isolation buffer  (New England Biolabs)

Journal: Cell Death & Disease

Article Title: Interaction of lncRNA LENT with DHX36 regulates translation and suppresses autophagy in melanoma

doi: 10.1038/s41419-025-08296-3

Figure Lengend Snippet: A Immunofluorescence by confocal microscopy showing DHX36 localization in different cell lines. HSP60 is used as a mitochondria marker and DAPI to stain the nucleus. Scale bars = 10 µM. B DHX36 signal was quantified in the whole cell and divided by the signal in mitochondria or in the nucleus. Each measured cell is represented by one point, and groups are compared by one-way ANOVA (Dunnett test). * P < 0.033; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001. C Immunoblot of cytosolic or mitochondrial fractions of different cell lines. D Purified mitochondria were digested with increasing concentrations of digitonin, and retained proteins were analyzed by western blot. E Immunogold staining coupled with electron microscopy. Representative stained particles for DHX36 or COX IV in the cytoplasm (C) or mitochondria (M) are indicated with arrows. Scale bars are indicated on images. F LENT levels quantified by RT-qPCR in cytosolic or mitochondrial fractions of different cell lines.

Article Snippet: Then, purified mitochondria were digested on ice for 15 min with increasing concentrations of Digitonin (Invitrogen) in mitochondria isolation buffer (210 mM Mannitol, 70 mM Sucrose, 1 mM EDTA, 10 mM HEPES and protease inhibitors cocktail).

Techniques: Immunofluorescence, Confocal Microscopy, Marker, Staining, Western Blot, Purification, Electron Microscopy, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Interaction of lncRNA LENT with DHX36 regulates translation and suppresses autophagy in melanoma

doi: 10.1038/s41419-025-08296-3

Figure Lengend Snippet: A Transmission electron microscopy of 501Mel cells 48 h following transfection of control or LENT-targeting ASO. Autophagosomes and degraded melanosomes were observed in LENT-silenced cells. Scale bars are indicated on the images. B Quantification of cytosolic LC3B (LC3-I) and lipid-associated LC3B (LC3 II) by western blot in 501Mel or MM117 cells. Vinculin is used as a loading control. C LC3B immunoblot in extracts of IGR37 CDX tumors. D Confocal microscopy of unfixed 501Mel cells stained with lysotracker and mitotracker in control or LENT-silenced conditions. The numbers of co-localizing lysosomes and mitochondria was determined and compared between the two conditions by Welch’s test. Scale bars = 10 µM. E Oxygen consumption rate was determined upon LENT or DHX36 silencing. Reserve capacity was obtained by subtracting the maximal capacity with the basal capacity. Comparisons were done by one-way ANOVA (Dunnett test). * P < 0.033; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001.

Article Snippet: Then, purified mitochondria were digested on ice for 15 min with increasing concentrations of Digitonin (Invitrogen) in mitochondria isolation buffer (210 mM Mannitol, 70 mM Sucrose, 1 mM EDTA, 10 mM HEPES and protease inhibitors cocktail).

Techniques: Transmission Assay, Electron Microscopy, Transfection, Control, Western Blot, Confocal Microscopy, Staining