Journal: iScience

Article Title: Confined migration induces heterochromatin formation and alters chromatin accessibility

doi: 10.1016/j.isci.2022.104978

Figure Lengend Snippet: Confined migration decreases intergenic chromatin accessibility near centromeres and telomeres, and global transcription (A) Distribution of the distance between each “down” peak and TSS of its associated genes, calculated by GREAT annotated region-gene associations. Y -axis: the fraction of all associated genes. X -axis: binned distances to TSS in kb. The number on the top of each bar: the number of peak-associated genes with distances in that bin. (B) Genome browser shots of three representative genes associated with “down” peaks, (i) TERT , (ii) COL23A1 , and (iii) MUC2 ATAC-seq signals in low-, medium-, and high-concentration collagen samples. Red arrows: ATAC-seq peaks that are negatively correlated with increasing collagen concentrations. (C) The percentage of “up” and “down” peaks located within 3 Mb from centromeres or telomeres. ∗∗∗ p < 0.001, two-way ANOVA with Tukey’s multiple comparison test. (D) Representative karyoplot of DA peaks on chromosome 12. Black lines: locations of “up” peaks (top) and “down” peaks (bottom). Red areas: centromeres. Green areas: telomeres. Blue arrows: clustering of “down” peaks near centromeres and telomeres. Scale bar: 20 Mb. (E) Left panel: representative images of pSer2-RNAPII (red) and DAPI (blue) in HT1080 cells during confined migration. Scale bar: 10 μm. Right panel: quantification of pSer2-RNAPII intensities in cells migrating through control or confined channels. All values are normalized to control channels “before” cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-way ANOVA with Tukey’s multiple comparison test. (F) Left panel: representative images of 5-EU (green) and DAPI (blue) in HT1080 cells during confined migration. Scale bar: 10 μm. Right panel: quantification of 5-EU intensities in cells migrating through control or confined channels. All values are normalized to control channels “before” cells. ∗∗∗ p < 0.001, two-way ANOVA with Tukey’s multiple comparison test. Data are presented as the mean ± SEM, based on n DA peaks or n cells (listed in each graph) pooled from three biological replicates, except for data in (E) that are based on two biological replicates. Please see also Figure S7 .

Article Snippet: Cells migrating in microfluidic devices were pulsed with 1 mM 5-ethynyl uridine (EU, Jena Bioscience) in complete DMEM for 4 h, and then fixed with 4% PFA in PBS for 30 min. After two PBS washes, the devices were carefully removed as described in the IF staining section.

Techniques: Migration, Concentration Assay, Comparison, Control

Journal: Advanced Science

Article Title: Dipyridamole Acts as Clinical Ferroptosis Inhibitor to Prevent from Tissue Injury

doi: 10.1002/advs.202500566

Figure Lengend Snippet: Identification of candidate clinical ferroptosis inhibitors. a) Schematic of identification of potential ferroptosis inhibitors for disease treatment, using HT1080 cells pretreated with clinical drugs (10 µ m ) followed by RSL3 treatment for 24 h. b) Volcano plots showing the top hits of clinical drug screen in HT1080 cells. c,d) Dose‐dependent toxicity of RSL3‐induced cell death of HT1080 (c) and A375 cells (d) after pre‐treatment with DMSO, dipyridamole (10 µ m ), or MF (10 µ m ) for 12 h. Cell viability was measured 12 h post‐treatment after using CCK8. e) Dose‐dependent toxicity of RSL3‐induced cell death in OVCAR8 cells after pre‐treatment with dipyridamole (10 and 20 µ m ) for 12 h. Cell viability was assessed 12 h post‐treatment after using CCK8. f,g) Cell death measurement of HT1080 (f) and OVCAR8 (g) cells treated with RSL3 (500 n m ), dipyridamole (10 µ m ), Fer‐1 (4 µ m ) for 6 h. Dead cells were labeled with SYTOX Green. h) Cell death measurement of HT1080 cells treated with RSL3 (500 n m ), MF (10 µ m ) and Lipro‐1(4 µ m ) for 6 h. Dead cells were labeled with SYTOX Green. i,j) BODIPY 581/591 C11 staining of lipid peroxidation in HT1080 cells treated with RSL3 (250 n m ), Lipro‐1(4 µ m ) or dipyridamole (10 µ m ) for 4 h. Data and error bars are mean ± SEM, n = 3 biologically independent experiments in b–h and j. All P values were calculated using a two‐tailed, unpaired Student's t ‐test.

Article Snippet: HT1080 DHODH KD cells need Uridine (TargetMol, T2221, 50 μ m ) supplementation daily to culture.

Techniques: Labeling, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Dipyridamole Acts as Clinical Ferroptosis Inhibitor to Prevent from Tissue Injury

doi: 10.1002/advs.202500566

Figure Lengend Snippet: Dipyridamole‐mediated ferroptosis depends on SLC7A11. a,b) Immunoblot assays of classical ferroptosis‐related genes in HT1080 (a) and H9c2 (b) cells with dipyridamole (20 µ m ) treatment at indicated times. c‐e) Immunoblot assays of SLC7A11 expression in HT1080 (c), OVCAR8 (d), and 786‐O (e) cells with dipyridamole (20 µ m ) treatment at indicated times. f) Immunoblot assays of SLC7A11 expression in HT1080 WT and SLC7A11 KO cells. g) Cell viability assay in HT1080 WT and SLC7A11 KO cells treated with DMSO, dipyridamole (20 µ m ), or RSL3 at the indicated concentration. h) The GSH levels in HT1080 cells treated with RSL3 (250 n m ), Fer‐1(4 µ m ), and dipyridamole (10 µ m ) for 4 h. i) Immunoblot assays of exogenous SLC7A11 expression in HT1080 SLC7A11 KO cells stably expressing with the Mock and SLC7A11‐Flag vector. j) Cell viability assay in HT1080 SLC7A11 KO cells stably expressing with the Mock and SLC7A11‐Flag vector treated with DMSO, RSL3 (5 µ m ), and dipyridamole (10 µ m ) at the indicated concentration. k) The cell death of HT1080 SLC7A11 KO cells stably expressing the Mock and SLC7A11‐Flag vector was measured. These cells were treated with DMSO, RSL3 (5 µ m ), or dipyridamole (10 µ m ) for 6 h. Dead cells were labeled with SYTOX™ Green. Data and error bars are mean ± SEM, n = 3 biologically independent experiments in g, h, j, and k. All P values were calculated using a two‐tailed, unpaired Student's t‐test.

Article Snippet: HT1080 DHODH KD cells need Uridine (TargetMol, T2221, 50 μ m ) supplementation daily to culture.

Techniques: Western Blot, Expressing, Viability Assay, Concentration Assay, Stable Transfection, Plasmid Preparation, Labeling, Two Tailed Test

Journal: Advanced Science

Article Title: Dipyridamole Acts as Clinical Ferroptosis Inhibitor to Prevent from Tissue Injury

doi: 10.1002/advs.202500566

Figure Lengend Snippet: Dipyridamole stabilizes SLC7A11 via regulation of RNF126. a) Immunoblot assays were conducted for SLC7A11 in HT1080 cells which were treated with dipyridamole (20 µ m ) and CHX (20 mg mL −1 ) for the indicated times (shown in the up ). Then, the densitometry quantification of SLC7A11 protein levels was calculated by using ImageJ software (NIH), and the results were plotted in the bottom panel for the determination of the half‐life. The statistical results are normalized with the data at 0 h in each group. b) Immunoprecipitation assays of exogenous SLC7A11‐Flag and HA‐ubiquitin in HEK 293T cells. c) Pattern diagram of mass spectrometry in HEK 293T cells transfected with SLC7A11‐Flag with or without dipyridamole (10 µ m ) treatment. Anti FLAG M2 affinity gel was used for immunoprecipitation. d) Immunoblot assays of endogenous SLC7A11 in HT1080 cells transfected with the Mock, RBX1‐HA or RNF126‐HA vector. e) Immunoblot assays of endogenous RNF126 and exogenous SLC7A11 in HEK 293T cells with dipyridamole (20 µ m ) treatment at 0, 6, and 12 h. f) Immunoblot assays of co‐immunoprecipitation of exogenous RNF126‐HA and SLC7A11‐Flag in HEK 293T cells. g) Immunoblot assays of exogenous RNF126 and SLC7A11 in HEK 293T cells with dipyridamole (20 µ m ) treatment for 16 h. h) Immunoblot assays of exogenous RNF126‐HA in HT1080 cells transfected with the Mock and RNF126‐HA vector. i) RSL3 (500 n m )‐induced cell death of HT1080 transfected with the Mock and RNF126‐HA vector. Cell viability was assessed 12 h post‐treatment after using CCK8. j) Immunoblot assays of endogenous RNF126‐HA in HT1080 cells infected with the sh Ctrl and sh RNF126 virus. k) Dose‐dependent toxicity of RSL3‐induced cell death of HT1080 sh Ctrl and sh RNF126 cells for 12 h. Cell viability was assessed 12 h post‐treatment after using CCK8. Data and error bars are mean ± SEM, n = 3 biologically independent experiments in i and k. P values were calculated using a two‐tailed, unpaired Student's t‐test. Grayscale analysis of the images was performed using ImageJ software.

Article Snippet: HT1080 DHODH KD cells need Uridine (TargetMol, T2221, 50 μ m ) supplementation daily to culture.

Techniques: Western Blot, Software, Immunoprecipitation, Ubiquitin Proteomics, Mass Spectrometry, Transfection, Plasmid Preparation, Infection, Virus, Two Tailed Test