Merck Life Science).

Techniques: Control, Quantitative RT-PCR, Expressing, Purification, Transfection, Co-Culture Assay, Labeling, Cell Culture, Luminescence Assay, Derivative Assay

Journal: Cell death & disease

Article Title: YAP1 expression is associated with survival and immunosuppression in small cell lung cancer.

doi: 10.1038/s41419-023-06053-y

Figure Lengend Snippet: Fig. 3 Efficacy of immunotherapy in small cell lung cancer (SCLC) cell lines. A mRNA expression levels of YAP1 in different SCLC cell lines. B Protein expression level of YAP1 in different SCLC cell lines. Scale bars indicate median fluorescence intensity. C Cell viability of SCLC by Cell Counting Kits-8 (CCK8) assay in the control group (equivalent volume of PBS), the EC group (etoposide plus cisplatin), the EC/Atezolizumab group (atezolizumab plus etoposide and cisplatin), and the Atezolizumab group. D Gating strategy of early and late apoptotic-tumor cells after drug exposure. E The proportions of early and late apoptotic-tumor cells in different groups. EC etoposide plus cisplatin, FVD Fixable Viability Dye, ns not significant, E/T cells effector and target cells, YAP1 yes-associated protein 1; *p < 0.05.

Article Snippet: To establish the etoposide and cisplatin (EC) group, SCLC cells were treated with medium containing 0.25 μM etoposide (S1225, Selleck Chemicals) and 0.5 μM cisplatin (S1166, Selleck Chemicals).

Techniques: Expressing, Cell Counting, CCK-8 Assay, Control

Journal: Cell death & disease

Article Title: YAP1 expression is associated with survival and immunosuppression in small cell lung cancer.

doi: 10.1038/s41419-023-06053-y

Figure Lengend Snippet: Fig. 4 YAP1 inhibitor potentiates immunotherapy in small cell lung cancer (SCLC)-Y subtype. A Cell viability of SCLC by Cell Counting Kits- 8 (CCK8) assay. B, C The proportions of early and late apoptotic-tumor cells in the chemoimmunotherapy (atezolizumab plus etoposide and cisplatin) group and the VP/chemoimmunotherapy (verteporfin plus atezolizumab, etoposide and cisplatin) group. D Median fluorescence intensity of Fas in different groups. FVD Fixable Viability Dye, ns not significant, VP Verteporfin.

Article Snippet: To establish the etoposide and cisplatin (EC) group, SCLC cells were treated with medium containing 0.25 μM etoposide (S1225, Selleck Chemicals) and 0.5 μM cisplatin (S1166, Selleck Chemicals).

Techniques: Cell Counting, CCK-8 Assay

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Defects in DSB repair and mitosis in topbp1 mutant. ( a ) Diagram with the mean number of leaves per seedling in the WT and topbp1 mutant grown just in MS medium or supplemented with cisplatin (30 μM) or cisplatin + etoposide (5 μM). Data collected from three independent experiments ( n = 100 per treatment and day). ( b ) Mitotic anaphases of the WT and topbp1 . Statistical differences between the WT and topbp1 for each treatment analysed by Mann–Whitney test, *** p < 0.001; ns = not significant. Comparisons among treatments within the same genetic background are shown in . Scale bar is 5 μm.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Results of the pairwise comparison of the mean number of leaves per seedling untreated (MS), treated with cisplatin, and treated with cisplatin + etoposide (Cis + Etop) by the Kruskal–Wallis test followed by Dunn’s post-hoc test in the WT and topbp1 at days 7, 12, and 16.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques: Comparison

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Meiotic stages of WT plants treated with different topoisomerase II inhibitors. ( a ) Plants were treated with TOPII inhibitors in two ways: (i) a 2 h pulse (P) or (ii) continuous (C). In both cases, flower buds were fixed at 12 h, 28 h, or 38 h after treatment. ( b – m ) Images of pollen mother cells at different stages of meiosis of the WT treated with TOPII inhibitors. ( b ) Anaphase I treated with merbarone 1 μM (P) fixed at 38 h showing an anaphase bridge. ( c ) Anaphase I treated with merbarone 1 μM (C) fixed at 38 h showing a chromosome fragment. ( d ) Anaphase II treated with merbarone 10 μM (P) fixed at 38 h showing chromosome mis-segregation. ( e ) Telophase II treated with merbarone 1 μM (C) fixed at 38 h showing micronuclei. ( f ) Anaphase I treated with etoposide 0.05 μM (P) fixed at 38 h showing a broken anaphase bridge. ( g ) Anaphase II treated with etoposide 0.05 μM (C) fixed at 38 h showing chromosome mis-segregation. ( h ) Telophase II treated with etoposide 0.05 μM (P) fixed at 28 h showing a micronucleus. ( i ) Telophase II treated with etoposide 5 μM (P) fixed at 28 h showing a micronucleus. ( j ) Anaphase I treated with ICRF-187 0.1 μg/mL (P) fixed at 38 h showing an anaphase bridge. ( k ) Anaphase I treated with ICRF-187 100 μg/mL (C) fixed at 28 h showing two anaphase bridges. ( l ) Metaphase II/anaphase II treated with ICRF-187 100 μg/mL (C) fixed at 28 h showing an anaphase bridge. ( m ) Telophase II treated with ICRF-187 0.1 μg/mL (P) fixed at 38 h showing a micronucleus. Arrows indicate errors in meiotic divisions. Scale bar 10 μm.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques:

Journal: PLOS Biology

Article Title: Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells

doi: 10.1371/journal.pbio.3003096

Figure Lengend Snippet: (A) Effect of IRF4, IRE1, or XBP1 silencing on in vitro spheroid growth of AMO1. Cells were stably transfected with plasmids encoding Dox-inducible shRNAs against either IRF4 (purple) or non-targeting control (blue). Growth of these cells in the absence (closed symbols) or presence (open symbols) of Dox (0.2 μg/mL) was compared to that of cells expressing shRNAs against IRE1 or XBP1. Spheroid growth, depicted as FC confluence, was monitored by time-lapse microscopy in an IncuCyte instrument and values represent mean ± SEM. (B) Effect of IRF4, IRE1, or XBP1 silencing on number of cell divisions. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were stained with CFSE-type dye and incubated in the absence (filled curves) or presence (open curves) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dashed line) was used as a non-proliferative control. Representative experiment out of 3 independent replicates. (C) Effect of IRF4, IRE1, or XBP1 silencing on DNA replication. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were pulsed with BrdU (10 μM) and incubated in the absence (filled bars) or presence (open bars) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dotted line) was used as a non-proliferative control. Data represented as mean ±SEM. (D) Effect of IRE1 or IRF4 silencing on cell cycle progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence (filled symbols) or presence (open symbols) of Dox (0.2 μg/mL) for the indicated timepoints, EtOH-fixated and PI stained before analyzed by flow cytometry. The indicated cell cycle phases were determined according to univariate (DNA content) modeling. Representative experiment out of at least 3 independent replicates. (E) Effect of IRE1 or IRF4 silencing on the rate of G2/M progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were pre-incubated with 9 μM RO-3306 CDK1 inhibitor (synchronization to G2/M phase) in the absence or presence of Dox (0.2 μg/mL). Cells in G2/M phase were collected and their cell cycle progression during indicated time points post-sorting was analyzed by flow cytometry as before. The indicated cell cycle phases were determined according to DNA content and EdU incorporation to accurately decipher S phase. (F) Effect of IRE1 or IRF4 silencing on CDK2 activation. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence or presence of Dox (0.2 μg/mL) for 24 h. CDK2 was purified by immunoprecipitation. The top band is inactive CDK2 and the bottom band is the active form . Additionally, binding of the CDK2 substrate, Rb, is reduced by IRE1 or IRF4 silencing while binding of p21, the CDK inhibitor, is increased. Ig represents an isotype control for Ig detection. (G) Effect of IRE1 or IRF4 silencing on subcellular abundance of CDK2. Samples from and samples from AMO1 shIRF4 Cl.1 cells were analyzed by IB for CDK2 protein. Subcellular fractions: C—cytoplasmic, M—Membrane, SN—Soluble Nuclear, CN—Chromatin-bound Nuclear. Nuclear fractions were analyzed by IB for IRE1 and IRF4 while Cofilin, Histone H3, and Lamin B2 served as fractionation internal controls. The blots for IRE1, IRF4, Cofilin, and Lamin B2 from are shown here again for direct comparison. Data underlying this figure can be found in and .

Article Snippet: Etoposide (25 μM, #1226) was from R&D systems.

Techniques: In Vitro, Stable Transfection, Transfection, Control, Expressing, Time-lapse Microscopy, Staining, Incubation, Flow Cytometry, Activation Assay, Purification, Immunoprecipitation, Binding Assay, Membrane, Fractionation, Comparison

Journal: Experimental cell research

Article Title: Distinct regulation of p53-mediated apoptosis by protein kinase Cα, δ, ε and ζ: Evidence in yeast for transcription-dependent and -independent p53 apoptotic mechanisms.

doi: 10.1016/j.yexcr.2011.02.007

Figure Lengend Snippet: Fig. 4 – PKCδ and ε phosphorylate p53 at Ser376-378 in H2O2-treated yeast cells. Yeast cells expressing only p53 and co-expressing p53 and PKCδ/ε were incubated in galactose selective medium to 0.45 OD600 before treatment with 5 mM H2O2 for 1 h at 30 °C. p53 phosphorylation was analysed using (A) phospho-p53(Ser15), (B) phospho-p53(Ser20), (C) phospho-p53(Ser46) and (D) PAb421 (Ser376–378) antibodies. Detection of p53 with DO-1 antibody was used as loading control. In A–C, the etoposite-treated MCF7 cell lysate was used as positive control (C+); in D, p53 from yeast expressing only p53 (YEplac/p53), which expression was previously confirmed with the anti-p53 DO-1 antibody, was used as positive control. (E) Quantification of band intensities obtained in D.

Article Snippet: The etoposide- treated MCF7 cell lysate (Santa Cruz Biotechnology) was used as positive control.

Techniques: Expressing, Incubation, Phospho-proteomics, Control, Positive Control