Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A , B The viability of ES cells, specifically the A673 and RDES cell lines, was assessed following treatment with the top 10 anticancer compounds. C Bone marrow stem cells (BMSC) served as a normal control group. Both ES and normal BMSC cells were exposed to UNC0379 at the indicated concentration for 48 h, after which cell viability was measured. D The colony formation assay demonstrated a significant reduction in the ability of A673, RDES, and SKNMC cells to form colonies after treatment with UNC0379 at 2 μM, as compared to the DMSO control group. E The impact of SETD8 knockdown using short hairpin RNA (shRNA) on cell proliferation was evaluated through a colony formation assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Control, Concentration Assay, Colony Assay, Knockdown, shRNA

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A WB analysis was conducted to assess the expression levels of the SETD8 protein in BMSC and across four ES cell lines. The original blots corresponding to these experiments are displayed in Fig. . B IHC provided representative images of SETD8 protein expression in ES patient samples at a magnification of ×200. C , D Kaplan–Meier plots depicted the overall survival (OS) and event-free survival (EFS) for ES patients with high versus low SETD8 expression levels. E The impact of UNC0379 (2 μM) on the clonogenic capacity of ES cells, in the context of various cell death inhibitors (10 μM Z-VAD-FMK, necrostatin-1, Ferrostatin-1, deferoxamine, 5 mM 3-Methyladenine and 200 μM Methyladenine), was evaluated through a colony formation assay. F The viability of A673 and RDES cells following 48 h of exposure to UNC0379 (2 μM) with or without the aforementioned cell death inhibitors was measured using the CCK-8 assay. G A column chart illustrated the variation in apoptosis rates among the ES cell lines following SETD8 inhibition by UNC0379. H The relative levels of intracellular iron (Fe2+) in ES cells were measured after SETD8 was inhibited by UNC0379. I The levels of reactive oxygen species (ROS) within ES cells were determined post-SETD8 inhibition by UNC0379. J The lipid ROS levels in ES cells were assessed after SETD8 inhibition by UNC0379. Data are presented as the mean ± standard deviation from three separate experiments. ‘ns’ denotes no statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Expressing, Colony Assay, CCK-8 Assay, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A The clonogenic potential of SETD8 knockdown in ES cells was evaluated in the presence or absence of specific cell death inhibitors, including 10 μM Z-VAD-FMK, and 10 μM Ferrostatin-1, using a colony formation assay. B A673 and RDES cells underwent SETD8 knockdown and were treated with the aforementioned inhibitors for 48 h, after which their viability was measured using the CCK-8 assay. C A column chart displayed the shifts in apoptosis rates among the ES cell lines subsequent to SETD8 knockdown. D The levels of intracellular ROS in ES cells were quantified following SETD8 knockdown. E Intracellular lipid ROS levels were similarly measured in ES cells post-SETD8 knockdown. F Relative changes in intracellular Fe2+ levels were observed in ES cells after SETD8 knockdown. The data are expressed as the mean ± standard deviation derived from three individual experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Knockdown, Colony Assay, CCK-8 Assay, Standard Deviation, Derivative Assay

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A , B The KEGG pathway analysis enriched by DEGs from A673 and RDES after SETD8 suppression by siRNA. C , D mRNA expression levels of DEGs in the MAPK pathway validated in ES cells after SETD8 genetic knockdown by siRNA. E Results of WB analysis to confirm the protein expression changes of RAC3, ERK1/2, and p-ERK1/2 in the MAPK pathway after SETD8 inhibition. The original blots corresponding to these experiments are displayed in Fig. . F Results of WB analysis to confirm the protein expression changes of ERK1/2 and p-ERK1/2 after RAC3 knockdown. The original blots corresponding to these experiments are displayed in Fig. . G Colony formation assay after RAC3 knockdown in ES cells. H Colony formation assay determined the reproductive ability of ES cells with RAC3 knockdown in the absence or presence of indicated cell death inhibitors (10 µM Z-VAD-FMK and 10 µM Ferrostatin-1). I A673 and RDES cells with RAC3 knockdown in the absence or presence of indicated cell death inhibitors (10 µM Z-VAD-FMK and 10 µM Ferrostatin-1) for 48 h, cell viability was assayed with CCK-8. J Changes of apoptosis rates after RAC3 inhibition in ES cells. K Changes of the intracellular ROS levels after RAC3 inhibition in ES cells. L Changes of the intracellular lipid ROS levels after RAC3 inhibition in ES cells. M Changes of the relative intracellular Fe2+ levels in ES cells after RAC3 inhibition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Expressing, Knockdown, Inhibition, Colony Assay, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A Silver staining demonstrated co-immunoprecipitation using an antibody against SETD8 in A673 cells. B Mass spectrometry data confirmed the physical association between SETD8 and YBX1 proteins. C Immunoprecipitation results validated the binding interaction between SETD8 and YBX1 in ES cells. The original blots corresponding to these experiments are displayed in Fig. . D WB analysis detected changes in the expression levels of phosphorylated YBX1 at serine102 (p-Ser102 YBX1) and total YBX1 in ES cell lines following SETD8 knockdown. The original blots corresponding to these experiments are displayed in Fig. . E Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed alterations in YBX1 mRNA levels in ES cell lines post-SETD8 knockdown. F The clonogenic capacity of ES cells post YBX1 knockdown was illustrated through a colony formation assay. G The reproductive potential of YBX1 knockdown in ES cells was evaluated in the context of specific cell death inhibitors (10 μM Z-VAD-FMK and Ferrostatin-1) using a colony formation assay. H A column chart, based on CCK-8 assay results, depicted cell viability following YBX1 knockdown with or without the specified cell death inhibitors for 48 h. I A column chart showed the shifts in apoptosis rates across ES cell lines after YBX1 was suppressed using siRNA. J Relative changes in intracellular Fe2+ levels were observed in ES cells after YBX1 inhibition. K Intracellular ROS levels were measured in ES cells following YBX1 inhibition. L Intracellular lipid ROS levels were assessed in ES cells after YBX1 inhibition. Data are presented as mean ± standard deviation from three separate experiments. ‘ns’ signifies no statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Silver Staining, Immunoprecipitation, Mass Spectrometry, Binding Assay, Expressing, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Colony Assay, CCK-8 Assay, Inhibition, Standard Deviation

Journal: Cell Death & Disease

Article Title: SETD8 inhibits apoptosis and ferroptosis of Ewing’s sarcoma through YBX1/RAC3 axis

doi: 10.1038/s41419-024-06882-5

Figure Lengend Snippet: A qRT-PCR analysis verified the expression of RAC3 mRNA subsequent to the inhibition of YBX1. B ChIP-PCR confirmed the presence of YBX1 bound to the RAC3 promoter region in ES cells. C ChIP-qPCR was utilized to assess the alterations in YBX1 binding at the RAC3 promoter in A673 and RDES cells with or without SETD8 knockdown. D , E WB analysis determined the distribution of YBX1 in the cytoplasmic and nuclear fractions after SETD8 knockdown for a duration of 48 h. LaminB1 served as a nuclear fraction loading control, while GAPDH was used as a cytoplasmic fraction loading control. The original blots corresponding to these experiments are displayed in Fig. . F Immunofluorescence (IF) assays revealed the cellular localization of the YBX1 protein in ES cells following SETD8 knockdown for 48 h. Data are presented as mean ± standard deviation from three separate experiments. * p < 0.05, ** p < 0.01.

Article Snippet: The RDES and SKNMC human ES cell lines, along with BMSC, were procured from Jennio Biotech Co., Ltd. Additionally, the A673 and SK-NEP-1 human ES cell lines were sourced from Procell, Wuhan, China.

Techniques: Quantitative RT-PCR, Expressing, Inhibition, Binding Assay, Knockdown, Control, Immunofluorescence, Standard Deviation

Journal: Nature Communications

Article Title: Prion-like domain mediated phase separation of ARID1A promotes oncogenic potential of Ewing’s sarcoma

doi: 10.1038/s41467-024-51050-0

Figure Lengend Snippet: a Expression levels of ARID1A protein in different types of cancer obtained from the cancer cell line encyclopedia. b Representative immunoblot image measuring ARID1A protein levels in various cancer cell lines. The quantification represents ARID1A/β-actin protein density ratio. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. c Immunohistochemistry results showing ARID1A staining in normal bone tissue and two Ewing’s sarcoma patient tissues. Scale bars: 10 μm. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. d Immnuocytochemistry image illustrating endogenous ARID1A localization in WT, ARID1A −/− , ARID1A −/− + WT and ARID1A −/− + ΔDD cells. Scale bars: 5 μm. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. e Left: wound healing assay conducted on WT, ARID1A −/− , ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines. Right: quantification of the wound healing assay. Bars represent the SEM; ** p < 0.01, *** p < 0.001, NS non-significant. n = 10 technical replicate of wound closures. Statistical analysis performed using a two-tailed Wilcoxon signed rank test on 48 h samples of ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines. Scale bar: 500 μm. f Left: spheroid formation assay performed for four cell lines over 4 days. Right: quantification of the spheroid formation assay. Bars represents the mean ± SEM; ** p < 0.01, *** p < 0.001, NS non-significant. n = 10 technical replicates of spheroids. Statistical analysis performed using a two-tailed Wilcoxon signed rank test on day 4 samples of ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines. Scale bar: 500 μm. g Left: spheroid invasion assay conducted on four cell lines over 2 days. Right: quantification of the spheroid invasion assay. Bars represents the mean ± SEM; ** p < 0.01, *** p < 0.001, NS non-significant. n = 10 technical replicates of spheroids. Statistical analysis performed using a two-tailed Wilcoxon signed rank test on day 4 samples of ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines. Scale bar: 500 μm. h Left: in vivo xenograft assay performed using four cell lines. Nude mice and extracted tumors are shown. Top right: quantification of the volume of the extracted tumors. Bottom right: quantification of the weight of the extracted tumors. Bars represents the mean ± SEM; ** p < 0.01, *** p < 0.001, NS non-significant. n = 10 tumor extracts. Statistical analysis performed using a two-tailed Wilcoxon signed rank test. ARID1A −/− , ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines were individually compared to WT. i Representative immunohistochemistry images of extracted tumors formed by the four cell lines. Immunostaining was performed using an anti-ARID1A antibody. Scale bars: 10 μm. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. Source data are provided as a Source Data file.

Article Snippet: The A673, SK-N-MC cell lines were purchased from the Korean Cell Line Bank.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining, Wound Healing Assay, Two Tailed Test, Tube Formation Assay, Invasion Assay, In Vivo, Xenograft Assay, Immunostaining

Journal: Nature Communications

Article Title: Prion-like domain mediated phase separation of ARID1A promotes oncogenic potential of Ewing’s sarcoma

doi: 10.1038/s41467-024-51050-0

Figure Lengend Snippet: a A heatmap illustrating expression of DEGs (FDR < 0.05) obtained from the RNA-seq results of WT, ARID1A −/− , five ARID1A −/− + WT, five ARID1A −/− + ΔDD, and ARID1A −/− + PrLD(Y/S) A673 cell lines. The colors indicate normalized gene expression. The dendrogram above the heatmap indicates the hierarchical clustering result of the samples. b Tornado plots illustrating ±800 bp regions from each dysregulated cREs (FDR < 0.05) obtained from ATAC-seq of WT, ARID1A −/− , five ARID1A −/− + WT, five ARID1A − / − + ΔDD, and ARID1A −/− + PrLD(Y/S) A673 cell lines. The colors indicate normalized read counts (left, red) and log2 (LLPS-positive/LLPS-negative) read counts (right, yellow, and cyan). c Top 10 enriched gene ontologies in ARID1A LLPS-dependent upregulated DEGs. The cancer-related terms are marked with an asterisk. d The rank of transcription factor motifs overrepresented in the ARID1A LLPS-dependent upregulated cREs. The top two enriched motifs are highlighted. e Tornado plots illustrating published A673 EWS/FLI1 ChIP-seq signal on the ARID1A LLPS-dependent upregulated cREs, ARID1A LLPS-dependent downregulated cREs, and other randomly selected cREs, respectively. The colors indicate normalized EWS/FLI1 ChIP-seq signal over the input signal.

Article Snippet: The A673, SK-N-MC cell lines were purchased from the Korean Cell Line Bank.

Techniques: Expressing, RNA Sequencing, Gene Expression, ChIP-sequencing

Journal: Nature Communications

Article Title: Prion-like domain mediated phase separation of ARID1A promotes oncogenic potential of Ewing’s sarcoma

doi: 10.1038/s41467-024-51050-0

Figure Lengend Snippet: a A heatmap showing significantly ( P value < 0.05) altered long-range chromatin contacts between the DEGs and the dysregulated cREs in an ARID1A LLPS-dependent manner. b A barplot illustrating the number of linkages between upregulated DEGs and upregulated, downregulated, and control cREs, respectively. c Left: a tornado plot illustrating published A673 EWS/FLI1 ChIP-seq signal on the upregulated cREs connecting to ARID1A LLPS-dependent upregulated genes. The colors indicate normalized EWS/FLI1 ChIP-seq signal over the input signal. Middle and right: a heatmap illustrating the upregulated cREs (middle) and the upregulated genes connected to the upregulated cREs (right). The colors indicate normalized read count in the regions and normalized gene expression, respectively. The dashed line indicates linkages between EWS/FLI1-bound upregulated cREs and the upregulated genes. d The normalized Hi-C contact frequencies around the TFAP2B gene promoter are illustrated as a virtual 4C plot. The genome tracks of ATAC-seq and published EWS/FLI1 ChIP-seq signal are shown below. The dashed vertical line indicates the viewpoint of the 4C plot and the asterisk indicates the transcription start site of the TFAP2B gene. The shaded regions highlight the linkages between the TFAP2B gene and the EWS/FLI1-bound upregulated cREs via the proximal colocalization or the altered long-range chromatin contacts. e Odds ratio that an activated gene is included in the cancer-related GO terms shown in Fig. , comparing the genes linked to the upregulated cREs versus unlinked genes ( P values for the enrichment of the linked genes versus the unlinked genes: migration = 0.034, cell adhesion = 0.038, two-sided Fisher’s exact test). f A heatmap comparing normalized Hi-C contact frequencies of ARID1A LLPS-positive, negative, and published EWS/FLI1 knockdown (KD) A673 cells, respectively. Only the contacts between EWS/FLI1-bound upregulated cREs and their linked upregulated genes are shown.

Article Snippet: The A673, SK-N-MC cell lines were purchased from the Korean Cell Line Bank.

Techniques: Control, ChIP-sequencing, Gene Expression, Hi-C, Migration, Knockdown

Journal: Nature Communications

Article Title: Prion-like domain mediated phase separation of ARID1A promotes oncogenic potential of Ewing’s sarcoma

doi: 10.1038/s41467-024-51050-0

Figure Lengend Snippet: a Binding site mapping of FLAG-EWS/FLI1 and GFP-ARID1A recombinant proteins by co-immunoprecipitation assay. Tested proteins include PrLD1, ARID, PrLD2, Pfam, PrLD(Y/S) mutant, and full-length ARID1A. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. b Confocal image of an in vitro co-droplet assay demonstrating colocalization of purified GFP-ARID1A and mCherry-EWS/FLI1. Scale bars: 5 μm. The representative images supported by the relevant statistics have been chosen upon three independent preparations with similar outcomes. c Representative confocal images of ARID1A −/− + WT and ARID1A −/− + ΔDD A673 cell lines immunostained with anti-FLI1 and anti-ARID1A antibodies. Scale bars: 5 μm. d Quantification of number of FLI1 puncta per cell lines in ( c ). n = 32 technical replicates of cells; bars represents mean ± SEM; ** p < 0.01, *** p < 0.001, NS non-significant. Statistical analysis performed using a two-tailed t -test. ARID1A −/− , ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines were individually compared to WT. e ChIP assays performed on EWS/FLI1-bound enhancers in ARID1A −/− + WT and ARID1A −/− + ΔDD A673 cell lines using antibodies against IgG, FLI1, H3K27ac, and SMARCC1. Bars represents mean ± SEM; n = 3 technical replicates; ** p < 0.01, *** p < 0.001, NS non-significant. Statistics by two-tailed t -test using ARID1A −/− + WT, and ARID1A −/− + ΔDD A673 cell lines as comparison. Source data are provided as a Source Data file.

Article Snippet: The A673, SK-N-MC cell lines were purchased from the Korean Cell Line Bank.

Techniques: Binding Assay, Recombinant, Co-Immunoprecipitation Assay, Mutagenesis, In Vitro, Purification, Two Tailed Test, Comparison