ht1080 fibrosarcoma cells  (ATCC)

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: HT1080 cells stably expressing pHLuorin_M153R-CD63-mScarlet were seeded onto glass bottom MatTek plates and imaged live. Images were taken every 10 s.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques:

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: The indicated HT1080 cell types were induced to form spheroids and then mixed into 3D type I collagen. Spheroids were imaged every 30 min for 8 hr. Scale bar = 100 mm.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques:

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Representative confocal image of HT1080 cells stained with phalloidin-Alexa fluor 488 and CD63 shown in red. The red channel has been edited using brightness and contrast tools for ease of visibility. Note the localization of the exosome marker CD63 in extracellular deposits and at or near the tips of filopodia (arrowheads). Representative of 20 images. Scale bar is 10 mm in each panel. ( B ) Time series of pHluorin-M153R-CD63-mScarlet movie in HT1080 cells. Yellow arrowheads indicate fusion sites and yellow arrows indicate filopodia. Note a filopodium forming shortly after MVE fusion. ( C ) Representative kymographs showing MVE docking (red), fusion (yellow), and filopodia formation in HT1080 cells. Yellow arrowheads denote MVE fusion events, and black arrowheads denote the formation of a filopodium. Each pixel is 10 s x 0.2857 mm. ( D ) Quantification of the time elapsed between MVE fusion and filopodia formation. n=420 kymographs from 46 cells from three independent experiments (biological replicates). ( E ) Primary cortical neurons were co-transfected with GFP-Rab27b (green) and mCherry as a filler to visualize filopodia (red) on DIV 5 and fixed for imaging on DIV 6. SV2 negative staining (no signal) identifies these structures as filopodia instead of dendritic spines. Arrows in merged images indicate localization of GFP-Rab27b to tips and bases of filopodia. Scale bars = 5 µm. ( F ) Percent GFP-Rab27b localization to tips and bases of filopodia in 70 individual cortical neurons from three independent experiments (biological replicates). Red line indicates the median. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Staining, Marker, Transfection, Imaging, Negative Staining

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) WB of Rab27a KD in HT1080 cell lysates. ( B ) TEM of SEVs (purified by cushion DG) and LEVs from HT1080 cells. Scale bar = 200 nm in each image. ( C ) Secretion rates of SEVs from HT1080 cell lines (N=3). Nanoparticle tracking analysis traces of SEVs from shScr and shRab27a HT1080 cells showing size (diameter) distribution of SEVs and particles/mL/cell. ( D ) Representative images showing filopodia in control and Rab27a-KD H1080 cells. Images have been edited with brightness and contrast tools for ease of visibility. Scale bars in wide field and zoom insets = 10 mm. ( E ) Quantification of filopodia in control and Rab27a-KD HT1080 cell lines. ≥20 cells per condition per biological replicate, from three biological replicates. ( F ) Data from graph in E displayed as filopodia per cell. ( G ) Data in displayed as filopodia per cell. ( H ). Data from displayed as filopodia per cell. ( I ) Data from displayed as filopodia per cell. ( J ) Data from displayed as filopodia per cell. ( K ) Cell areas of cells used for quantification in . Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 2—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Purification, Control, Western Blot

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot of Endoglin KD in HT1080 cells. ( B ) Nanoparticle tracking analysis traces of SEVs purified from shScr and shEng HT1080 cells showing size distribution (diameter) of SEVs and particles/mL/cell (N=3 biological replicates). ( C ) SEV secretion rates of HT1080 shScr and shEng HT1080 cells. ( D ) Representative images of HT1080 shScr and shEng cells. Images have been edited with brightness and contrast for ease of visibility. Scale bar in wide field and zoom insets = 10 mm. ( E ) Quantitation of filopodia density for control and shEng HT1080 cells.≥20 cells per condition per biological replicate, from four biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Western Blot, Purification, Quantitation Assay, Control

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Cartoon diagram of metastatic colony assay in avian embryos. On day 0, fluorescent HT1080 cells were injected (100,000 cells per egg) into the vein of the chicken embryo. On day 4, the egg was opened, the embryo was sacrificed, and a circular tool was used to punch holes through the shell. The chorioallantoic membrane (CAM) was peeled away from the shell, placed on a glass slide with a coverslip, and immediately imaged. The cartoon was created using BioRender.com . ( B ) Representative low power wide field images of colony formation in the CAM. Scale bar = 200 mm. ( C ) Representative high-power wide field images of colony formation in the CAM. Scale bar = 100 mm. ( D, E ) Quantification of CAM colony number ( D ) and size ( E ) from high-power images as in C. 4–7 eggs were harvested per replicate for each condition for three biological replicates. ( D ) Colony number is graphed per field of view using 25–30 fields of view per egg. ( E ) Quantification of the percent of large (≥5000 mm 2 ) colonies formed by control and shEng HT1080 cells. ( F ) 3D invasion in collagen. HT1080 cell spheroids were seeded in collagen gels and imaged for 8 hr. Invasion is quantified as fold area increase in the size of each spheroid over 8 hr. Scale bar = 100 mm. Error bars, SEM. ns, not significant; *p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Colony Assay, Injection, Membrane, Control

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Native gel Western blot of B16F1 SEVs. ( B ) Standard western blot of HT1080 SEVs. ( C ) Western blot of cortical neuron total cell lysate (TCL) and SEVs. ( D ) Representative images and quantitation of filopodia number in control (lipofectamine) and THSD7A-mScarlet-transfected HT1080 cells. Arrowheads indicate THSD7A at the ends of filopodia (white arrowheads) or in extracellular deposits (red arrowheads). Scale bars in wide field and zoom insets = 10 mm. ( E ) (Left) Western blot of control shRNA (NTC) and shTHSD7A (C-04, C05, C-06) - expressing HT1080 cell lines. Vinculin is used as a loading control and numbers below the blot indicate normalized THSD7A levels. (Right) Filopodia counts in control and shTHSD7A HT1080 cells. ≥20 cells per condition per biological replicate, from three biological replicates. ( F ) THSD7A coated coverslips rescue filopodia defect in shEng B16F1 and HT1080 cells.≥20 cells per condition per biological replicate, from three biological replicates. ( G, H ) Cortical neurons were transfected with a FLAG-THSD7A expression vector or vector control, fixed, and stained with an antibody against THSD7A, and imaged by confocal microscopy. ( G ) Representative images. Arrows indicate THSD7A localization to the tips of filopodia. Scale bar = 5 mm. ( H ) Quantification of filopodia in neurons expressing FLAG-THSD7A or control vector. n=42 neurons from three separate experiments (biological replicates). ( I ) Rescue of filopodia numbers in shHrs neurons plated on dishes coated with various concentrations of recombinant human THSD7A, as indicated. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 6—source data 1. PDF file containing the original western blots and Ponceau stain from , indicating the relevant bands. Figure 6—source data 2. Original files for western blot and Ponceau analysis displayed in . Figure 6—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 4. Original files for western blot analysis displayed in . Figure 6—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 6. Original files for western blot analysis displayed in . Figure 6—source data 7. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 8. Original files for western blot analysis displayed in .

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Western Blot, Quantitation Assay, Control, Transfection, shRNA, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Recombinant

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot analysis of total cell lysates (TCL) and SEVs from HT1080 control and shEng cells +/-rescue with WT endoglin or control expression vectors. The figure was made from cropped images of membranes to remove irrelevant lanes. ( B ) Quantification of endoglin expression (normalized to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( C ) Quantification of THSD7A expression (relative to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( D ) Quantification of filopodia in HT1080 control cells and shEng cells rescued with WT endoglin expression. N=3, at least 30 total cells per condition. ( E ) Representative confocal images of THSD7A-mScarlet-expressing control and shEng HT1080 cells immunostained for CD63. Box 1 shows extracellular THSD7A and CD63 deposits. Box 2 shows intracellular CD63-positive MVEs. For both boxes, the zoomed images have been adjusted for brightness and contrast (to equivalent levels for control and shEng cells) for easy visualization. Note that the overlap of THSD7A (magenta) and CD63 (green) gives a white signal, pointed out with white arrowheads in the shEng merged image in Zoom 2. Scale bar is 10 mm in wider field view and 5 mm in zoom insets. ( F ) Quantification of colocalization of internal CD63 and mScarlet signals in HT1080 cells from nonadjusted images.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 7—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 7—source data 2. Original files for western blot analysis displayed in .

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Western Blot, Control, Expressing

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: Control and endoglin-KD HT1080 cells were plated on coverslips coated with poly-D-lysine (PDL) or THSD7A. In some cases, cells were treated with the Cdc42 inhibitor ML141 (10 µM) or transfected with the dominant active Cdc42 mutant Q61L, as indicated.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Control, Transfection, Mutagenesis

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARP inhibitors increase MMEJ at ISceI-mediated deDSBs. ( A, B ) Schematic ( A ) and experimental timeline ( B ) of MMEJ reporter used in panels (C)–(F), Figs and , , , and E, and , and and . Cutting the reporter with ISceI and repair by microhomology annealing to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry + cells (MMEJ+) within the BFP+ (ISceI+) population. ( C–E ) MMEJ quantification using reporter and timeline from panel (A) and (B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µM or as indicated), DNA-PKcsi (NU7441, 1 µM), Polθi (ART558 10 µM), niraparib (2.5 µM), rucaparib (2.5 µM), and talazoparib (2.5 µM). ( F ) MMEJ quantification in HT1080 parental cells compared to isogenic POLQ -KO cells following olaparib treatment. Values are normalized to wild-type DMSO. Statistical analyses for panels (C)–(F): Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, *** P <.001, **** P <.0001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Functional Assay, Flow Cytometry, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARPi-mediated MMEJ increase is dependent on PARP1, not PARP2. ( A, B ) Immunoblot of Actin, PARP1, PARP2, and PAR in parental HT1080 cells and isogenic PARP1 -KO ( A ) and PARP2 -KO clones. ( C, D ) MMEJ quantification using reporter and experimental timeline from Fig. and in HT1080 parental cells and indicated isogenic PARP1 -KO ( C ) or PARP2 -KO clones ( D ). Values are normalized to each cell line DMSO. Drug used was olaparib (5 µM). Statistical analyses for panels (C) and (D): Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one-way ANOVA with multiple comparison correction. ns: nonsignificant, ** P <.01, **** P <.0001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Western Blot, Clone Assay, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARPi-dependent MMEJ increase depends on HR. ( A ) MMEJ quantification using the reporter and experimental timeline from Fig. and in HT1080 cells following treatment with olaparib (5 µM), DNA-PKcsi (NU7441, 1 µM), or both (combo). Values are normalized to DMSO. Immunoblot of 53BP1 and histone H3 in parental HT1080 cells and isogenic TP53BP1 -KO cells. ( C ) MMEJ quantification in HT1080 parental cells and indicated isogenic 53BP1 -KO cells. Values are normalized to wild-type DMSO. Drug used: olaparib (5 µM). ( D ) Immunoblot of BRCA2 and actin in parental HT1080 cells and two isogenic BRCA2 -KO clonal lines. ( E ) MMEJ quantification in HT1080 parental cells and BRCA2 -KO clones. Values are normalized to wild-type DMSO. Drug used: olaparib (5 µM). Statistical analyses for panels (A), (C), and (E). Data represent three independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, **** P <.0001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Western Blot, Clone Assay, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARPi-dependent MMEJ increase does not occur at Cas9-induced DSBs. ( A, B ) Schematic ( A ) and experimental timeline ( B ) of MMEJ reporter used in panels (C)–(E), Figs and . Repair of the Cas9-induced cut with microhomology annealing leads to an in-frame, functional mCherry gene. Flow cytometry was used to identify the mCherry + cells (MMEJ+) within the GFP+ (Cas9+) population. ( C–E ) MMEJ quantification using the reporter and timeline from panels (A) and (B) in HT1080 cells. Values are normalized to DMSO. Drugs used were olaparib (5 µM or indicated dose), DNA-PKcsi (NU7441, 1 µM), Polθi (ART558, 10 µM). ( F ) HR quantification using the DR-GFP reporter in HT1080 cells after an ISceI- or Cas9-induced DSB, ± olaparib treatment (5 µM). Statistical analyses ( C–F ): Data represent three (D–F) or four ( C ) independent experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, *** P <.001, **** P <.0001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Functional Assay, Flow Cytometry, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: PARP1 and PARP2 are dispensable for MMEJ at deDSBs. ( A, B ) Immunoblot of Actin, PARP1, PARP2, and PAR in HT1080 cells and isogenic PARP1 -KO with or without complemented GFP-PARP1 ( A ) or PARP2 -KO with or without complemented PARP2 . ( C, D ) MMEJ quantification using the reporter with ISceI cut (from Fig. and ) in HT1080 parental cells and indicated isogenic PARP1 -KO with or without complemented GFP-PARP1 ( C ) or PARP2 -KO with or without complemented PARP2 ( D ). Values are normalized to wild-type DMSO. Drug used was olaparib (5 µM). ( E ) MMEJ quantification using reporter with Cas9 cut (from Fig. and ) in HT1080 cells and isogenic PARP1 -KO cells with or without complemented GFP-PARP1. Values are normalized to wild-type DMSO. Statistical analyses ( C–E ): Data represent four ( C ) or three independent ( D, E ) experiments, each the average of three technical replicates. Data are mean ± SEM. Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, ** P <.01, *** P <.001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Western Blot, Comparison

Journal: Nucleic Acids Research

Article Title: PARP1 and PARP2 are dispensable for DNA repair by microhomology-mediated end-joining at double-ended DSBs

doi: 10.1093/nar/gkaf1437

Figure Lengend Snippet: MMEJ is predominantly active in mitosis, when PARP1 is dispensable for DSB repair. ( A ) Experimental timeline for panel (B). Sixteen hours prior to transfection, cells were synchronized in G1 with palbociclib (250 nM), drugs were added 5 h after transfection and flow cytometry was performed 48 h later. A control was performed with the same timeline but without palbociclib synchronization. ( B ) MMEJ quantification using the MMEJ reporter with ISceI or Cas9 cut in asynchronous or G1-synchronized HT1080 cells. Drugs used: olaparib (5 µM), Polθi (ART558, 10 µM), DNA-PKcsi (NU7441, 1 µM). Data represent three independent experiments. ( C ) MMEJ quantification using the reporter with ISceI cut in HT1080 parental cells and indicated isogenic RHNO1 -KO or POLQ- KO clonal lines, ± olaparib 5 µM. Data represent three independent experiments, each the average of three technical replicates. ( D ) Experimental timeline for panel (E). HT1080 cells are arrested in RO3306 (9 µM) for 16 h DMSO (0.1%), olaparib (5 µM), or Polθi (ART558, 10 µM) were added 15 h after RO3306. After 16 h in RO3306, RO3306 was removed and cells were maintained in respective DMSO, olaparib, or Polθi treatment. Cells were irradiated (2 Gy) 40 min after RO3306 release and fixed for micronuclei staining 5 h later. ( E ) Representative images (left) and quantification (right panel) of IF from HT1080 cells from panel (D) in cells 5 h post irradiation compared to no irradiation. Cells were stained for DAPI and the percentage of cells with micronuclei was quantified. Data represent three independent experiments. For each replicate, at least 300 cells were analysed. Statistical analyses for panels (B), (C), and (E): Statistical test, one way ANOVA with multiple comparison correction. ns: nonsignificant, * P <.05, ** P <.01, *** P <.001, **** P <.0001.

Article Snippet: Fibrosarcoma HT1080 cells were purchased from ATCC (CCL-121).

Techniques: Transfection, Flow Cytometry, Control, Irradiation, Staining, Comparison

Journal: Genes

Article Title: REV1 Loss Triggers a G2/M Cell-Cycle Arrest Through Dysregulation of Mitotic Regulators

doi: 10.3390/genes17010044

Figure Lengend Snippet: REV1 inhibition suppresses tubulin expression. ( A ) mRNA levels of β-Tubulin genes in HT1080 cells treated with REV1 inhibitor JH-RE-06 were measured using qPCR. Results are shown as mean fold change ± SEM (N = 3 biological replicates) compared to untreated cells. * p < 0.02, calculated using unpaired Student’s t -test, ns denotes non-significant. ( B ) Representative Western blot of β-tubulin expression relative to β-actin in HT1080 cells treated with JH-RE-06 for 8, 16, 24, and 48 h. Quantification of data is in the . ( C ) mRNA levels of Tubb6 in WT mouse embryonic fibroblasts (MEF) and REV1 KO MEF cells using qPCR. Results are shown as mean fold change ± S.E.M. (N = 6 biological replicates) compared to untreated cells. *** p < 0.0002, calculated using unpaired Student’s t -test. Statistical test done using Graphpad Prism 10. ( D ) Representative Western blot of REV1 and β-Tubulin protein expression in WT mouse embryonic fibroblasts (MEF) and REV1 KO MEF cell lines relative to β-actin. N = 3 biological replicates, quantification values in the . Gene names are in the mouse species nomenclature.

Article Snippet: Similarly, human fibrosarcoma HT1080 cells (American Type Culture Collection (ATCC), MA, Virginia, USA) were cultured under the same conditions in RPMI with 4.5 g/L D-glucose (Gibco, Thermo Fisher Scientific, NY, USA), supplemented with 10% (vol/vol) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% PenStrep (Gibco, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Expressing, Western Blot

Journal: Nature Communications

Article Title: AAK1 activation-mediated iron trafficking drives ferroptotic cell death

doi: 10.1038/s41467-025-67523-9

Figure Lengend Snippet: A Protein levels associated with iron metabolism in various cancer cell lines treated with erastin for 12 h. A549, 8 μM; CAL51, 30 μM; HN6, 30 μM; HT1080, 4 μM; MDA-MB-231, 8 μM; MCF7, 30 μM. B , C Total ( B ) and divalent ( C ) iron levels of various cancer cell lines treated with erastin for 12 h. A549, 4 μM; CAL51, 20 μM; HN6, 20 μM; HT1080, 1 μM; MDA-MB-231, 2 μM; MCF7, 20 μM . D Schematic of the endocytosis assays. TFR1 in the cell surface were labelled with TFR1-specific antibodies at 4 °C followed by transferring cells to 37 °C to allow TFR1 internalization. The internalization levels of TFR1 will be established by antibody-labelled TFR1 from the cell surface evaluated by flow cytometry. This figure was created in BioRender. Lichao, L. (2025) https://BioRender.com/p1ntlvc . E Endocytosis assays of TFR1 in various cancer cell lines treated with erastin for 12 h. A549, 4 μM; CAL51, 20 μM; HN6, 20 μM; HT1080, 1 μM; MDA-MB-231, 2 μM; MCF7, 20 μM. F PKCβ was knocked out using single guide RNAs (sgRNAs) in MDA-MB-231 (left) and HT1080 (right) cells. G Total (left) and divalent (right) iron levels in the indicated MDA-MB-231 cells treated with erastin for 12 h. H , I Endocytosis assays of TFR1 in the indicated MDA-MB-231 ( H ) and HT1080 ( I ) cells treated with 2 μM or 1 μM erastin for 12 h, respectively. The cells with blue highlight were kept in 4 °C for dormancy. The cells with red highlight were transferred to 37 °C for internalization. The cells with grey highlight were labelled with non-specific antibody as isotype control. J , K Plasmids of PKCβⅠ or PKCβⅡ were transfected into PKCβ -knockout MDA-MB-231 ( J ) and HT1080 ( K ) cells, respectively. Endocytosis assays of TFR1 were performed in these cells treated with 2 μM or 1 μM erastin for 12 h, respectively. L Total (left) and divalent (right) iron levels in the indicated MDA-MB-231 cells treated with erastin for 12 h. A , F , J , K Data are representative of n = 3 biologically independent experiments. B , C , E , J , K Data are presented as means ± SD, n = 3 biologically independent experiments, unpaired two-tailed Student’s t test. G–I , L Data are presented as means ± SD, n = 3 biologically independent experiments, one-way ANOVA test.

Article Snippet: Human breast cancer cell MDA-MB-231 and MCF7, human fibrosarcoma cell HT1080, human Non-Small Cell Lung Cancer cell A549 were obtained from American Type Culture Collection.

Techniques: Transferring, Flow Cytometry, Control, Transfection, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: AAK1 activation-mediated iron trafficking drives ferroptotic cell death

doi: 10.1038/s41467-025-67523-9

Figure Lengend Snippet: A Phosphorylation level and gene score for individual genes were analyzed in the phosphoproteome using three individual wild-type and PKCβ -KO MDA-MB-231 cells. Potential genes exhibiting lower phosphorylation level have a negative score and genes exhibiting higher phosphorylation level have a positive score. Red highlights AAK1 . Blue highlights ACSL4 . Data was analyzed by unpaired two-tailed Student’s t test. B AAK1 was knocked out by single guide RNAs (sgRNAs) in MDA-MB-231 (top) and HT1080 (bottom) cells. C , D Endocytosis of TFR1 in the indicated MDA-MB-231 ( C ) and HT1080 ( D ) cells treated with 2 μM or 1 μM erastin for 12 h, respectively. The cells with blue highlight were kept in 4 °C for dormancy. The cells with red highlight were transferred to 37 °C for internalization. The cells with grey highlight were labelled with non-specific antibody as isotype control. E AAK1 -knockout MDA-MB-231 cells were transfected with sgRNA-resistant AAK1 . Endocytosis of TFR1 were performed in the indicated cells treated with erastin with/without dynasore. erastin, 2 μM for 12 h; dynasore, 150 μM for 1 h. F AAK1 -knockout HT1080 cells were transfected with sgRNA-resistant AAK1 . Endocytosis of TFR1 were performed in the indicated cells treated with erastin with/without dynasore. erastin, 1 μM for 12 h; dynasore, 150 μM for 1 h. G Total (left) and divalent (right) iron levels were assayed in the indicated MDA-MB-231 cells treated with erastin for 12 h. H Endogenous PKCβⅡ was immunoprecipitated from MDA-MB-231 (left) and HT1080 (right) cells treated with erastin for 12 h, followed by immunoblots using a AAK1-specific antibody to establish the interaction of endogenous PKCβⅡ with endogenous AAK1. I Endogenous AAK1 was immunoprecipitated from MDA-MB-231 (left) and HT1080 (right) cells treated with erastin for 12 h, followed by immunoblots using a PKCβⅡ-specific antibody to establish the interaction of endogenous AAK1 with endogenous PKCβⅡ. B–F , H , I Data are representative of n = 3 biologically independent experiments. C , D , G Data are presented as means ± SD, n = 3 biologically independent experiments, one-way ANOVA test. E , F , H , I Data are presented as means ± SD, n = 3 biologically independent experiments, unpaired two-tailed Student’s t test.

Article Snippet: Human breast cancer cell MDA-MB-231 and MCF7, human fibrosarcoma cell HT1080, human Non-Small Cell Lung Cancer cell A549 were obtained from American Type Culture Collection.

Techniques: Phospho-proteomics, Two Tailed Test, Control, Knock-Out, Transfection, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: AAK1 activation-mediated iron trafficking drives ferroptotic cell death

doi: 10.1038/s41467-025-67523-9

Figure Lengend Snippet: A Total phosphorylation levels of AAK1 in MDA-MB-231 cells treated with erastin, with/without 5 μM Go6983. B Total phosphorylation levels of AAK1 in MDA-MB-231 cells treated with erastin, with/without 5 μM Enza (enzastaurin). C , D Total phosphorylation levels of AAK1 in the indicated MDA-MB-231 cells treated with erastin. E Purified recombinant AAK1 proteins were incubated with/without recombinant activated PKCβII kinase or λ-phosphatase for 0.5 h in kinase buffer with ATP in vitro. F HEK293T cells were transfected with Flag-AAK1 plasmid, followed by isolating from HEK293T cells using anti-Flag antibody. The isolated AAK1 proteins were incubated with/without recombinant activated PKCβII kinase or λ-phosphatase for 0.5 h in kinase buffer with ATP in vitro. G Schematic of the potential phosphorylation sites of AAK1 indicated by phosphoproteomic analysis. H , I AAK1 -knockout MDA-MB-231 ( H ) and HT1080 ( I ) cells were transfected with plasmids of wild-type or various mutant AAK1 . The phosphorylation inactivated mutation was performed by mutating Ser/Thr into Ala, while the activated mutation was performed by mutating Ser/Thr into Glu. Single or double mutants of both of the activated or inactivated mutation of potential phosphorylation sites S670/T674 on AAK1 were included. J Endocytosis assays of TFR1 in the indicated MDA-MB-231 (left) and HT1080 (right) cells treated with 2 μM or 1 μM erastin, respectively for 12 h. K HEK293T cells were transfected with wild-type Flag-AAK1 or mutant Flag-AAK1-S670/T674Ala plasmid, followed by isolating from HEK293T cells using anti-Flag antibody. The isolated AAK1 proteins were incubated with/without recombinant activated PKCβII kinase for 0.5 h in kinase buffer with ATP in vitro. The phosphorylation levels of AAK1 were established by a phospho-S670/T674-AAK1-specific (p-AAK1 (S670/T674)) antibody. L , M The phosphorylation levels of AAK1 in MDA-MB-231 ( L ) and HT1080 ( M ) cells treated with erastin with/without Go6983, Enza, or Fer-1 for 12 h by Immunoblots using phospho-S670/T674-AAK1-specific antibody. erastin, Go6983, Enza, Fer-1, 10 μM for MDA-MB-231 and 5 μM for HT1080. A – F , H , I , K – M Data are representative of n = 3 biologically independent experiments. J Data are presented as means ± SD, n = 3 biologically independent experiments, unpaired two-tailed Student’s t test.

Article Snippet: Human breast cancer cell MDA-MB-231 and MCF7, human fibrosarcoma cell HT1080, human Non-Small Cell Lung Cancer cell A549 were obtained from American Type Culture Collection.

Techniques: Phospho-proteomics, Purification, Recombinant, Incubation, In Vitro, Transfection, Plasmid Preparation, Isolation, Knock-Out, Mutagenesis, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: AAK1 activation-mediated iron trafficking drives ferroptotic cell death

doi: 10.1038/s41467-025-67523-9

Figure Lengend Snippet: A The phosphorylation levels of AP2M1 in PKCβ -knockout MDA-MB-231 (top) and HT1080 (bottom) cells treated with erastin at different concentrations for 12 h. B The phosphorylation levels of AP2M1 in MDA-MB-231 (top) and HT1080 (bottom) cells treated with erastin at different concentrations with/without 5 μM Go6983 for 12 h. C The phosphorylation levels of AP2M1 in the indicated MDA-MB-231 (top) and HT1080 (bottom) cells treated with/without 10 μM or 5 μM erastin, respectively for 16 h. D The phosphorylation levels of AP2M1 in AAK1 -knockout MDA-MB-231 (top) and HT1080 (bottom) cells treated with erastin at different concentrations for 12 h. E The phosphorylation levels of AP2M1 in MDA-MB-231 (top) and HT1080 (bottom) cells treated with erastin at different concentrations with/without 10 μM SGC-AAK1-1 for 12 h. F The phosphorylation levels of AP2M1 in the indicated MDA-MB-231 (top) and HT1080 (bottom) cells treated with/without 10 μM or 5 μM erastin, respectively for 16 h. G Knockout of AP2M1 was performed in MDA-MB-231 cells using single guide RNAs (sgRNAs). Plasmids of AP2M1-WT , AP2M1-T156A , and AP2M1-T156E were transfected into AP2M1 -knockout cells. These cells were verified by immunoblots. H Endocytosis assays of TFR1 in the indicated MDA-MB-231 cells treated with 2 μM erastin for 12 h. A–G Data are representative of n = 3 biologically independent experiments. H Data are presented as means ± SD, n = 3 biologically independent experiments, unpaired two-tailed Student’s t test.

Article Snippet: Human breast cancer cell MDA-MB-231 and MCF7, human fibrosarcoma cell HT1080, human Non-Small Cell Lung Cancer cell A549 were obtained from American Type Culture Collection.

Techniques: Phospho-proteomics, Knock-Out, Transfection, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: AAK1 activation-mediated iron trafficking drives ferroptotic cell death

doi: 10.1038/s41467-025-67523-9

Figure Lengend Snippet: A , B AAK1 depletion by specific siRNA in MDA-MB-231 ( A ) and HT1080 ( B ) cells and verified by real-time qPCR (left) and western blotting (right). Data are representative of n = 3 biologically independent experiments. C , D Cell-death ( C ) and lipid-peroxidation ( D ) measurements for the indicated MDA-MB-231 (left) and HT1080 (right) cells treated with 10 μM or 5 μM erastin, respectively with/without 10 μM Fer-1 for 12 h. E , F Cell-death ( E ) and lipid-peroxidation ( F ) measurements for the indicated MDA-MB-231 cells treated with erastin (left) or cystine deprivation (right) with/without Fer-1 for 12 h. erastin, 10 μM; Fer-1, 10 μM. -Cys, cystine deprivation. G , H Cell-death ( G ) and lipid-peroxidation ( H ) measurements for the indicated MDA-MB-231 cells treated with erastin (left) or cystine deprivation (right) with/without various cell death inhibitors for 16 h. erastin, Fer-1 and DFO, 10 μM; -Cys, cystine deprivation; NAC, 5 mM N-acetyl-cysteine; Z-V, 10 μM Z-VAD-FMK; Nec, 2 μM necrostatin-1s. I , J Cell-death ( I ) and lipid-peroxidation ( J ) measurements for the indicated MDA-MB-231 (left) and HT1080 (right) cells treated with 10 μM or 5 μM erastin, respectively for 16 h (cell death) or 12 h (lipid-peroxidation). K , L Cell-death ( K ) and lipid-peroxidation ( L ) measurements for the indicated MDA-MB-231 (left) and HT1080 (right) cells treated with 10 μM or 5 μM erastin, respectively for 12 h (cell death) or 12 h (lipid-peroxidation). A–F Data are presented as means ± SD, n = 3 biologically independent experiments, one-way ANOVA test. G–L Data are presented as means ± SD, n = 3 biologically independent experiments, unpaired two-tailed Student’s t test.

Article Snippet: Human breast cancer cell MDA-MB-231 and MCF7, human fibrosarcoma cell HT1080, human Non-Small Cell Lung Cancer cell A549 were obtained from American Type Culture Collection.

Techniques: Western Blot, Two Tailed Test