Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: The crucial role of LSH in ATT-induced ferroptosis in HCT-116. HCT-116 cells were pre-treated with siLSH (A) or Flag-LSH (B) for 24 h and then treated with or without ATT (10 μM) for 24 h. Cytotoxicity of ATT in cells was tested. Flow cytometry was performed to evaluate oxidation-dependent DCF fluorescence (C) , C11-BODIPY-detected lipid peroxidation (D) , and intracellular Ca 2+ levels (E) in CRC cells with LSH knockdown after ATT treatment. HCT-116 cells were pre-treated with Flag-LSH for 24 h and then treated with or without ATT (10 μM) for 24 h. Flow cytometry was used to analyze the effects of ATT on intracellular oxidative activity (DCFH-DA) (F) , lipid peroxidation (C11-BODIPY) (G) , and calcium levels (H) in CRC cells with LSH overexpression. Effects of ATT on target protein LSH and downstream protein CYP24A1 upon LSH knockdown (I) and LSH overexpression (J) . Data are expressed as mean ± SD (n = 3). Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test. P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: Flow Cytometry, Fluorescence, Knockdown, Activity Assay, Over Expression, Labeling

Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: Identification of LSH-interacting proteins perturbed by ATT. HCT-116 cells were treated with ATT (10 μM) for 24 h. (A) LC-MS/MS analysis of LSH-immunoprecipitated proteins in HCT-116 cells (left). Representative peptide counts and molecular weights of indicated proteins are shown (right). (B) Co-immunoprecipitation (Co-IP) analysis of the interaction between LSH and EWSR1 in CRC cells. (C) Immunofluorescence co-localization analysis of EWSR1 and LSH in CRC cells. Scale bar: 5 μm. (D) HCT-116 cells were pre-treated with Flag-LSH for 24 h and then treated with or without ATT (10 μM) for 24 h. Co-IP analysis of interactions between Flag-tagged wild-type LSH, mutant, and EWSR1. (E) HCT-116 cells were treated with ATT (0, 5, 10, 20 μM) for 24 h. Protein levels of EWSR1 in CRC cells were determined. (F) HCT-116 cells were treated with siEWSR1 for 48 h. Effect of EWSR1 knockdown on HCT-116 cell viability. Influence of EWSR1 knockdown on lipid peroxidation (C11-BODIPY) (G) and intracellular calcium levels (H). Statistical significance was assessed using one-way ANOVA followed by Dunnett's post hoc test. Impact of EWSR1 knockdown on (I) RNA and (J) protein expression of LSH and CYP24A1. Statistical significance was assessed using two-way ANOVA followed by Dunnett's post hoc test. (K) HCT-116 cells were transfected with siEWSR1 for 48 h, and a dual-luciferase assay was performed to examine its effect on the promoter activity of CYP24A1 mediated by exogenous LSH. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test. Data are expressed as mean ± SD (n = 3). P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Mutagenesis, Knockdown, Expressing, Transfection, Luciferase, Activity Assay, Labeling

Journal: Redox Biology

Article Title: Artemisitene triggers calcium-dependent ferroptosis by disrupting the LSH-EWSR1 interaction in colorectal cancer

doi: 10.1016/j.redox.2025.103950

Figure Lengend Snippet: The effect of ATT on CRC progression in vivo . (A) Representative images of tumor samples from each group (n = 6). (B) Measurement of tumor volume and (C) weight (n = 6). (D) HE staining of tumor tissues (Scale bar: 100 μm) and IHC detection of Ki-67, CYP24A1, SCD, and LSH expression in tumor tissues (Scale bar: 50 μm). (E) Protein expression levels of CYP24A1, SCD, and LSH in tumor tissues were determined. (F) Impact of ATT on the interaction of LSH and EWSR1 in vivo was assayed by Co-IP assay. Statistical significance was assessed using one-way ANOVA followed by Dunnett's post hoc test. P -values are labeled directly in figure. P < 0.05 was considered statistically significant, and P ≥ 0.05 was considered non-significant.

Article Snippet: The antibodies used are as follows: LSH (Santa Cruz, sc-46665, USA), EWSR1 (ABclonal, A9640, China), CYP24A1 (Origene, TA368684S, USA), and SCD (ABclonal, A26246, China).

Techniques: In Vivo, Staining, Expressing, Co-Immunoprecipitation Assay, Labeling

Journal: Genome Biology

Article Title: HELLS is required for maintaining proper DNA modification at human satellite repeats

doi: 10.1186/s13059-025-03681-9

Figure Lengend Snippet: Human satellite repeats are key targets for HELLS activity. A Protein structure of HELLS including known functional domains and our guide RNA target sites. Below is the raw Sanger sequencing result of the homozygous HELLS KO iPSC clone (ZIP8K8, clone #B3) with a 31-bp deletion and the western blot verification. B Violin plots showing mean methylation over 1 kb tiles of whole genome bisulfite sequencing (WGBS) data generated for WT, the KO clones (HELLS: ZIP8K8, clone #B3, DNMT3B: ZIP34K14, clone #C1, and DNMT3A/B: ZIP34K14, clone #C3). Plots show median (horizontal line) and 25% and 75% quantiles (stronger and weaker vertical lines, respectively). n = 2,488,423 tiles. C Boxplots showing mean methylation over genomic features for WT and each of the knockout clones (HELLS, DNMT3B, and DNMT3A/B). The horizontal bar shows the median per feature, and boxes and whiskers reflect the quartiles. From left to right: n represents the number of regions included for calculating the DNA methylation distribution for each feature, with values of 16,201; 23,263; 1,411,943; 2,184. D Representative IGV browser tracks of the centromeric regions of three different chromosomes (1, 10, and 16) showing WGBS data for WT and the knockout clones (HELLS, DNMT3B, and DNMT3A/B) with satellite repeat class annotation (light gray), highlighting active alpha satellites (red), classical human satellite II (blue), and beta satellites (pink). E Heatmap visualizing the DNA methylation levels of the CenSat classes for WT and each of the knockout clones (HELLS, DNMT3B, and DNMT3A/B). The CenSat classes include: ribosomal DNA (rDNA), other centromeric satellites (censat), centromeric transition region (ct), monomeric alpha satellites (mon), beta satellites (bsat), classical human satellite III (hsat3), classical human satellite I type A (hsat1A), active alpha satellite (act. hor), classical human satellite I type B (hsat1B), divergent alpha satellite (dhor), inactive alpha satellite (incat. hor), gamma satellites (gsat), and classical human satellite II (hsat2). F Cumulative distribution plot of the delta DNA methylation between WT and the HELLS KO. The y -axis shows the cumulative fraction, representing the proportion of data points that are less than or equal to the corresponding value on the x -axis. G Split violin plots showing mean methylation over 1 kb tiles of WGBS generated for arrested (left half) and proliferating (right half) WT and HELLS KO. Plots show median (horizontal line) and 25% and 75% quantiles (stronger and weaker vertical lines, respectively). n = 2,488,423 tiles. H IGV browser track of chromosome 1 showing the change in DNA methylation between cycling as well as arrested WT and HELLS KO iPSCs. I Representative IGV browser tracks of loci across different chromosomes showing ATAC-seq for WT and HELLS KO and the change in DNA methylation between WT and HELLS KO. J Change in DNA methylation between arrested HELLS KO and WT cells as a function of change in DNA methylation between proliferating HELLS KO and WT cells

Article Snippet: Western blots were performed using an anti-HELLS antibody (Santa Cruz, #sc-46665) at a 1:2000 dilution, an anti-DNMT3A antibody (Abcam, #ab188470) at a 1:2000 dilution, an anti-DNMT3B antibody (Cell Signaling Technologies, #67259) at a 1:1000 dilution, and an anti-GAPDH antibody (Cell Signaling #2118S).

Techniques: Activity Assay, Functional Assay, Sequencing, Western Blot, Methylation, Methylation Sequencing, Generated, Clone Assay, Knock-Out, DNA Methylation Assay

Journal: Genome Biology

Article Title: HELLS is required for maintaining proper DNA modification at human satellite repeats

doi: 10.1186/s13059-025-03681-9

Figure Lengend Snippet: Enhancer elements do not require HELLS for differentiation-associated chromatin dynamics and DNA methylation regulation. A Directed differentiation of WT and HELLS KO iPSCs into endoderm (day 5, D5) imaged by brightfield at 40 ×. The white scale bar reflects 25 µm. B Smooth scatter plots comparing WT and HELLS KO delta iPSC and endoderm ATAC-seq signal (left panel) and CpG methylation (right panel) across all identified ATAC-seq peaks. The color scale indicates the density of CpGs with blue representing higher density. C Representative IGV browser tracks of a locus highlighting one static and one dynamic region. Tracks show DNA methylation and ATAC-seq signal of undifferentiated iPSCs and differentiated endoderm cells (WT and HELLS KO). D Smooth scatter plots comparing delta iPSC and endoderm ATAC-seq with delta iPSC and endoderm CpG methylation for WT (left panel) and HELLS KO (right panel) across all identified ATAC-seq peaks. The color scale indicates the density of CpGs with blue representing higher density. E PCA of ENCODE-rE2G enhancer predictions in WT and HELLS KO iPSCs and endoderm (Diff.). F Heatmap visualizing the DNA methylation levels of putative somatic regulatory elements for WT and each of the knockout clones (HELLS, DNMT3B, and DNMT3A/B iPSCs), as well as WT and HELLS KO endoderm

Article Snippet: Western blots were performed using an anti-HELLS antibody (Santa Cruz, #sc-46665) at a 1:2000 dilution, an anti-DNMT3A antibody (Abcam, #ab188470) at a 1:2000 dilution, an anti-DNMT3B antibody (Cell Signaling Technologies, #67259) at a 1:1000 dilution, and an anti-GAPDH antibody (Cell Signaling #2118S).

Techniques: DNA Methylation Assay, CpG Methylation Assay, Knock-Out, Clone Assay