Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: A The number of differentially expressed genes was screened by iTRAQ-based proteomics after overexpression of USP32 in H1299 cells. B H1299 cells were transiently transfected with Myc vector or Myc-USP32 expression plasmid for 48 h. Proteins were extracted from the cells and separated on a gel, which was then stained with Coomassie brilliant blue R-250. A framed protein band was evaluated by mass spectrometry analysis. C Representative mass spectral peaks of BAG3 interacting with Myc-USP32. D Docking model of USP32 and BAG3 proteins and surface map of their interfacial residues (USP32, blue; BAG3, yellow; hydrogen-bonding interactions, dotted line). E Co-localization studies of three cells of NSCLC using anti-USP32 antibody (1:100, green) and anti-BAG3 antibody (1:100, red) followed by DAPI nuclear counterstaining (blue). Merged images of USP32 (green) and BAG3 (red) with DAPI (blue) are also shown. Scale bar:50 μm. F , G Exogenous interaction of USP32 with BAG3. HEK293T cells were cotransfected with Myc-USP32 and/or Flag-BAG3 plasmids. Cell lysates were immunoprecipitated with the indicated antibodies and then immunoblotted with anti-Flag antibody ( F ) or anti-Myc antibody ( G ). H , I Endogenous interaction of USP32 with BAG3. A549 and H1299 cell lysates were immunoprecipitated with anti-USP32/BAG3 antibody or IgG antibody, followed by immunoblotting with anti-BAG3/USP32 antibody. J , K USP32 and BAG3 protein levels were detected by immunoblotting in USP32 overexpressed and knockdown overexpressed cells. ** p < 0.01; *** p < 0.001.

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Knockdown

Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: A , B After transfection of overexpression plasmid Myc-USP32 or small interfering siUSP32 into H1299 and A549 cells for 48 h, afterwards they were treated with 50 μg/mL cycloheximide (CHX), and the cells were collected at the indicated times, and then subjected to Western blotting analysis of BAG3 protein levels. C – F NSCLC cells after overexpression and interference with USP32 were treated with or without MG132 for 6 h, respectively, and BAG3 levels were determined by immunoblotting. G In the presence of Myc-vector or Myc-USP32 plasmid, HA-labeled ubiquitin and Flag-BAG3 were co-transfected with H1299 cells for 48 h, after which the cells were harvested by adding MG132 treatment for 6 h, and cell lysates were immunoprecipitated experimentally with anti-Flag antibody, and ubiquitination was detected by HA antibody. H H1299 cells were co-transfected with Myc- USP32/Flag- BAG3/ HA-Ubiquitin-K48/HA-Ubiquitin-K63 plasmids and treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Flag antibody, and ubiquitylation was detected by HA antibody. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Labeling, Immunoprecipitation

Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: A , B Overexpression of BAG3 in A549 and H1299 cells after knockdown of USP32 was performed for rescue experiments, followed by detection of USP32 and BAG3 expression by Western blot. C – F BAG3 overexpression in A549 and H1299 cells restored cell proliferation and migration capacity after being knocked down by USP32. Scale bar:100 μm. G Changes in the expression of EMT-related proteins Vimentin and E-cadherin were detected after rescue experiments in A549 and H1299 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, Migration

Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: A GSEA enrichment of the MAPK signaling pathway in lung cancer patient-associated differential genes. Data were obtained from the TCGA database. B GSEA analysis shows that high expression of USP32 in lung cancer patients is associated with the MAPK signaling pathway. Data were obtained from the TCGA database. NES means normalized enrichment score. C , D Overexpression of USP32 and empty plasmid were transfected in H460 and H1299 cells, and Western blot was performed to detect the protein levels of USP32 and members of the RAF/MEK/ERK signaling pathway (RAF1, p-RAF1, MEK-1/2, p-MEK1/2, ERK1/2, and p-ERK1/2). E , F siUSP32#2 was transfected into A549 and H1299 cells, and Western blotting was performed to detect the expression of USP 32, RAF1, p-RAF1, MEK-1/2, p-MEK1/2, ERK1/2 and p-ERK1/2. G, H Overexpression of BAG3 activates the RAF/MEK/ERK pathway in A549 and H1299 cells of USP32 knockdown * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Over Expression, Plasmid Preparation, Transfection, Western Blot, Knockdown

Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: A Positive correlation between USP32 of LUAD/LUSC and BAG3 analyzed from the TCGA database. B Validation of USP32 and BAG3 expression using Western blot in multiple cell lines of NSCLC. C USP32 positively correlates with BAG3 in various non-small cell lung cancer cell lines. D Representative immunohistochemical (IHC) staining for USP32 and BAG3 in human NSCCL tissues. E The number of cases with negative and positive results for BAG3 and USP32 expression in NSCLC tissues were counted. F After 34 scoring immunostaining of NSCLC tissues, the correlation between USP32 and BAG3 expression was analyzed using scatterplots.

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Immunohistochemistry, Immunostaining

Journal: Oncogenesis

Article Title: USP32 facilitates non-small cell lung cancer progression via deubiquitinating BAG3 and activating RAF-MEK-ERK signaling pathway

doi: 10.1038/s41389-024-00528-z

Figure Lengend Snippet: USP32 activates the RAF/MEK/ERK signaling pathway through interaction with BAG3 and promotes the proliferation, migration and EMT of non-small cell lung cancer cells. (Figure was created with Biorender.com).

Article Snippet: The expression plasmid Flag-BAG3 (empty vector: p3xFLAG-CMV-10) was purchased from GeneChem (Shanghai, China).

Techniques: Migration