Journal: Oncotarget

Article Title: Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway.

doi: 10.18632/oncotarget.2140

Figure Lengend Snippet: Figure 1: BRAF regulates Usp5 activity. A. BRAF mutant (SK-Mel28, A375) and non-mutant (SK-Mel147) melanoma cells were treated with 5 µM vemurafenib for the 24 hr before cell lysates were subjected to immunoblotting for the protein indicated. B. BRAF mutant and non-mutant cells were treated with DMSO (-) or 5 μM vemurafenib (+) for 24 h before cell lysates were subjected to immunoblotting for total ubiquitin. The mobility of mono-, di-, tri- and tetra-Ub is denoted. C. Melanoma cells were incubated with 5 µM vemurafenib for 24 hours before DUB activity was assessed in lysates by HA-UbVS labeling followed by HA blotting (top). Migration of HA-Ub-Vs labeled Usp5 is denoted (top). Immunoblotting of the same membrane for Usp5 is shown at the bottom. D. Left – SK-Mel28 cells were incubated with or without vemurafenib for 24 hr before lysates were labeled by incubation with HA-UbVS. Usp5 was immunoblotted as a measure of its activation. Activated Usp5 appears as doublet above the Usp5 band. pERK and actin immunoblots are also shown. Right – Similar analysis was conducted with A375 cells. Usp7 was immunoblotted as a control. E. Control or BRAF KD SK-Mel28 cells were assessed for BRAF, pERK and Usp5-specific DUB activity by HA-UbVS labeling followed by Usp5 blotting. F. HEK293T cells transfected with control or BRAFV600E expression vector were assessed for BRAF and pERK by immunoblotting. Usp5-specific DUB activity was assessed as described in D.

Article Snippet: Antibodies used in this study were purchased from the following sources: anti-actin and FLAG (SigmaAldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/ mouse/rat IgG-conjugated horseradish peroxidase, p21, ERK and Mcl-1 (Santa Cruz Biotechnology); USP7, USP5, BRAF and p73 (Bethyl Laboratories); anti– poly(ADP-ribose) polymerase (PARP), pERK, Caspase8, Caspase3, Bid, Bax, Bak (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science); CD95/Fas (Clone EPR5700; Epitomics); anti-NOXA, p53 (DO-1; CalBiochem); anti-DR4, DR5 (ProSci incorporated) and FAS monoclonal antibody (CH11; Millipore).

Techniques: Activity Assay, Mutagenesis, Western Blot, Ubiquitin Proteomics, Incubation, Labeling, Migration, Membrane, Activation Assay, Control, Transfection, Expressing, Plasmid Preparation

Journal: Oncotarget

Article Title: Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway.

doi: 10.18632/oncotarget.2140

Figure Lengend Snippet: Figure 2: Usp5 regulates melanoma cell growth. A. BRAF mutant (red) and non-mutant (green) control and Usp5 KD melanoma cells were plated at 5,000 cells per plate (Day 0) and cell counts were conducted daily. Each value represents the average +/- S.D. of 3 independent counts. The p value for each grouping (control vs. Usp5) was <0.05 (*). B. Control or Usp5 KD melanoma cells (as noted) were immunoblotted for Usp5, p21 and actin. C. Phase contrast and eGFP fluorescent images of A375 cells with stable Usp5 knockdown (KD) and control cells grown on matrigel for 7 days. The bar graph shows the number of single round acinar structures +/- S.D. obtained by counting 100 structures from 3 separate wells. D. Control or FLAG-Usp5 A375 cells (inset), were plated at 5,000 cells per culture dish and counted daily for 4 days. Each value represents the average +/- S.D. of 3 independent counts. * p<0.05.

Article Snippet: Antibodies used in this study were purchased from the following sources: anti-actin and FLAG (SigmaAldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/ mouse/rat IgG-conjugated horseradish peroxidase, p21, ERK and Mcl-1 (Santa Cruz Biotechnology); USP7, USP5, BRAF and p73 (Bethyl Laboratories); anti– poly(ADP-ribose) polymerase (PARP), pERK, Caspase8, Caspase3, Bid, Bax, Bak (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science); CD95/Fas (Clone EPR5700; Epitomics); anti-NOXA, p53 (DO-1; CalBiochem); anti-DR4, DR5 (ProSci incorporated) and FAS monoclonal antibody (CH11; Millipore).

Techniques: Mutagenesis, Control, Knockdown

Journal: Oncotarget

Article Title: Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway.

doi: 10.18632/oncotarget.2140

Figure Lengend Snippet: Figure 3: Usp5 regulates apoptotic responsiveness to kinase inhibition. A. Control and Usp5 KD melanoma cells with wild- type p53 and mutant or non-mutant BRAF expression (as indicated) were immunoblotted for Usp5, p53, FAS and actin. B. Control and Usp5 KD SK-Mel28 cells were immunoblotted for Usp5, p73 and actin. C. Control or Usp5 KD SK-Mel28 and A375 cells were treated with 5 µM vemurafenib for the interval noted before cell lysates were immunoblotted for the protein indicated. D. Control and Usp5 KD A375 cells were treated with 5 µM MI219 for 24 hr before cell lysates were immunoblotted for the protein indicated. E. Control and Usp5 KD SK-Mel 147 cells were treated with or without 1 µM PD 0325901 for 6 hr. Cell lysates were subjected to immunoblotting for the protein indicated.

Article Snippet: Antibodies used in this study were purchased from the following sources: anti-actin and FLAG (SigmaAldrich); anti-ubiquitin clone P4D1, goat, anti-rabbit/ mouse/rat IgG-conjugated horseradish peroxidase, p21, ERK and Mcl-1 (Santa Cruz Biotechnology); USP7, USP5, BRAF and p73 (Bethyl Laboratories); anti– poly(ADP-ribose) polymerase (PARP), pERK, Caspase8, Caspase3, Bid, Bax, Bak (Cell Signaling Technology); anti-HA (clone 3F10; Roche Applied Science); CD95/Fas (Clone EPR5700; Epitomics); anti-NOXA, p53 (DO-1; CalBiochem); anti-DR4, DR5 (ProSci incorporated) and FAS monoclonal antibody (CH11; Millipore).

Techniques: Inhibition, Control, Mutagenesis, Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of PFS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for PFS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. (F, G) Kaplan-Meier plots of PFS for CRC patients undergoing exclusive first-line chemotherapy (F) and chemotherapy combined with bevacizumab (G) , stratified by BRAF mutation status. PFS, progression-free survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of OS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for OS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. OS, overall survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Construction of a four-gene mutation signature prediction disease progression and prognosis in FPHYP cohort. (A, B) Univariate and multivariate analyses were performed to assess the impact of clinicopathological features, individual somatic gene mutations, and the four-gene mutation signature on PFS (A) and OS (B) in CRC. (C) The Kaplan-Meier survival analysis for PFS in CRC patients between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (D) The Kaplan-Meier survival analysis for OS in CRC cases between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (E, F) ROC curves for PFS (E) and OS (F) that dependent on time were generated to evaluate the prognostic model’s performance, which is based on the gene mutation status within the FPHYP cohort. PFS, progression-free survival; OS, overall survival; MT, mutation type; WT, wiled type; ROC, receiver operating characteristic.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis, Biomarker Discovery, Generated

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Immunohistochemical analysis of BRAF and ARID2 in CRC. (A, B) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of the BRAF -MT (A) and BRAF -WT (B) groups. (C) Comparison of the IOD/Area value between BRAF -MT and BRAF -WT groups. (D, E) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of stage I (D) and stage IV (E) groups. (F) Comparison of the IOD/Area value between stage I and IV groups. (G, H) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of the ARID2 -MT (G) and ARID2 -WT (H) groups. (I) Comparison of the IOD/Area value between ARID2 -MT and ARID2 -WT groups. (J, K) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of stage I (J) and stage IV (K) groups. (L) Comparison of the IOD/Area value between stage I and IV groups. MT, mutation type; WT, wiled type; IOD, cumulative optical density.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Immunohistochemical staining, Expressing, Comparison, Mutagenesis