Journal: Communications Biology

Article Title: USP24 promotes autophagy-dependent ferroptosis in hepatocellular carcinoma by reducing the K48-linked ubiquitination of Beclin1

doi: 10.1038/s42003-024-06999-5

Figure Lengend Snippet: A The relationship of USP24 expression with autophagy markers was investigated via Gene Expression Profiling Interactive Analysis (GEPIA). B qRT-PCR was performed to measure the mRNA expression of ULK1, Atg5 and Beclin1 after silencing USP24 ( n = 3). C qRT-PCR was performed to measure the mRNA expression of ULK1, Atg5 and Beclin1 after overexpressing USP24 ( n = 3). D The expression of ULK1, Atg5, Beclin1, LC3B-II and p62 proteins in USP24 knockdown HCC cells were measured by western blot. E The expression of autophagy related proteins after overexpressing USP24 was detected by western blot. F Transmission electron microscopy (TEM) was used to detect the formation of autophagic structures. Red arrows denote representative autolysosome. Scale bar, 5 μm (left), 500 nm (right). G The number of autophagic vesicles was counted and presented in the image ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After being washed by PBS three times, the cells were incubated with Dylight 594 (red for USP24) and Dylight 488 (green for Beclin1) conjugated secondary antibodies (Abbkine, Wuhan, China) for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Transmission Assay, Electron Microscopy

Journal: Communications Biology

Article Title: USP24 promotes autophagy-dependent ferroptosis in hepatocellular carcinoma by reducing the K48-linked ubiquitination of Beclin1

doi: 10.1038/s42003-024-06999-5

Figure Lengend Snippet: A , B Reciprocal Co-IP experiments were performed to detect the interaction between USP24 and Beclin1. An IgG antibody was used as the control. C The cell double immunofluorescence assay was carried out using anti-USP24 and anti-Beclin1 antibodies. The colocalization of USP24 (red) and Beclin1 (green) was observed under a confocal microscope (n = 3). Scale bar, 10 μm. D The colocalization was evaluated by Mander’s colocalization coefficients in ten regions of interest ( n = 10). E SMMC-7721 cells were treated with 10 μg/ml MG132 before harvesting. An anti-Beclin1 antibody was used to immunoprecipitate the Beclin1 protein, then an anti-Ubi antibody was used for western blot. F, G The levels of K48 and K63-linked ubiquitination were measured by western blot. H–K Control and USP24 overexpressed HCCLM3 and SMMC-7721 cells were treated with CHX (100 μg/ml) for indicated time. The expression level of Beclin1 was determined by western blot ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After being washed by PBS three times, the cells were incubated with Dylight 594 (red for USP24) and Dylight 488 (green for Beclin1) conjugated secondary antibodies (Abbkine, Wuhan, China) for 1 h at room temperature.

Techniques: Co-Immunoprecipitation Assay, Control, Immunofluorescence, Microscopy, Western Blot, Expressing

Journal: Communications Biology

Article Title: USP24 promotes autophagy-dependent ferroptosis in hepatocellular carcinoma by reducing the K48-linked ubiquitination of Beclin1

doi: 10.1038/s42003-024-06999-5

Figure Lengend Snippet: A – C The cells overexpressed USP24 were treated with Bafilomycin A1 (10 nM) for 24 h. A CCK8 assay was used to detect the cell viability ( n = 5). B–C Transwell assay was performed to evaluate the migration ability of HCC cells, and the migration rate was calculated ( n = 3). D The efficiency of Beclin1 knockdown was measured by western blotting. E CCK8 assay was performed to determine the cell viability after USP24 overexpression or Beclin-1 knockdown ( n = 3). F–G Transwell assays of HCC cells with USP24 overexpression or Beclin1 knockdown were performed to evaluate the ability of migration, and the migration rate was calculated ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After being washed by PBS three times, the cells were incubated with Dylight 594 (red for USP24) and Dylight 488 (green for Beclin1) conjugated secondary antibodies (Abbkine, Wuhan, China) for 1 h at room temperature.

Techniques: CCK-8 Assay, Transwell Assay, Migration, Knockdown, Western Blot, Over Expression