microscopy channel slide ibidi μ-slide  (ibidi GmbH)

Journal: Cell Communication and Signaling : CCS

Article Title: Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

doi: 10.1186/s12964-024-01507-y

Figure Lengend Snippet: Impairment of the endocytic pathway in chemoresistant cells. A EC50 was determined in A2780 and in A2780 CIS cells after 72 h of treatment with CDDP using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide) assay. B , C Relative protein abundance of RAB4, RAB5A, RAB7, RAB9, RAB27, RILP, LAMP-1, and ATP6V1G1 was assessed by Western blot analysis and quantified by densitometry normalizing against α-Tubulin. D Late endocytic acid compartments were live stained with LysoTracker DND-99 dye and images were acquired with confocal microscopy. E , F The number and size of LysoTracker-positive organelles were determined by ImageJ software. G Quantification of Green DQBSA puncta per cell was determined by ImageJ software. Data represent the mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Cells were seeded into microscopy chambers (8 well μ-slide, Ibidi GmBh, Martinsried, Germany) and after 24 h incubated with MitoTracker Red CM-H 2 XROS (50 nM), LysoTracker Green DND-26, MitoTracker Green FM (50 nM), and/or LysoTracker Red DND-99 (1 μM) (ThermoFisher Scientific) as previously reported [ , ].

Techniques: Quantitative Proteomics, Western Blot, Staining, Confocal Microscopy, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

doi: 10.1186/s12964-024-01507-y

Figure Lengend Snippet: Analysis of EV content. A EV morphology was determined by Transmission Electron Microscopy (TEM). Scale bar = 100 nm. B , C The total amount of extracellular proteins released by A2780 and A2780 CIS cells normalized on total cell proteins and without normalization. D EVs were purified by ultracentrifugation and RAB7 content in cell lysates and EVs were evaluated by Western blot analysis. The purity of EVs was ascertained according to the ISEV guidelines by looking at different negative and positive markers as indicated. E - H Expression levels of CD81, CD9, CD63, AND RAB7 were determined in cellular lysates and in EVs by densitometry normalizing against β-Actin. I - J A2780 CIS were treated with GW4869 EV secretion inhibitor and intracellular levels of RAB7 and CD81 (as a positive control of inhibition) were measured by western blotting analysis normalizing against HSP90. K - P EVs were purified by immunoisolation. Positive and negative controls were used to verify the purity of EVs. Mitochondrial protein expression was evaluated in EVs by Western blot and the relative abundance of RAB7, ATP5A, NDUFS3, SDHA, and SDHB was determined through densitometric analysis normalizing against vinculin. Q Total DNA was extracted from A2780 cells and A2780 CIS EVs. MitoALL resequencing kit was used to analyze mitochondrial DNA (mtDNA) contained in EVs. Mock 1 and Mock 2 represent two conditions of PCR purity control in which PCR mix with amplicon 19 and 21 primers were used as controls. Data in graphs represent the mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Cells were seeded into microscopy chambers (8 well μ-slide, Ibidi GmBh, Martinsried, Germany) and after 24 h incubated with MitoTracker Red CM-H 2 XROS (50 nM), LysoTracker Green DND-26, MitoTracker Green FM (50 nM), and/or LysoTracker Red DND-99 (1 μM) (ThermoFisher Scientific) as previously reported [ , ].

Techniques: Transmission Assay, Electron Microscopy, Purification, Western Blot, Expressing, Positive Control, Inhibition, Control, Amplification

Journal: Cell Communication and Signaling : CCS

Article Title: Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

doi: 10.1186/s12964-024-01507-y

Figure Lengend Snippet: Treatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) and analysis of EV content. A A2780 cells were treated with CCCP (10 μm) and EVs were purified by immunoisolation. Mitochondrial protein expression was evaluated in EVs by Western blot analysis and B - H the relative abundance of RAB7, ATP5A, UQCRC2, SDHA, SDHB, NDUFS3, and NDUFB8 was determined through densitometric analysis normalizing against β-Actin. Parkin protein was used as control of CCCP treatment. I , J A2780 cells were treated with CCCP (10 μm) and EVs were purified by ultracentrifugation. RAB7 levels were determined, and their relative abundance was measured with densitometry normalizing against β-Actin. K A2780 CIS cells were transfected with empty vector (Mock) and with GFP-RAB7, and L OCR was determined with Seahorse Mito stress kit assay. M OCR and Extracellular Acidification Rate (ECAR) obtained with Seahorse instruments allowed to determine the energetic map. N - S Basal respiration, maximal respiration, ATP-production coupled respiration, proton leak, spare respiration capacity, and non-mitochondrial oxygen consumption were determined by Seahorse data elaboration. T - V A2780 CIS cells were transfected with GFP-RAB7, and DQBSA assay and LAMP-1 immunostaining were performed and analyzed by confocal microscopy. Data represent the mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Cells were seeded into microscopy chambers (8 well μ-slide, Ibidi GmBh, Martinsried, Germany) and after 24 h incubated with MitoTracker Red CM-H 2 XROS (50 nM), LysoTracker Green DND-26, MitoTracker Green FM (50 nM), and/or LysoTracker Red DND-99 (1 μM) (ThermoFisher Scientific) as previously reported [ , ].

Techniques: Purification, Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Immunostaining, Confocal Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

doi: 10.1186/s12964-024-01507-y

Figure Lengend Snippet: Analysis of mitochondrial and lysosomal deficit and EVs in SKOV-3 sensitive and resistant cells. A EC50 was determined in SKOV-3, SKOV-3 CIS-1, and SKOV-3 CIS-2 after 72 h of treatment with CDDP using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide) assay. B Late endocytic acid compartments were live stained with LysoTracker DND-26 dye and images were acquired with confocal microscopy. C , D The number and size of LysoTracker-positive organelles were determined by ImageJ software. E , F Relative protein abundance of RAB7, RAB27, and LAMP-1 was assessed by Western blot analysis and quantified by densitometry normalizing against α-Tubulin. G Oxygen rate consumption (OCR) was determined in SKOV-3 and in SKOV-3 CIS-1 and CI-2 with Seahorse Mito stress kit assay. H - M Basal respiration, maximal respiration, ATP-production coupled respiration, proton leak, and spare respiration capacity were determined by Seahorse data elaboration. N - R EVs were purified by immunoisolation. Positive and negative controls were used to verify the purity of EVs. Mitochondrial protein expression was evaluated in EVs by Western blot and the relative abundance of RAB7, UQCRC2, NDUFB8, and three tetraspanins (CD9, CD63, and CD81) was determined through densitometric analysis normalizing against α-Tubulin.* p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar = 10 μm

Article Snippet: Cells were seeded into microscopy chambers (8 well μ-slide, Ibidi GmBh, Martinsried, Germany) and after 24 h incubated with MitoTracker Red CM-H 2 XROS (50 nM), LysoTracker Green DND-26, MitoTracker Green FM (50 nM), and/or LysoTracker Red DND-99 (1 μM) (ThermoFisher Scientific) as previously reported [ , ].

Techniques: Staining, Confocal Microscopy, Software, Quantitative Proteomics, Western Blot, Purification, Expressing