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
![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 <t>Δ[3–10]BRAFV600E</t> 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](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0044/pmc05410044/pmc05410044__12943_2017_645_Fig6_HTML.jpg)
![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](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0044/pmc05410044/pmc05410044__12943_2017_645_Fig7_HTML.jpg)
