Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 upregulates RRBP1 protein level. (A) Flowchart of proteomic analysis was illustrated. (B) Twenty‐six overlapped proteins were identified to be increased in wild‐type USP35 overexpression group (USP35 WT) and decreased in shUSP35‐2 group through Venn diagrams software (available online: http://bioinformatics.psb.ugent.be/webtools/Venn/ ). (C) The indicated DEPs were confirmed by western blot. (D–F) Expression of RRBP1 was detected by western blot in USP35 wild‐type or catalytically inactive mutant overexpression (WT and C450A) A549 and PC9 cells (D), shRNAs mediated USP35 knockdown A549 and PC9 cells (E) and gRNA mediated USP35 knockdown H1299 cells (F). All data are presented by mean ± SD. ** P < 0.01, *** P < 0.001 based on the Student t ‐test. All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Over Expression, Software, Western Blot, Expressing, Mutagenesis, Knockdown

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 deubiquitinates and stabilizes RRBP1. (A, B) H1299 cells stably expressing wild‐type USP35 (USP35 WT) and catalytically inactive form, USP35 C450A as well as empty vector (EV) (A), and PC9 cells with stably expressing USP35‐specific shRNAs (shUSP35‐1, shUSP35‐2) and scramble RNA (B) were treated with 50 μg·mL −1 cycloheximide (CHX) for 0, 2, 4, 6, 8, 10 h (A) or for 0, 0.5, 1, 2, 4, and 6 h (B). Expressions of RRBP1 were detected by western blot. Quantitative analyses of CHX chase data were shown in the graphs. (C, D) USP35 overexpressed (USP35 WT) H1299 cells and the control group (EV) (C), and USP35 knockdown (shUSP35‐1 and shUSP35‐2) PC9 cells and the control group (Scramble) (D) were treated with or without 10 µ m MG132 for 6 h. Expressions of RRBP1 were detected by western blot. Quantitative analyses were shown in the graphs. (E) H1299 cells were transfected or cotransfected with Flag‐RRBP1 alone or along with HA‐USP35 WT or HA‐USP35 C450A. Cell lysates were immunoprecipitated with anti‐Flag antibody, followed by immunoblotting with anti‐Ub antibody. (F) PC9 cells were transfected or cotransfected with Flag‐RRBP1 alone or along with HA‐USP35 WT or in combination with HA‐USP35 WT and 2 or 4 µg shUSP35‐2. Cell lysates were immunoprecipitated with anti‐Flag antibody, followed by immunoblotting with anti‐Ub antibody. All data are presented by mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 based on the Student t ‐test. All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Knockdown, Transfection, Immunoprecipitation

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 interacts with RRBP1. (A, B) HEK293T cells were cotransfected with Flag‐RRBP1 and Myc‐USP35. Cell lysates were immunoprecipitated with anti‐Myc antibody, followed by immunoblotting with anti‐Flag antibody (A) or immunoprecipitated with anti‐Flag antibody, followed by immunoblotting with anti‐Myc antibody (B). (C, D) Cell lysates of H1299 (C) and A549 (D) were immunoprecipitated with anti‐USP35 antibody or IgG antibody, followed by immunoblotting with anti‐RRBP1 antibody. (E) H1299 and A549 cells were stained with anti‐USP35 and anti‐RRBP1 antibodies, followed by corresponding TRITC‐conjugated and FITC‐conjugated secondary antibodies staining. Merged image (yellow) shows the overlap of USP35 and RRBP1 staining. Images were captured respectively via confocal microscopy (H1299) and fluorescence microscopy (A549). (F, G) H1299 cells were costained anti‐USP35 or anti‐RRBP1 antibodies with the ER‐resident protein calnexin, followed by corresponding TRITC‐conjugated and FITC‐conjugated secondary antibodies staining. Merged images (yellow) showed the overlapped areas of USP35 or RRBP1 with calnexin. Images were captured respectively via confocal microscopy. Scale bars indicate 50 µm (E–G) and 10 µm in the inserts (E–G). All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Immunoprecipitation, Western Blot, Staining, Confocal Microscopy, Fluorescence, Microscopy

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 overexpression inhibits TM‐induced cell apoptosis. (A) H1299 cells with stable overexpression of USP35 (USP35 WT) and their control (EV) cells were treated with 2 µ m TM for 0, 24, 48, and 72 h. The indicated proteins were detected by western blot. (B) A549 cells transfected with USP35‐specific siRNAs (siUSP35‐1 and siUSP35‐2) and scramble siRNA (siNC) were treated with or without 2 µ m TM for 48 h. The indicated proteins were detected by western blot. (C, D) USP35 WT and its control H1299 cells (C) and siUSP35‐1, siUSP35‐2, and siNC A549 cells (D) were treated with or without 2 µ m TM for 48 h. The cells were subsequently stained with Annexin V‐FITC and propidium iodide (PI) and analyzed by flow cytometry. Quantitative analyses were shown in the graphs. (E, F) USP35 WT and its control H1299 cells (E), siUSP35‐1 and siNC A549 cells (F) were treated with or without 2 µ m TM for 48 h. The apoptotic cells were detected using TUNEL staining. Quantitative analyses were shown in the graphs. Scale bars indicate 50 µm (E). All data are presented by mean ± SD. * P < 0.05, *** P < 0.001 based on the Student t ‐test. All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Over Expression, Control, Western Blot, Transfection, Staining, Flow Cytometry, TUNEL Assay

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 overexpression attenuates TM‐induced cell apoptosis through up‐regulating RRBP1. (A) RRBP1‐specific siRNA (siRRBP1‐1) or control siRNA was introduced into USP35 overexpression (USP35 WT) H1299 cells. Then, the cells were treated with or without 2 µ m TM for 48 h. The indicated proteins were detected by western blot. (B) USP35 overexpressed (USP35 WT) H1299 cells were treated with Radezolid (RRBP1 inhibitor, 10 µ m ) or TM (2 µ m ) alone or in combination for 48 h. The indicated proteins were detected by western blot. (C, D) The apoptotic rates of aforementioned H1299 cells were detected by flow cytometry. Quantitative analyses were shown in the graphs. All data are presented by mean ± SD. *** P < 0.001 based on the Student t ‐test. All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Over Expression, Control, Western Blot, Flow Cytometry

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: USP35 silencing boosts TM‐induced cell apoptosis through down‐regulating RRBP1. (A) Flag‐RRBP1 expression plasmid or control plasmid was introduced into USP35 silenced (siUSP35‐1) A549 cells. Then, the cells were treated with or without 2 µ m TM for 48 h. The indicated proteins were detected by western blot. (B) The apoptotic rates of aforementioned A549 cells were detected by flow cytometry. Quantitative analyses were shown in the graphs. All data are presented by mean ± SD. *** P < 0.001 based on the Student t ‐test. All results are representatives of three independent experiments.

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Flow Cytometry

Journal: Molecular Oncology

Article Title: USP35 mitigates endoplasmic reticulum stress‐induced apoptosis by stabilizing RRBP1 in non‐small cell lung cancer

doi: 10.1002/1878-0261.13112

Figure Lengend Snippet: RRBP1 expression positively correlates with USP35 expression in NSCLC tissues. (A) The representative immunohistochemistry staining of USP35 and RRBP1 in NSCLC tissues were shown. The box plot indicated the relative RRBP1 level in USP35‐low and USP35‐high patients (median USP35 or RRBP1 expression was defined as cutoff point). Scale bars indicate 250 and 50 µm in the inserts. (B, C) The correlation between the expression of USP35 and RRBP1 in NSCLC tissues ( n = 45) (B) and the correlations between the expression of USP35 and RRBP1 in NSCLC tissues in TCGA database from GEPIA ( n = 969) (C) were presented in scatter plot. (D) The Kaplan–Meier Plotter database (229198_at) analyzed the USP35 levels in relation to the overall survival of patients with lung adenocarcinoma (Cutoff value used in analysis: 142). (E) The Kaplan–Meier Plotter database (201204_s_at) analyzed the RRBP1 levels in relation to the overall survival of patients with lung adenocarcinoma (cutoff value used in analysis: 681).

Article Snippet: Specific gRNAs for USP35 were synthesized and cloned into H6825 pLenti‐U6‐spgRNA v2.0‐CMV‐sfGFP‐P2A‐3Flag‐spCas9 vector (Obio Technology, Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Staining