ym 53601  (MedChemExpress)

Journal: The EMBO Journal

Article Title: LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

doi: 10.1038/s44318-024-00130-4

Figure Lengend Snippet: ( A ) Schematic detailing the unbiased drug screen. Luciferase readouts of cocultured wells were normalized to their respective monocultured wells, both in the presence of the tested drug (2 µM), to determine the relative viability. ( B ) Dataset summarizing the results of the unbiased drug screen. Relative viabilities of each drug were normalized to the DMSO control groups. ( C ) Cancer cell viability determined using luciferase readouts for LN229 TAZ(4SA) cells cocultured with dHL-60 cells with DMSO, simvastatin (2.5 µM), or fluvastatin (2.5 µM). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with drugs ( n = 3 independent experiments), one-way ANOVA. ( D ) Diagram summarizing the MVA pathway and the targets of drugs tested. ( E ) Luciferase assay results for LN229 TAZ(4SA) cells cocultured with dHL-60 cells with GGTI-298 ( n = 3 independent experiments). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with drugs. One-way ANOVA. ( F ) Cancer cell viability determined using luciferase readouts for LN229 TAZ(4SA) cells cocultured with TAN with DMSO, simvastatin (2.5 µM), or fluvastatin (2.5 µM). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with drugs ( n = 3 independent experiments), one-way ANOVA. ( G ) Luciferase assay results for LN229 TAZ(4SA) cells cocultured with TAN with GGTI-298 (10 µM) and YM-53691 (10 µM) ( n = 3 independent experiments). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with drugs. One-way ANOVA. ( H , I ) Cell death evaluated using YOYO-3 Iodide stain for LN229 TAZ(4SA) cells cultured with ( H ) IKE (0.75 µM) or ( I ) RSL3 (0.125 µM) with DMSO, fluvastatin (2.5 µM), simvastatin (2.5 µM), GGTI-298 (5 µM), YM-53601 (10 µM), or FTI-277 (10 µM) ( n = 3 independent experiments), one-way ANOVA. ( J ) Representative images showing immunofluorescent PKH26, MPO and DAPI signal for LN229 TAZ(4SA) cells cocultured with PKH26-labeled dHL-60 cells with DMSO, simvastatin (5 µM), fluvastatin (5 µM), GGTI-298 (10 µM), or YM-53601 (10 µM) ( n = 3). Scale bar, 10 µm. ( K ) Quantification of MPO puncta in each tumor cell in the experiments as shown in ( J ). Two fields of view were used for each group in each experiment ( n = 3 independent experiments). One-way ANOVA. ( L ) Quantification of PKH26 puncta in each tumor cell in the experiments as shown in ( J ). Two fields of view used for each group in each experiment ( n = 3 independent experiments). One-way ANOVA. Error bars, s.e.m. .

Article Snippet: Compounds used to treat cells: Prestwick Chemical FDA Library (provided by the Drug Discovery, Development and Delivery core of PSU College of Medicine), simvastatin (10010344, Cayman Chemicals), fluvastatin (10010334, Cayman Chemicals), GGTI-298 (16176, Cayman Chemicals), YM-53601 (18113, Cayman Chemicals), FTI-277 (S7465, SelleckChem), RGDS (A9041, Sigma-Aldrich), Vps34-IN1 (17392, Cayman Chemicals), Liproxstatin-1 (SML1414, Sigma), 4-Aminobenzoic hydrazide (4-ABHA, 103200050, Acros Organics), IKE (S8877, SelleckChem), RSL3 (19288, Cayman Chemicals).

Techniques: Luciferase, Control, Staining, Cell Culture, Labeling

Journal: The EMBO Journal

Article Title: LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

doi: 10.1038/s44318-024-00130-4

Figure Lengend Snippet: ( A ) Representative images showing the morphology of LN229 TAZ(4SA) cells after 12 h treatment with DMSO, simvastatin (2.5 µM), fluvastatin (2.5 µM), GGTI-298 (10 µM), or YM-53601 (10 µM). Scale bar, 50 µm. ( B ) Luciferase assay results for LN229 TAZ(4SA) cells cocultured with dHL-60 cells with Rho inhibitor 1 (Rho-In1) (500 nM) or NSC23766 (100 µM) ( n = 4 independent experiments). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with drugs. One-way ANOVA. ( C ) Depictions of different tiers of neutrophil (red oval) internalization by LN229 TAZ(4SA) tumor cells: attached (Tier 1), entering (Tier 2), and internalized (Tier 3). ( D ) Representative images showing the internalization tiers depicted in ( C ). Scale bar, 50 µm. ( E ) Quantification of frequency of dHL-60 internalization tiers during coculture with LN229 TAZ(4SA) cells in the conditions described in ( A ). ~300 cells surveyed per repeat ( n = 3 independent experiments). One-way ANOVA. ( F ) Representative immunofluorescent images showing LAMP1 staining, PKH26 signal, and DAPI staining for LN229 TAZ(4SA) cells cocultured with PKH26-labeled dHL-60 cells ( n = 3). Scale bar, 20 µm. ( G ) Cancer cell viability determined using luciferase readouts for LN229 TAZ(4SA) cells cocultured with dHL-60 cells with DMSO, Bafilomycin A1 (400 nM), or pepstatin A-methyl ester (25 µM). Luminescence readouts of cocultured wells were normalized to their respective LN229 TAZ(4SA) cells monocultured wells, both with indicated drugs ( n = 4 independent experiments), one-way ANOVA. ( H ) Representative immunofluorescent images showing LC3B staining, PKH26 signal, and DAPI staining for LN229 TAZ(4SA) cells cocultured with PKH26-labeled dHL-60 cells ( n = 3). The outlined areas are enlarged and shown in the insets. Scale bar, 20 µm. Error bars, s.e.m. .

Article Snippet: Compounds used to treat cells: Prestwick Chemical FDA Library (provided by the Drug Discovery, Development and Delivery core of PSU College of Medicine), simvastatin (10010344, Cayman Chemicals), fluvastatin (10010334, Cayman Chemicals), GGTI-298 (16176, Cayman Chemicals), YM-53601 (18113, Cayman Chemicals), FTI-277 (S7465, SelleckChem), RGDS (A9041, Sigma-Aldrich), Vps34-IN1 (17392, Cayman Chemicals), Liproxstatin-1 (SML1414, Sigma), 4-Aminobenzoic hydrazide (4-ABHA, 103200050, Acros Organics), IKE (S8877, SelleckChem), RSL3 (19288, Cayman Chemicals).

Techniques: Luciferase, Staining, Labeling

Journal: The EMBO Journal

Article Title: LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

doi: 10.1038/s44318-024-00130-4

Figure Lengend Snippet: ( A ) Representative images showing LN229 TAZ(4SA) cells cocultured with PKH26-labeled dHL-60 cells with DMSO, simvastatin (2.5 µM), fluvastatin (2.5 µM), GGTI-298 (10 µM), or YM-53601 (10 µM). Scale bar, 100 µm. ( B ) Quantification of LN229 TAZ(4SA) cells with overlapping PKH26-labeled dHL-60 cells in the conditions described in ( A ) ( n = 3 independent experiments). One-way ANOVA. ( C ) Representative immunofluorescent images showing LAMP1 staining, PKH26 signal, and DAPI staining for LN229 TAZ(4SA) cells cocultured with PKH26-labeled d32Dcl3cl3 cells ( n = 3). Scale bar, 20 µm. Error bars, s.e.m.

Article Snippet: Compounds used to treat cells: Prestwick Chemical FDA Library (provided by the Drug Discovery, Development and Delivery core of PSU College of Medicine), simvastatin (10010344, Cayman Chemicals), fluvastatin (10010334, Cayman Chemicals), GGTI-298 (16176, Cayman Chemicals), YM-53601 (18113, Cayman Chemicals), FTI-277 (S7465, SelleckChem), RGDS (A9041, Sigma-Aldrich), Vps34-IN1 (17392, Cayman Chemicals), Liproxstatin-1 (SML1414, Sigma), 4-Aminobenzoic hydrazide (4-ABHA, 103200050, Acros Organics), IKE (S8877, SelleckChem), RSL3 (19288, Cayman Chemicals).

Techniques: Labeling, Staining