imr90 cells  (Tocris)

Journal: Nature Communications

Article Title: TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases

doi: 10.1038/s41467-025-59129-y

Figure Lengend Snippet: a Schematic representation of the lentiviral expression vectors used to modify RKO Cas9 cells for knock-out and cell competition assays. b RKO cells harbouring Dox-inducible Cas9 were transduced with sgRNA vectors targeting TRIM52 or the safe-harbour locus AAVS1 . Cas9 was induced by Dox treatment for 5 days. Whole cell lysates (WCE) were analysed by WB. c Relative cell fitness of sg TRIM52 -transduced cells compared to untransduced cells was determined by measuring the percentage of iRFP fluorescent cells over the indicated period and normalized to the fraction of sg AAVS1 -transduced cells relative to untransduced cells. Data represent biological replicates as mean values +/- SD, n = 3. d Schematic representation of genetic modifier screen, grey and blue circles represent individual cells transduced with different sgRNAs. e RKO cells expressing Cas9 and transduced with a lentiviral genome-wide sgRNA library were further transduced with individual sgRNAs targeting TRIM52 or AAVS1 . Following Cas9 induction, cells were grown for 12 doublings and integrated sgRNA-coding sequences were analysed by NGS analysis of gDNA, sgRNA enrichment calculated by MAGeCK analysis, and log2-fold change and adjusted p-value plotted relative to library representation determined in the unselected cell population. Factors on the left-hand side of the plot are identified as genes which lead to synthetic lethality upon ablation in TRIM52 -targeted cells. Non- TRIM52 -specific genes were filtered out by comparison with data from DGCR8 -targeted cells. Factors involved in DNA-damage repair are marked in pink and labelled by name. f Filtered TRIM52 ablation-specific genes were selected and their Log2 fold change compared to AAVS1 , plotted in a heatmap, and grouped by their function. g Relative cell fitness of sg TRIM52 , sg NHEJ1 and sg TRIM52 /sg NHEJ1 transduced cells compared to untransduced cells was determined by measuring the percentage of iRFP fluorescent cells over the indicated period. sg TRIM52 fractions were normalized to sg AAVS1/CCR5 -transduced cells, relative to untransduced cells. Data represent biological replicates as mean values +/- SD, n = 3. Data analysed by unpaired two-sided t-test comparing means of TRIM52/AAVS1 and NHEJ1/TRIM52, with FDR adjustment using Benjamini, Krieger, and Yekutieli. Source data are provided as a Source Data file.

Article Snippet: In brief, 1 × 10 6 RKO Cas9 cells were treated with etoposide (10 μg/ml) for 2 h., washed with PBS and lysed by adding 1 mL DNAzol (Thermo Fisher Scientific, 11558626).

Techniques: Expressing, Knock-Out, Transduction, Genome Wide, Comparison

Journal: Nature Communications

Article Title: TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases

doi: 10.1038/s41467-025-59129-y

Figure Lengend Snippet: a A TurboID-TRIM52 fusion was expressed, cells treated with proteasome inhibitor for 5 h., and biotin for the last 15 min. Biotinylated proteins were purified under denaturing conditions and analysed by mass-spectrometry. Factors involved in DNA-damage repair are marked in pink and labelled by name. Data represent biological replicates, n = 3. b Putative interactors of TRIM52 with p value < 0.05 and Log2 fold change > 2.5 were selected, analysed by gene ontology enrichment analysis, and top hits per ontology category plotted in a heatmap by their Log2 fold change enrichment compared to the TurboID-EGFP control. Statistical analysis was conducted using moderated t-statistics via the limma-trend method in R and applying the Benjamini–Hochberg multiple testing correction. Data represent biological replicates as mean values +/- SD, n = 3. c Schematic representation of TOP2 cleavage complex resolution. d RKO-Cas9 cells transduced with sgRNAs targeting safe harbour loci AAVS1 / CCR5, TRIM52 , TDP2 or both were treated with Dox for 5 days to induce Cas9. Cells were treated with etoposide (ETO) for 2 h. Samples were harvested and subjected to RADAR analysis. Isolated protein-DNA covalent complexes were analysed by slot blot, e quantified and normalized to dsDNA levels. Data represent biological replicates as mean values +/- SD, n = 4. Data were analysed by 2-way ANOVA. f RKO-Cas9 cells transduced with sgRNAs targeting the safe harbour locus AAVS1 or TRIM52 were treated with Dox for 5 days to induce Cas9. Cells were treated with DMSO or ETO for 2 h., fixed, and stained with anti-γH2AX antibody. The number of γH2AX foci was determined by immunofluorescence microscopy, and g plotted, and h plotted normalized to the surface area of the corresponding nuclei. Scale bar: 10 µm. The number of foci were counted for each biological sample. Data represent n = 3 technical replicates, analysed by 2-way ANOVA. Source data are provided as a Source Data file.

Article Snippet: In brief, 1 × 10 6 RKO Cas9 cells were treated with etoposide (10 μg/ml) for 2 h., washed with PBS and lysed by adding 1 mL DNAzol (Thermo Fisher Scientific, 11558626).

Techniques: Purification, Mass Spectrometry, Control, Transduction, Isolation, Dot Blot, Staining, Immunofluorescence, Microscopy

Journal: Nature Communications

Article Title: TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases

doi: 10.1038/s41467-025-59129-y

Figure Lengend Snippet: a Schematic representation of the expression vectors in the screening cell line. mCherry and EGFP-TRIM52 are expressed in an equimolar manner, yet EGFP-TRIM52 accumulates at low steady-state levels, resulting from its rapid proteasomal turn-over. b Schematic representation of the genetic screen to identify TRIM52 regulators. EGFP high cells (blue circles) with potential knock-outs in factors involved in TRIM52 degradation were collected by FACS, and their integrated sgRNA CDSs quantified by NGS, relative to those from unsorted cell pools. c Screening cells were transduced with an sgRNA library targeting ubiquitin-related genes and treated with Dox for 3 or 6 days. Cells expressing the highest and lowest 1-2% of each fluorophore were collected by FACS, their integrated sgRNA CDSs quantified by NGS, and sgRNA enrichment calculated by MAGeCK analysis, and log2-fold change and adjusted p-value plotted. d Heatmap displaying the Log2 fold change for genes enriched in the EGFP-TRIM52 high sorted population after exclusion of genes enriched in mCherry high population on days 3 and 6. e RKO-Cas9 cells expressing MYC-mCherry-P2A-Ollas-EGFP-TRIM52 (teal) or mCherry-cMYC-P2A-EBFP (purple) as a control were transduced with lentiviral vectors expressing the indicated sgRNAs. Cas9 expression was induced for 6 days, after which EGFP-TRIM52 and mCherry-cMYC protein levels were quantified by flow cytometry ( f ), or analysed by WB ( g – h ). f MFI plotted for flow cytometry samples. Data represent biological replicates as mean values +/- SD, n = 3. Data were analysed by 1-way ANOVA. g WB samples, and h quantification by densitometry. Source data are provided as a Source Data file.

Article Snippet: In brief, 1 × 10 6 RKO Cas9 cells were treated with etoposide (10 μg/ml) for 2 h., washed with PBS and lysed by adding 1 mL DNAzol (Thermo Fisher Scientific, 11558626).

Techniques: Expressing, Transduction, Ubiquitin Proteomics, Control, Flow Cytometry

Journal: Nature Communications

Article Title: TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases

doi: 10.1038/s41467-025-59129-y

Figure Lengend Snippet: a HEK-293T cells were transfected with plasmids expressing the indicated TwinStrep-mCherry-tagged TRIM52 expression constructs and an internal EBFP as a control. Cells were treated with proteasomal inhibitor epoxomicin (EPO) for 5 h., after which protein levels were quantified by flow cytometry, and normalized to EBFP levels and to corresponding untreated (UNT) samples and plotted. Data represent biological replicates as mean values +/- SD, n = 3. Data were analysed by 1-way ANOVA. b HEK-293T cells were transfected to express HA-tagged WT TRIM52, TRIM52 neutral loop2 (Loop2-neut) or TRIM52 in which the loop 2 region is reduced to the size of other RING proteins (ΔLoop2), treated with EPO for 5 h, TRIM52 immunoprecipitated, and ubiquitination levels analysed by WB. c HEK-293T cells constitutively expressing Cas9 and sgRNAs targeting the indicated genes were transfected with plasmids expressing TwinStrep-mCherry-tagged TRIM52, TRIM52 (ΔLoop2), RING, or RING (ΔLoop2), as well as an internal control EBFP. Protein levels were quantified by flow cytometry, normalized to EBFP and the sg AAVS1 control, and plotted. Data represent biological replicates as mean values +/- SD, n = 3. d RKO cells expressing a TurboID-TRIM52 fusion protein were incubated with biotin for 15 min., after which biotinylated proteins were purified under denaturing conditions, and analysed by mass-spectrometry. Statistical analysis was conducted using moderated t-statistics via the limma-trend method and applying the Benjamini–Hochberg multiple testing correction. Data represent n = 3 biological replicates. e Heatmap displaying the Log2 fold change of selected interactors of TRIM52. f HEK-293T cells expressing 3xHA-tagged TRIM52 were treated with EPO for 5 h., after which TRIM52 was immunoprecipitated, and analysed by WB for co-immunoprecipitation with HUWE1. g HEK-293T cells expressing 3xHA-tagged TRIM52 were treated with EPO for 5 h, after which TRIM52 was immunoprecipitated, and analysed by WB for co-immunoprecipitation with HUWE1, BIRC6, and UBR4. h Purified TwinStrep-tagged EGFP-TRIM52, EGFP-RING and EGFP-RINGΔLoop2 were incubated with purified human HUWE1 for 3 h. Twin-Strep-EGFP-TRIM52 was then immunoprecipitated from the samples using EGFP-trap beads and analysed for complex formation with HUWE1 by WB. Source data are provided as a Source Data file.

Article Snippet: In brief, 1 × 10 6 RKO Cas9 cells were treated with etoposide (10 μg/ml) for 2 h., washed with PBS and lysed by adding 1 mL DNAzol (Thermo Fisher Scientific, 11558626).

Techniques: Transfection, Expressing, Construct, Control, Flow Cytometry, Immunoprecipitation, Ubiquitin Proteomics, Incubation, Purification, Mass Spectrometry

Journal: Aging Cell

Article Title: Identification of Senomorphic miRNAs in Embryonic Progenitor and Adult Stem Cell‐Derived Extracellular Vesicles

doi: 10.1111/acel.70071

Figure Lengend Snippet: Evidence that multiple types of stem cell EVs function as senomorphics. (A) Representative image of SA‐β‐gal staining of liver sections from Ercc1 − /Δ mice treated with AC83 EVs. Scale bar equals 2.5 mm. (B) Quantification of SA‐β‐gal staining of liver samples from Ercc1 − /Δ mice treated with AC83 EVs. (C) qPCR analyses of p16 Ink4a , p21 Cip1 , IL‐1β , IL‐6 , and Mcp‐1 of the livers from nature aging mice treated with AC83 EVs. (D) Quantification of total cell count and percentage of senescent IMR90 cells following treatment with four types of young stem cell‐derived EVs. Senescence was assessed by C12FDG SA‐β‐gal staining at 48 and 96 h posttreatment. Total cells were assessed using DAPI staining. Cell numbers are expressed as a percentage relative to untreated controls. (E, F) Relative mRNA expression of p16 Ink4a , p21 Cip1 , IL‐1β , and IL‐6 in senescent IMR90s as measured by qPCR. Data are shown as the mean ± SEM. P values are indicated with *p < 0.05 and **p < 0.01.

Article Snippet: The miRNAs were transfected into etoposide‐induced senescent IMR90 cells to determine their senotherapeutic activity. mirVana miRNA mimics were purchased from Ambion.

Techniques: Staining, Cell Counting, Derivative Assay, Expressing

Journal: Aging Cell

Article Title: Identification of Senomorphic miRNAs in Embryonic Progenitor and Adult Stem Cell‐Derived Extracellular Vesicles

doi: 10.1111/acel.70071

Figure Lengend Snippet: Identification of a senomorphic miRNA cocktail. (A) Screening of miRNA cocktails in senescent IMR90 cells. The table shows the concentration (nM) of each miRNA in different combinations (E3–E48). The heat map represents the expression levels of senescence markers (p16 Ink4a , p21 Cip1 ) and SASP factors (IL‐1β, IL‐6, Mcp‐1) by qPCR as well as the percentage of SA‐β‐gal‐positive cells at 48 and 96 h posttransfection. Blue indicates downregulation, and red indicates upregulation, with the scale showing fold‐change. (B) Expression of p16 Ink4a , p21 Cip1 , and SASPs as well as the percent of SA‐β‐gal‐positive IMR90 cells after transfection of the E5 cocktail of miRNAs. (C) IL‐6 levels in the supernatant of senescent IMR90s 96 h posttransfection with the E5 cocktail. Data are shown as the mean ± SEM. p values are indicated with *p < 0.05 and * *p < 0.01.

Article Snippet: The miRNAs were transfected into etoposide‐induced senescent IMR90 cells to determine their senotherapeutic activity. mirVana miRNA mimics were purchased from Ambion.

Techniques: Concentration Assay, Expressing, Transfection

Journal: Aging Cell

Article Title: Identification of Senomorphic miRNAs in Embryonic Progenitor and Adult Stem Cell‐Derived Extracellular Vesicles

doi: 10.1111/acel.70071

Figure Lengend Snippet: Possible senomorphic mechanism of E5. (A) Relative expressions of PCAF and HIPK2 in senescent IMR90 cells quantitated by qPCR. (B) Expression of PCAF and HIPK2 at 24, 48, and 96 h posttransfection with E5 by qPCR. (C) Representative immunoblots showing the protein levels of PCAF, HIPK2, p53 S46 (phosphorylated p53 at Ser46), and p53 K379 (acetylated p53 at Lys379) at 24 and 96 h posttransfection with E5. (D, E) Densitometry quantification of Western blot bands. (F) A model for how the E5 miRNA cocktail inhibits p53 activity. Data are shown as the mean ± SEM. P values are indicated with *p < 0.05 and **p < 0.01.

Article Snippet: The miRNAs were transfected into etoposide‐induced senescent IMR90 cells to determine their senotherapeutic activity. mirVana miRNA mimics were purchased from Ambion.

Techniques: Expressing, Western Blot, Activity Assay

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Schematic of the screen. ( B ) Representative images for IL-6 and IL-8 immunofluorescence (IF) of IMR90 ER:RAS cells transfected with the indicated siRNAs. Scale bars, 100 μm. ( C ) Percentage of IMR90 ER:RAS cells positive for IL-6 expression beyond a predetermined threshold following transfection with the indicated siRNAs. Data represent means ± SD ( n = 3). ** P < 0.01 and *** P < 0.001; ordinary one-way analysis of variance (ANOVA; Dunnett’s multiple comparisons test). ( D ) Results of the pooled siRNA screen for SASP superinducers. Normalized B -score values of IL-6 versus IL-8 for each replicate sample siRNA are shown. The dashed lines represent the thresholds (+2 SD of the mean of the negative non-targeting siRNA controls). Hits were selected if siRNA pools showed a normalized B -score value for both IL-6 and IL-8 beyond the specified threshold in both replicates. ( E ) Screen for IL-6 superinducers. Normalized score for the control siRNAs (left) and samples (right). Dotted line denotes threshold. **** P < 0.0001; ordinary one-way ANOVA (Tukey’s multiple comparisons test). ( F ) Summary of the SASP superinducer siRNA screens. The Venn diagram of the secondary screen shows the distribution of 124 hits across the indicated SASP readouts. ( G ) Percentage of IMR90 ER:RAS cells positive for the indicated SASP components 7 days after siRNA transfection. Two distinct siRNAs targeting FURIN are shown as well as the non-targeting control. Data represent means ± SD ( n = 3). *** P < 0.001 and **** P < 0.0001; two-way ANOVA (Tukey’s multiple comparisons test). ( H ) Representative IF images of the indicated SASP components following transfection of siRNAs targeting FURIN . Scale bar, 100 μm. The results of the primary SASP siRNA screen [shown in (D) and (E)] are presented in table S1. The results of the secondary SASP siRNA screen [summarized in (F)] are presented in table S2.

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: Immunofluorescence, Transfection, Expressing, Control

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Schematic of the coculture of IMR90 ER:RAS and NK92-MI (NK) cells. ( B ) Quantification and representative images of NK-mediated killing of IMR90 ER:RAS cells, measured as the percentage change in cell count after 48-hour coculture using 4′,6-diamidino-2-phenylindole (DAPI) staining. Control wells contained dimethyl sulfoxide (DMSO) or 4OHT-treated IMR90 ER:RAS cells only. Scale bar, 300 μm. Data represent means ± SEM ( n = 5). **** P < 0.0001; two-way ANOVA (Tukey’s multiple comparisons test). ( C ) Schematic of the experiment testing the effect of indisulam on NK-mediated killing. ( D ) Quantification and representative images of NK coculture with control and senescent IMR90 ER:RAS cells [2:1 effector–to–target cell (E:T) ratio]. Scale bar, 300 μm. Data represent means ± SEM ( n = 4). ** P < 0.01; two-way ANOVA (Tukey’s multiple comparisons test). ( E ) Schematic of the siRNA screen to identify siRNAs potentiating NK-mediated killing of senescent cells. ( F ) Screen results showing normalized NPA scores for NK-mediated killing of senescent cells. The teal dotted line represents the cutoff (NPA ≥ 0.7). Hits were selected if at least two of the four siRNAs targeting a gene scored above the cutoff in at least three of the six replicates. siRNAs against SMARCA4 are highlighted. Screen results are presented in table S3. ( G ) Summary workflow of the screens for siRNAs potentiating NK-mediated killing of senescent cells. ( H ) Quantification of the percentage change in IMR90 ER:RAS cell counts in the indicated conditions. Data represent means ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ordinary one-way ANOVA (Dunnett’s multiple comparisons test). ( I ) Representative images of IMR90 ER:RAS cells transfected with the indicated siRNAs and cocultured with NK cells (2:1 E:T ratio) for 48 hours. Scale bar, 300 μm.

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: Cell Counting, Staining, Control, Transfection

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Quantification and images of IL-8 expression in control and senescent IMR90 ER:RAS cells at day 7. AU-15330 was added on day 4. Scale bars, 300 μm. Data represent means ± SEM ( n = 3). ( B ) Coculture of senescent AU-15330–treated IMR90 ER:RAS cells and NK cells at 2:1 E:T ratio. Cell numbers were measured using IncuCyte software and normalized to time 0. Data represent means ± SEM ( n = 3). a.u., arbitrary units. ( C ) Quantification of percentage change in AU-15330–treated IMR90 ER:RAS cells cocultured with NK cells (2:1 E:T ratio). Data represent means ± SEM ( n = 5). ( D ) Representative images of (C). Scale bar, 300 μm. ( E ) NK-mediated killing of etoposide-induced senescent AU-15330–treated IMR90 cells were cocultured with NK cells (2:1 E:T ratio). Data represent means ± SEM ( n = 4). ( F ) NK-mediated killing of cisplatin-induced senescent AU-15330–treated OVCAR4 cells cocultured with NK cells at day 6. Data represent means ± SD ( n = 3). ( G ) NK-mediated killing of senescent AU-15330–treated IMR90 ER:RAS cells cocultured with cord blood (CB)– or peripheral blood (PB)–derived NK cells. Data represent means ± SD. ( H ) Coculture of senescent AU-15330–treated IMR90 ER:RAS cells and peripheral blood–derived NK cells. Data represent means ± SEM. Ordinary one-way ANOVA (Tukey’s multiple comparisons test) was performed for (A), while two-way ANOVA (Tukey’s multiple comparisons test) was used in (B), (C), (E), (F), (G), and (H). n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: Expressing, Control, Software, Derivative Assay

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Heatmap representing average (of n = 3 replicates) NK-mediated killing of senescent IMR90 ER:RAS cells transfected with indicated siRNAs. ( B ) Percentage change in cell numbers of senescent IMR90 ER:RAS cells transfected with the indicated siRNAs following coculture with NK cells at a 2:1 E:T ratio. Data represent means ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; ordinary one-way ANOVA (Dunnett’s multiple comparisons test). ( C ) Representative images from (B). Scale bar, 300 μm. ( D ) Heatmap of RNA sequencing (RNA-seq) data showing a SWI/SNF signature. ( E ) IMR90 ER:RAS cells were treated with AU-15330 on day 4. Representative IF images at day 7. Scale bars, 100 μm. ( F ) Quantification of IMR90 ER:RAS cells positive for SMARCA4 from (E). Data represent means ± SEM ( n = 3). **** P < 0.0001; ordinary one-way ANOVA (Tukey’s multiple comparisons test). ( G ) Model of OIS in the liver. ( H ) Representative IF images of SMARCA4 and NRAS in liver samples. ( I ) SMARCA4 fluorescence intensity in NRAS + cells and NRAS − cells (left). A representative experiment out of the six mice ( n = 1000 cells). Data represent means ± SD. **** P < 0.0001; unpaired t test. Percentage of SMARCA4-positive cells in NRAS + and NRAS − cells (right). Data represent means ± SD ( n = 6). *** P < 0.001; unpaired t test. ( J ) SMARCA4 IF staining and quantification in ID8 Trp53 −/− cells treated with cisplatin (1 μM) or DMSO for 6 days. Scale bar, 100 μm. Data represent means ± SD ( n = 3). **** P < 0.0001; unpaired t test. ( K ) Schematic of in vivo intraperitoneal injection of ID8 Trp53 −/− cells and cisplatin treatment. ( L ) SMARCA4 IF staining and quantification in omental tumors. Scale bars, 50 μm. Data represent means ± SEM ( n = 8 mice per group). * P < 0.05; unpaired t test.

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: Transfection, RNA Sequencing, Fluorescence, Staining, In Vivo, Injection

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Gene set enrichment analysis (GSEA) plot of the SASP signature in 4OHT-induced IMR90 ER:RAS cells. NES, normalized enrichment score. ( B ) Heatmap of RNA-seq data showing up-regulation of SASP marker expression in senescent IMR90 ER:RAS cells transfected with siRNAs targeting SMARCA4 . ( C to F ) Representative IF images (left) and quantification (right) of AU-15330–treated IMR90 ER:RAS cells positive for cGAS (C), pSTING (D), pTBK1 (E), and pIRF3 (F) staining. Scale bars, 100 μm. Data represent means ± SEM ( n = 4). ** P < 0.01 and **** P < 0.0001; ordinary one-way ANOVA (Tukey’s multiple comparisons test). ( G ) Quantification of the percentage of IL-6– and IL-8–positive 4OHT-induced IMR90 ER:RAS cells in the presence or absence of AU-15330 following treatment with the indicated inhibitors (inh.). Data represent means ± SEM ( n = 3). *** P < 0.001 and **** P < 0.0001; two-way ANOVA (Dunnett’s multiple comparisons test).

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: RNA Sequencing, Marker, Expressing, Transfection, Staining

Journal: Science Advances

Article Title: SMARCA4 regulates the NK-mediated killing of senescent cells

doi: 10.1126/sciadv.adn2811

Figure Lengend Snippet: ( A ) Volcano plot showing fold change of repetitive element subfamilies in siSMARCA4 versus siNT IMR90 ER:RAS + 4OHT cells. The dashed line indicates P adj. < 0.05. ( B ) Volcano plot showing fold change of satellite element loci in siSMARCA4 versus siNT IMR90 ER:RAS + 4OHT cells. Blue dots indicate significantly different (DESeq2, P adj. < 0.05, fold change > 1.5). ( C ) IF images and quantification of AU-15330–treated IMR90 ER:RAS cells positive for ORF1. Scale bar, 100 μm. Data represent means ± SEM ( n = 4). ( D ) Representative IF images of cytoplasmic dsDNA staining (left), quantification of dsDNA intensity (center), and percentage of dsDNA-positive IMR90 ER:RAS cells (right) treated with and without AU-15330 as indicated. Data represent means ± SEM ( n = 5). ( E ) IF images of IL-6 (top)– or IL-8 (bottom)–positive IMR90 ER:RAS cells treated with AU-15330 and lamivudine (3TC) as indicated. Scale bars, 100 μm. ( F ) Quantification of IL-6– or IL-8–positive cells from (E). Data represent means ± SEM ( n = 3). ( G ) Representative IF images (left), quantification of dsRNA intensity (center), and percentage of dsRNA-positive IMR90 ER:RAS cells (right) treated with and without AU-15330 as indicated. Data represent means ± SEM ( n = 5). ( H ) Schematic of siRNA experiment in AU-15330–treated IMR90 ER:RAS cells. ( I ) Quantification of IL-6–positive IMR90 ER:RAS cells. Data represent means ± SEM ( n = 6). ( J ) Scheme showing SASP activation and the mechanism of NK cell recruitment following SMARCA4 inhibition in senescent cells. Ordinary one-way ANOVA (Tukey’s multiple comparisons test) was used for (C), ordinary one-way ANOVA (Dunnett’s multiple comparisons test) for (D) and (G), two-way ANOVA (Dunnett’s multiple comparisons test) for (F), and two-way ANOVA (Šídák’s multiple comparisons test) for (I). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For chemotherapy-induced senescence, IMR90 cells were treated with 50 μM etoposide (Tocris, 1226) for 48 hours.

Techniques: Staining, Activation Assay, Inhibition

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Etoposide is a novel inhibitor of EV71 2A pro . ( A ) EV71 2A pro B-factor. ( B ) EV71 2A pro inhibitor-binding pocket (PDB: 4fvd). The pocket centers (H21, D39, and C110) are shown as sticks. ( C ) Virtual screening schematic. ( D ) Inhibitory effect of 13 candidates (6 and 30 µM) on EV71 infection on RD cells. All assays were repeated three times.

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Binding Assay, Infection

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Etoposide effectively inhibited EV71 proliferation. ( A ) Concentration-dependent reduction of EV71 proliferation treated with Etoposide. RD cells were infected by EV71-GFP virus at MOI of 1, with or without treatment by various concentrations of Etoposide (1.25–80 μM) for 24 h. DAPI was used to visualize the nucleus. The GFP fluorescence signals were used to monitor viral growth. Scale bar, 75 µm. ( B ) The levels of expression of EV71 VP1 were inhibited by Etoposide in a dose-dependent reduction manner. The expression level of GAPDH was not affected by the treatment of Etoposide. ( C ) The expression of EV71 VP1 was normalized to the expression level of MOCK. Protein abundance was quantified using ImageJ software. Statistical significance of the differences between group means was evaluated by one-way analysis of variance (ANOVA) using the Tukey honestly significant difference test as a post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, N.S., not significant). All experiments were performed three times, and the representative results were shown.

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Concentration Assay, Infection, Virus, Fluorescence, Expressing, Quantitative Proteomics, Software

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Etoposide plays inhibitory role in the viral replication stage. ( A ) Schematic diagram of time of addition assay. ( B–D ) Inhibition of EV71 luciferase reporter virus infection of RD cells by Etoposide (10 µM), NK-1.8k (2 µM) and GPP3 (0.5 µM) at various addition times (0 hpi indicates the time supplied inhibitors and virus simultaneously). Chemical structural formula of Etoposide, NK-1.8k and GPP3 are displayed above the corresponding chart. All experiments were performed three times.

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Inhibition, Luciferase, Virus, Infection

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Antiviral activity of Etoposide on different cell lines. (A–C) Cytotoxicity of Etoposide on RD, HEK-293T, and Vero cell lines, respectively. (D–F) Quantification of EC 50 on RD, HEK-293T, and Vero cell lines, respectively. All the data are means ± SD ( n = 3).

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Activity Assay

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Antiviral activity of Etoposide on four viral strains. RD cells were infected with EV71 strains BrCr (A), SK-EV006 (B), and Fuyang (C), as well as CVA16 (D). The cells were subjected to a range of Etoposide concentrations, denoted as A, B, and C. The EC 50 values were ascertained utilizing quantitative real-time PCR (qRT-PCR).

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Conventional molecular dynamics simulations. ( A ) Conformational overlap of Etoposide at the 300 ns. ( B ) Root-mean-square deviation (RMSD) of protein backbone atoms. ( C ) Radius of gyration of the protein. ( D ) RMSD of Etoposide heavy atoms. ( E–F ) Root-mean-square fluctuations (RMSF) of the protein.

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques:

Journal: Microbiology Spectrum

Article Title: Etoposide targets 2A protease to inhibit enterovirus 71 replication

doi: 10.1128/spectrum.02200-24

Figure Lengend Snippet: Etoposide and EV71 2A pro binding mechanism. ( A ) The binding free energy decomposition of Etoposide and 2A pro . ( B ) The binding model of Etoposide and 2A pro . The 2A pro is shown as cartoon, and the critical residues in the binding pocket are shown as sticks. ( C ) Interactions between Etoposide and 2A pro . Schematics were generated by LigPlus. The hydrophobic contacts are indicated as an “eyelash” motif. Hydrogen bonds are indicated as green dashed lines. ( D ) Schematic diagram of 2A pro cleavage of a fluorescent peptide. ( E–F ) Fluorescence resonance energy transfer curves of 2A pro wild-type and mutant hydrolysis substrates in the absence and presence of Etoposide, respectively. All data were performed three times.

Article Snippet: The cytotoxicity of Etoposide was tested in three cell lines using a Cell Counting Kit-8 (CCK-8, Beyotime).

Techniques: Binding Assay, Generated, Fluorescence, Förster Resonance Energy Transfer, Mutagenesis

Journal: Theranostics

Article Title: Ultra-small quercetin-based nanotherapeutics ameliorate acute liver failure by combatting inflammation/cellular senescence cycle

doi: 10.7150/thno.103746

Figure Lengend Snippet: Cellular uptake and intracellular distribution of QFN. Representative images (A) and flow cytometry analysis (B) of AML12 cells incubated with different concentrations of QFN-Cy5.5 for 6 h. Scale bar = 10μm. (C) Percentage of Cy5.5-positive AML12 cells from (B). Representative images (D) and flow cytometry analysis (E) of AML12 cells incubated with 20 μg/mL QFN-Cy5.5 for different periods. Scale bar = 10 μm. (F) Percentage of Cy5.5-positive AML12 cells from (E). (G-I) AML12 cells were cooled to 4 °C or separately pretreated with endocytosis-related inhibitors at 37 °C for 1 h, followed by incubation with 20 μg/mL QFN-Cy5.5 for 6 h. Ami, amiloride; CPZ, chlorpromazine; MβCD, methyl-β-cyclodextrin. Fluorescent imaging (G) and flow cytometry analysis (H) of QFN-Cy5.5 in AML12 cells. Scale bar = 100 μm. (I) Percentage of Cy5.5-positive AML12 cells from (H). (J-O) AML12 cells were incubated with 20 μg/mL QFN-Cy5.5 for 6 h. Lysosomes and mitochondria were labeled with Lyso-Tracker Green and Mito-Tracker Green, respectively. Images were captured using a confocal microscope. Co-localization of QFN-Cy5.5 with lysosomes (J) and with mitochondria (K) . Scale bar = 20 μm. Pearson's correlation coefficient (PCC) and Mander's correlation coefficient (MCC) analyses of QFN-Cy5.5 with lysosomes (L) or mitochondria (N) , respectively. Plot profile analysis of QFN-Cy5.5 co-localization with Lyso-Tracker (M) or Mito-Tracker (O) , respectively. Data are presented as mean ± SEM, *P < 0.05, ****P < 0.0001 (one-way ANOVA test with Tukey's multiple comparisons test).

Article Snippet: AML12 cells were treated with 4 μM etoposide (ETO, HY-13629, MedChemExpress, USA) for 24 h, followed by incubation in ETO-free complete conditioned media for an additional 24 h to induce cellular senescence.

Techniques: Flow Cytometry, Incubation, Imaging, Labeling, Microscopy

Journal: Theranostics

Article Title: Ultra-small quercetin-based nanotherapeutics ameliorate acute liver failure by combatting inflammation/cellular senescence cycle

doi: 10.7150/thno.103746

Figure Lengend Snippet: The anti-senescent and hepatocyte protective effects of QFN in vitro . (A-B) Cell viability of AML12 cells (A) and HepG2 cells (B) after incubation with different concentrations of QFN for 24 h and 48 h. (C) Cell viability of AML12 cells after exposure to indicated concentrations of H 2 O 2 for 2 h. (D) Cell viability of AML12 cells after exposure to 200 μM H 2 O 2 for 2 h and incubation with different amounts of QFN. (E-H) AML12 cells were treated with 4 μM etoposide (ETO) for 24 h and then cultured in an ETO-free medium for another 24 h to induce senescence. Quercetin and QFN were added during the ETO intervention until the cells were collected. Immunofluorescent staining of γH2AX (E) and P21 (F) in AML12 cells. Scale bar = 100 μm. (G) Immunoblot analysis of the protein levels of γH2AX, P53, and Lamin B1. The experiment was repeated three times. (H) Gene expression of Cdkn1a and Cdkn2a (n = 3). Representative images (I) and flow cytometry analysis (J) of JC-1 in AML12 cells after being stimulated with 100 μM H 2 O 2 for 2 h which was pretreated with quercetin and QFN for 6 h. Scale bar = 200 μm. Data are presented as means ± SEM (ns: not significant, ***P < 0.001, ****P < 0.0001, one-way ANOVA test with Tukey's multiple comparisons test).

Article Snippet: AML12 cells were treated with 4 μM etoposide (ETO, HY-13629, MedChemExpress, USA) for 24 h, followed by incubation in ETO-free complete conditioned media for an additional 24 h to induce cellular senescence.

Techniques: In Vitro, Incubation, Cell Culture, Staining, Western Blot, Expressing, Flow Cytometry