Journal: Molecular Cancer

Article Title: The landscape of BRAF transcript and protein variants in human cancer

doi: 10.1186/s12943-017-0645-4

Figure Lengend Snippet: BRAF transcript variants in the context of acquired resistance to BRAF and MEK inhibitors. a Real-time PCR detection of total BRAF ( red ), BRAF -ref ( grey ), BRAF -X1 plus X2 ( black ), BRAF -X1 ( blue ), and BRAF -X2 ( green ) in 451Lu parental cells (P) and in 451Lu-MR resistant cells (MR). The latter show acquired resistance to BRAF and MEK inhibitors due to the focal amplification of the BRAF gene. b Cartoon summarizing the position of the primers and the siRNAs used to determine the presence and the level of the Δ[3–10] variant of BRAF . For details, please refer to Additional file : Figure S17. c Real-time PCR detection of total BRAF ( red ), full length BRAF ( brown ), Δ[3–10] BRAF ( orange , left panel ), BRAF -ref ( grey ), BRAF -X1 plus X2 ( black ), BRAF -X1 ( blue ), and BRAF -X2 ( green , right panel ) in A375 parental cells and in A375 C2 cells. The latter show acquired resistance to vemurafenib due to the presence of Δ[3–10]BRAFV600E splicing variant. d PCR amplification of the reference, X1, and X2 Δ[3–10] BRAF splicing variants from the cDNA of A375 C2 cells. Lane 1: 1 kbp ladder. Lane 2: Δ[3–10] BRAF -ref amplification was obtained using BRAF -E1/2 F primer and ref BRAF -STOP R primer (open red and grey arrows in b ). Lane 3: Δ[3–10] BRAF -ref CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-ref plasmid and used as positive control. Lane 4: the amplification of Δ[3–10] BRAF -X1 ( upper band ) and Δ[3–10] BRAF -X2 ( lower band ) was obtained using BRAF -E1/2 F primer and BRAF -X1-STOP R primer (open red and black arrows in b ). Lane 5: Δ[3–10] BRAF -X1 CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-X1 plasmid and used as positive control. Lane 6: Δ[3–10] BRAF -X2 CDS was amplified from pMSCVHygro-Δ[3–10]BRAFV600E-X2 plasmid and used as positive control. e - f Real-time PCR detection of full length BRAF , Δ[3–10] BRAF , BRAF -ref , BRAF- X1 plus X2, BRAF -X1, and BRAF -X2 24 h after the transfection of si-fl BRAF ( e ) and si-Δ[3–10] BRAF ( f ) in A375 C2 cells. g Real-time PCR detection of full length and Δ[3–10] BRAF 24 h after the transfection of si-ref BRAF and si- BRAF -E19-1 in A375 C2 cells. h Western blot of full length and Δ[3–10] BRAFV600E, as well as of pMEK 48 h after the transfection of the indicated siRNAs or siRNA mixes in A375 C2 cells. i Growth curve of A375 C2 cells after the transfection of the indicated siRNAs. Throughout the experiment, the cells were kept in DMSO ( left panel ) or in 2 uM vemurafenib ( right panel ). The arrows highlight the increased sensitivity displayed by A375 C2 cells to si-Δ[3–10] BRAF ( orange ) and si- BRAF -E19-1 ( black ), when grown in vemurafenib. The graph represents the mean only of 3 independent experiments. ( j ) Colony formation assay of A375 C2 cells after the transfection of the indicated siRNAs. Throughout the experiment, the cells were kept in DMSO ( clean bars ) or in 2 uM vemurafenib ( dashed bars ). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM (or mean ± SD in a and c ) of 3 independent experiments. * p < 0.05, ** p < 0.01

Article Snippet: They were then incubated overnight at 4 °C with the following primary antibodies: anti-BRAF (F-7, #5284, Santa Cruz Biotechnology; mouse monoclonal antibody, dilution 1:1000 in 5% milk in TBST); anti-human BRAFV600E (VE1, #E19290, Spring Bioscience; mouse monoclonal antibody, dilution 1:400 in 1% milk in TBST); anti-pMEK (#9154, Cell Signaling; rabbit monoclonal antibody, dilution 1:1000 in 3% BSA in TBST); anti-α-TUBULIN (#T9026, Sigma-Aldrich; mouse monoclonal antibody, dilution 1:20000 in 5% milk in TBST); anti-EGFP (#A111-22, Molecular Probes, rabbit polyclonal antibody, dilution 1:2000 in 5% milk in TBST).

Techniques: Real-time Polymerase Chain Reaction, Amplification, Variant Assay, Plasmid Preparation, Positive Control, Transfection, Western Blot, Colony Assay

Journal: Molecular Cancer

Article Title: The landscape of BRAF transcript and protein variants in human cancer

doi: 10.1186/s12943-017-0645-4

Figure Lengend Snippet: Identification and characterization of BRAF protein isoforms. a Schematic representation of the 3’ terminal region of reference, X1, and X2 BRAF mRNAs, as well as of the corresponding C-terminal regions of reference, X1, and X2 BRAF proteins. b Immunoprecipitation of BRAF protein in A375 cells. Endogenous BRAF was immunoprecipitated using a specific antibody that recognizes the N-terminal domain (IP-BRAF). As negative control, no antibody was used (No Ab). The basal level of BRAF in the cell lysate is shown in Input. c Identification by mass spectrometry of the C-terminal peptides of BRAF-ref and BRAF-X1. Immunoprecipitated BRAF was subjected to LC-MS analysis. The presence of both isoforms is revealed by the detection of isoform-specific peptides (in green ). d Best transitions (BRAF-ref: 352 and 904; BRAF-X1: 1046 and 1117) of the two BRAF protein isoforms by mass spectrometry (MRM based method). e - f Upon the transient transfection of PIG-BRAFV600E-ref, X1, and X2 plasmids in HEK293T cells, western blot indicates that only reference and X1 BRAFV600E are efficiently translated and able to phosphorylate MEK, while X2 is not ( e ). This occurs in spite of the fact that according to real-time PCR for total BRAF levels, all 3 mRNAs are transcribed at similar levels ( f ). g - i Upon the stable infection of pMSCVHygro-Δ[3–10]BRAFV600E-ref, X1, and X2 plasmids in A375 cells, real-time PCR for total BRAF indicates that all 3 mRNAs are transcribed at similar levels ( g ), but western blot indicates that reference and X1 Δ[3–10]BRAFV600E are efficiently translated and able to phosphorylate MEK even in the presence of vemurafenib, while X2 is not ( h ). Consistently, only Δ[3–10]BRAFV600E-ref and -X1 are able to decrease the sensitivity of A375 cells to vemurafenib ( i ). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments

Article Snippet: They were then incubated overnight at 4 °C with the following primary antibodies: anti-BRAF (F-7, #5284, Santa Cruz Biotechnology; mouse monoclonal antibody, dilution 1:1000 in 5% milk in TBST); anti-human BRAFV600E (VE1, #E19290, Spring Bioscience; mouse monoclonal antibody, dilution 1:400 in 1% milk in TBST); anti-pMEK (#9154, Cell Signaling; rabbit monoclonal antibody, dilution 1:1000 in 3% BSA in TBST); anti-α-TUBULIN (#T9026, Sigma-Aldrich; mouse monoclonal antibody, dilution 1:20000 in 5% milk in TBST); anti-EGFP (#A111-22, Molecular Probes, rabbit polyclonal antibody, dilution 1:2000 in 5% milk in TBST).

Techniques: Immunoprecipitation, Negative Control, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Infection

Journal: Molecular Cancer

Article Title: The landscape of BRAF transcript and protein variants in human cancer

doi: 10.1186/s12943-017-0645-4

Figure Lengend Snippet: The X2 isoform displays a faster decay due to increased proteosomal-mediated degradation. a Schematic representation of the chimerical protein derived from the fusion of EGFP coding sequence with the CR3 domain of BRAFV600E-ref, X1, and X2 within the pEGFP-C1 plasmid. The asterisk indicates the presence of the V600E mutation. b - e Upon the transient transfection of pEGFP-C1 empty (pEGFP-empty), pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids in A375 cells, real-time PCR performed with primers for EGFP and for total BRAF indicates that the chimerical mRNAs are all transcribed at similar levels ( b ), but western blot ( c ), flow cytometry ( d ) and confocal microscopy analysis ( e ) indicate that, when fused with CR3-X2, EGFP protein is expressed at lower levels. The dotted box shows a higher exposure of the anti-EGFP antibody. f - h When PIG-BRAFV600E-ΔCterm plasmid, which lacks the nucleotides encoding for the X2-specific C-terminal domain ( f ), is transiently transfected in HEK293T cells, not only BRAF mRNA ( g ), but also BRAF protein is detectable ( h ). e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; ΔCterm: PIG-BRAFV600E-ΔCterm. i - j Upon the transient transfection of pEGFP-empty, pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids, A375 cells were treated with 100 ug/ml cicloheximide (CHX) ( i ) or 20 uM MG132 ( j ) for 8 h. The CHX treatment indicates that the decay rate of CR3-X2 is faster than that of CR3-ref and CR3-X1, while the MG132 treatment suggests that this is due to higher degradation rate through the ubiquitin-proteasome pathway. k The prediction of potential proteasomal cleavage sites using 3 different algorithms retrieves the indicated X2-specific consensus peptide. ( l ) The mutagenesis of Lys739 into a proteasome-insensitive Arg rescues the expression of the X2 isoform of BRAF protein. e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; X2 K739R : PIG-BRAFV600E-X2 in which Lys(K)739 has been substituted with Arg(R) (AAA to AGA triplet change). m Cartoon that summarizes the main findings of this article (details in the text). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: They were then incubated overnight at 4 °C with the following primary antibodies: anti-BRAF (F-7, #5284, Santa Cruz Biotechnology; mouse monoclonal antibody, dilution 1:1000 in 5% milk in TBST); anti-human BRAFV600E (VE1, #E19290, Spring Bioscience; mouse monoclonal antibody, dilution 1:400 in 1% milk in TBST); anti-pMEK (#9154, Cell Signaling; rabbit monoclonal antibody, dilution 1:1000 in 3% BSA in TBST); anti-α-TUBULIN (#T9026, Sigma-Aldrich; mouse monoclonal antibody, dilution 1:20000 in 5% milk in TBST); anti-EGFP (#A111-22, Molecular Probes, rabbit polyclonal antibody, dilution 1:2000 in 5% milk in TBST).

Techniques: Derivative Assay, Sequencing, Plasmid Preparation, Mutagenesis, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Confocal Microscopy, Positive Control, Expressing

Journal: PLoS ONE

Article Title: Genetic basis of calcifying cystic odontogenic tumors

doi: 10.1371/journal.pone.0180224

Figure Lengend Snippet: Case summaries.

Article Snippet: Specimens were then probed according to using antibodies against BRAF Val600Glu (1:4000, Clone VE1, E19290, mouse monoclonal, Spring Bioscience, Pleasanton, CA, USA), beta-catenin (1:50, Clone beta-catenin-1, M3539, mouse monoclonal, Dako, Glostrup, Denmark), and hair cortex keratin (1:500, Clone AE13, sc-57012, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally stained with 3,3'-diaminobenzidine.

Techniques: Next-Generation Sequencing, Sequencing, Staining

Journal: PLoS ONE

Article Title: Genetic basis of calcifying cystic odontogenic tumors

doi: 10.1371/journal.pone.0180224

Figure Lengend Snippet: A , Electropherogram of a TCT>TGT substitution at c.98 in CTNNB1, resulting in a Ser33Cys missense mutation in case #4. B , Electropherogram of a GTG>GAG substitution at position c.1799 in BRAF, resulting in Val600Glu missense mutation in case #25. Guanine is indicated by a black line, cytosine is indicated by a blue line, adenine is indicated by a green line, and thymine is indicated by a red line.

Article Snippet: Specimens were then probed according to using antibodies against BRAF Val600Glu (1:4000, Clone VE1, E19290, mouse monoclonal, Spring Bioscience, Pleasanton, CA, USA), beta-catenin (1:50, Clone beta-catenin-1, M3539, mouse monoclonal, Dako, Glostrup, Denmark), and hair cortex keratin (1:500, Clone AE13, sc-57012, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally stained with 3,3'-diaminobenzidine.

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Genetic basis of calcifying cystic odontogenic tumors

doi: 10.1371/journal.pone.0180224

Figure Lengend Snippet: A, and D , Representative photomicrographs of CCOT (case #6) and ameloblastoma (case #25) specimens stained with hematoxylin and eosin. B, C, E, and F , Immunostaining for ( B and E ) BRAF Val600Glu (clone name VE1) and ( C and F ) beta-catenin. Scale bars; 20 μm.

Article Snippet: Specimens were then probed according to using antibodies against BRAF Val600Glu (1:4000, Clone VE1, E19290, mouse monoclonal, Spring Bioscience, Pleasanton, CA, USA), beta-catenin (1:50, Clone beta-catenin-1, M3539, mouse monoclonal, Dako, Glostrup, Denmark), and hair cortex keratin (1:500, Clone AE13, sc-57012, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and finally stained with 3,3'-diaminobenzidine.

Techniques: Staining, Immunostaining