Journal: The Journal of Immunology Author Choice

Article Title: NR4A nuclear receptor expression in human macrophages mediates apoptosis and controls Mycobacterium tuberculosis growth

doi: 10.1093/jimmun/vkaf252

Figure Lengend Snippet: Macrophage apoptosis in response to pharmacologic treatment is reduced by NR4A knockdown. (A–C) AML cells were transfected with 15 μM of either scrambled siRNA or siRNA targeting NR4A1, NR4A2, or NR4A3. After 24 h, they were left untreated or treated with 1 μM staurosporine for 8 h, 10 μM actinomycin D for 24 h, or 100 μM etoposide for 24 h. Caspase-3 and caspase-7 cleavage activity was measured by luminescent assay. (D) Representative Western blot showing total and cleaved PARP1 protein expression in AML cells following treatment with staurosporine. (E) Quantified Western blot data are shown from (D). P values are indicated as asterisks above the plots as obtained from one-way ANOVA (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001) from n = 3 (A–C) or n = 4 (D, E) unique donors. Data are shown as mean ± SEM.

Article Snippet: Transfected AML cells in 96-well plates were treated with InSolution Staurosporine (Thermo Fisher Scientific), actinomycin D (Tocris Bioscience), or etoposide (Thermo Fisher Scientific) to induce cell death, or infected with M.tb at MOI of 5 for 24 to 72 h. Cell death was assayed in triplicate with the CellTiter Glo Assay (Promega) following the manufacturer’s instructions.

Techniques: Knockdown, Transfection, Activity Assay, Luminescence Assay, Western Blot, Expressing

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: 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: 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: 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: Cancer biology & therapy

Article Title: A small molecule that disrupts Mdm2-p53 binding activates p53, induces apoptosis and sensitizes lung cancer cells to chemotherapy.

doi: 10.4161/cbt.7.6.5841

Figure Lengend Snippet: Figure 6. Sensitization of A549 cells to etoposide-induced apoptosis by MI-43: A549 cells were treated with DMSO control (Con), MI-43 (30 μM), etoposide (15 μM) or combination of MI-43 and etoposide for 24 hrs. Both attached and detached cell population were collected and subjected to western blotting (A) and FACS analysis (B). A549 cells were seeded in 96-well plate and subjected to treatment of MI-43 30mm or etoposide 10mm alone or in combination for 24 hrs, followed by caspase-3 activity assay (C). Shown is mean ± SEM from two independent experiments, each run in triplicate.

Article Snippet: The cells were treated with DMSO, MI-43 or in combination with etoposide for 24 hours and subjected to Western blotting using antibodies against p53 (Ab-6, Calbiochem), p21 (BD Pharmingen), Mdm2 (Ab-1, Calbiochem), Bax or caspase-3 (Santa Cruz), PUMA (Ab-1 Calbiochem), Noxa (Calbiochem) and PARP (Bio-Mol).

Techniques: Control, Western Blot, Caspase-3 Activity Assay

Journal: Blood Advances

Article Title: Myeloid translocation gene CBFA2T3 directs a relapse gene program and determines patient-specific outcomes in AML

doi: 10.1182/bloodadvances.2018028514

Figure Lengend Snippet: RUNX1-RUNX1T1 and GCN5 opposingly regulate CBFA2T3 transcription. Scatter plot showing fold changes of CBFA2T3 and GCN5 between primary and relapsed samples in both GSE66525 (A) and GSE83533 (B) patient cohorts. The red-colored dots in panel B denote 2 inv(16) patients. (C) Gene expression of CBFA2T3 and p21 in Kasumi-1, NOMO-1, and A549 cells treated with GCN5 inhibitor MB-3 vs dimethyl sulfoxide (DMSO). Results were analyzed from a public ERP003933 dataset.51 (D) RT-qPCR results showing the effects of CPTH2 and MB-3 treatment on CBFA2T3 and p21 expression in 2 primary AML patient samples. (E) ChIP-Seq intensities of RUNX1-RUNX1T1, HEB, E2A, GCN5, and acetyl-H3K9 at CBFA2T3 promoter sites in control (shControl) and RUNX1-RUNX1T1-depleted (shRUNX1-RUNX1T1) Kasumi-1 cells. (F) ChIP-qPCR quantification of the binding of the indicated proteins to −71 and −2021 CBFA2T3 regulatory loci in shControl and shRUNX1-RUNX1T1-treated Kasumi-1 cells. (G) RT-qPCR results of CBFA2T3 levels in control and RUNX1-RUNX1T1-knockdown Kasumi-1 cells with and without CPTH2 treatment. #P < .1, *P < .05, **P < .01; ***P < .001.

Article Snippet: 9 Antibodies include RUNX1-RUNX1T1 (in-house made), HEB (SC-357; Santa Cruz Biotechnology), E2A (SC-349; Santa Cruz Biotechnology), CBFA2T3 (SC-9741; Santa Cruz Biotechnology), GCN5 (SC-20698; Santa Cruz Biotechnology), p300 (SC-584; Santa Cruz Biotechnology), H3K18ac (ab1191; Abcam), and H3K9ac (ab32129; Abcam).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, ChIP-sequencing, Control, ChIP-qPCR, Binding Assay, Knockdown

Journal: bioRxiv

Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells

doi: 10.1101/2024.08.27.609980

Figure Lengend Snippet: a , FACS profiles showing de novo DNA synthesis (BrdU incorporation) in U2OS cells with greater than G2/M DNA content (as assessed by 7AAD staining) 48 or 72 h following exposure to 9 Gy. Cells were pulsed with BrdU for 1 h prior to harvest. b , Representative FACS profiles (PI staining) of U2OS cells 48 h following exposure to 9 Gy with or without nocodazole (Noc) treatment at the 24-48 h time point following IR exposure. Cells with genomic amplifications highlighted in red. Quantitation is shown in . c , Elisa-based quantitation of BrdU incorporation in the peak fractions of DNA (50 ng each of L:L, H:L, and H:H DNA) isolated from the CsCl ultracentrifugation gradient shown in . d , Workflow ( top ) and representative FACS profiles (PI staining; bottom ) of U2OS cells left untreated or exposed to IR for 48 h and treated with or without aphidicolin (Aph) added immediately following IR and washed 24 h after (0-24 h) or added 24 h post-IR (24-48 h). Cells with genomic amplifications are highlighted in red, and quantitation of the results is shown in . e , The appearance of H:H DNA in cells exposed to IR is aphidicolin-sensitive. The line histogram is an extension of the plot shown in , and shows additional treatment with aphidicolin (Aph) with or without IR treatment as depicted in the experimental workflow shown on top. f , Histogram showing the percentage of U2OS cells with genomic amplifications (as determined by PI-FACS) 72 h following treatment with etoposide (1 μg ml - ), doxorubicin (0.1 μM), or exposure to ultraviolet radiation (UV; 100 J m - ). Data represent the average of three independent experiments ± S.D. g , Work flow ( top ) and quantitation of the percentage of A si SI-ER-U2OS cells with genomic amplifications (as determined by PI-FACS) following the induction of DSBs by the addition of 4-OHT for 48 h. Cells were treated with or without aphidicolin (Aph) added together with 4-OHT and washed 24 h after (0-24 h), or with Aph added 24 or 48 h following treatment with 4-OHT and harvested 24 h after Aph (48 and 72 h following treatment with 4-OHT, respectively). Data represent the average of three independent experiments ± S.D. ** p < 0.01. h , Representative FACS profiles showing de novo DNA synthesis (BrdU incorporation) in A si SI-ER-U2OS cells with greater than G2/M DNA content (as assessed by 7AAD staining) 48, 72, or 96 h following treatment with 300 nM 4-hydroxy-tamoxifen (4-OHT). Cells were pulsed with BrdU for 1 h prior to harvest.

Article Snippet: The DNA LIG4 inhibitor SCR7 (#SML1546, Sigma Aldrich) was added 24 h before irradiation, and was replenished every 24 h. Analysis of rereplication induction by FACS (PI staining) following the induction of DNA damage by etoposide (1 μg ml - , TOCRIS), doxorubicin (0.1 μM, Sigma Aldrich), or by UV (100 J m - ) was performed 72 following treatment.

Techniques: DNA Synthesis, BrdU Incorporation Assay, Staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Oncology reports

Article Title: Zoledronic acid inhibits proliferation of human fibrosarcoma cells with induction of apoptosis, and shows combined effects with other anticancer agents.

doi: 10.3892/or_00000851

Figure Lengend Snippet: Figure 5. Growth inhibitory effects of concurrent treatment with ZOL and anticancer agents on human fibrosarcoma cell lines. The capacity of ZOL and several antitumor agents to inhibit the growth of HT1080 cells was determined by employing the trypan blue dye exclusion method. Data from three independent experiments were collected, and Student's t-test was used to evaluate the efficacy of concurrent treatment with ZOL and other agents and to compare the effects of each anticancer agent alone. P-values of <0.05 were considered statistically significant and derived from two-sided statistical tests. X-axis: a, control; b, 1.2 μM of ZOL alone; c, 0.5xIC50 of antitumor drug alone; d, combination of b with c; e, 1.0xIC50 of antitumor drug; f, combination of b with d. Y-axis is cell counts (x105). (A) doxorubicin; (B) cisplatin; (C) etoposide; (D) 5-fluorouracil; (E) docetaxel; (F) paclitaxel; (G) gemcitabine; (H) methotrexate.

Article Snippet: Doxorubicin (from Toronto Research Chemicals, Inc., Toronto, Canada), 5-fluorouracil (from Nacalai Tesque, Inc., Kyoto, Japan), cisplatin, paclitaxel, docetaxel, gemcitabine (from LKT laboratories, Inc., St. Paul, MN, USA), etoposide (from CalbiochemNovabiochem, Cor(Merck KGaA), Darmstadt, Germany), and methotrexate (from Sigma Aldrich, Tokyo, Japan) were purchased from commercial sources.

Techniques: Derivative Assay, Control