Journal: Cell chemical biology

Article Title: GDC-0879, a BRAF V600E inhibitor, protects kidney podocytes from death

doi: 10.1016/j.chembiol.2017.11.006

Figure Lengend Snippet: Basic information of SIV-infected Chinese rhesus macaques on antiretroviral therapy

Article Snippet: Rabbit monoclonal anti-BRAF , Cell Signaling Technology , Cat#14814, RRID: N/A.

Techniques: Recombinant, Cell Viability Assay, Microarray, shRNA, Software, Drug discovery

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Structures of LF-268 and 268-TCO ( top ) and biochemical IC 50 values (bottom) of LF-268, 268-TCO, and LXH-254 for purified Y301D/Y302D ARaf, WT BRaf, and Y340D/Y341D CRaf (LF-268: n=3, 268-TCO and LXH-254: n=1). b, Schematic of the competitive 268-TCO pulldown experiment performed with HCT-116 cells using tetrazine-linked beads for enrichment ( left ) and a heatmap ( right ) showing the proteins that were significantly competed (log 2 ratio > 2 and p < 0.05) by 5 μM LF-268 compared to DMSO-treated cells (n=3). c, Schematic depicting the competitive kinobead-based inhibitor profiling workflow. Lysates were either incubated with 10 μM LF-268 or DMSO and only EPHA2 was significantly competed with a log 2 ratio > 2 for 10 μM LF-268 relative to DMSO (n=2). d, Representative western blots showing pErk levels in mutant KRas-expressing cells treated with a range of LF-268 doses for 4 h (n=2, **n=3). e, Quantification of normalized pErk percentages and determination of pErk IC 50 values from the western blots obtained in (d). The percent pErk value shown at each LF-268 concentration relative to DMSO is the mean of all replicates performed. IC 50 curves were fit to the percent pErk values obtained from all replicates. pErk IC 50 values and 95% confidence intervals (Cis) for each cell line are listed in Table S1 . f, Correlation plot of LF-268 and LXH-254 pErk IC 50 values for eleven mutant KRas-expressing cell lines. Pearson’s r value is shown. Values shown in blue indicate the six cell lines that comprise the comparative panel of mutant KRas-expressing cell lines.

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Purification, Incubation, Western Blot, Mutagenesis, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Percent activity values for purified Y301D/Y302D ARaf, WT BRaf, and Y340D/Y341D CRaf in the presence of a range of LF-268, 268-TCO, or LXH-254 concentrations (LF-268 n=3, 268-TCO n=1, LXH-254 n=1). For LF-268, individual data points represent the average of n=3 replicates and error bars represent standard error of the mean (s.e.m.). Calculated IC 50 values are shown in . b, STRING protein:protein interaction network mapping proteins with at least one phospho-site that exhibits a >2-fold average decrease in HCT-116 cells treated for 4 h with 5 μM LF-268 relative to DMSO (n=3). The thickness of the line indicates the relative strength of data that indicates a protein:protein interaction. MAPK1 and MAPK3 (Erk1 and Erk2) are labeled in red. Phospho-peptide quantifications are in Table S3 . c, Histogram of log 2 ratios of quantified phospho-peptides for HCT-116 cells treated with 5 μM of LF-268 for 4 h relative to DMSO following kinobead enrichment (n=3). Log 2 ratios are plotted from lowest to highest and the dotted line indicates a log 2 ratio < −2. Phospho-peptide quantifications are in Table S4 .

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Activity Assay, Purification, Labeling

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blot ( top left ) of ARaf, BRaf, and CRaf expression levels across the comparative panel of mutant KRas-expressing cell lines. Correlation plots of GAPDH-normalized ARaf ( top right ), BRaf ( bottom left ), and CRaf ( bottom right ) levels as measured by western blot (n=1) versus LXH-254 pErk IC 50 values. b, Western blots of phosphorylation levels of BRaf and CRaf regulatory phospho-sites (n=1). All images are from individual western blots. c, The ratio of CRaf co-immunoprecipitated with BRaf (normalized to immunoprecipitated BRaf) from CIAR-293 cells pre-treated with A115 relative to DMSO for 1 h, followed by incubation with LXH-254 for 3 h. CRaf co-immunoprecipitation levels for DMSO and A115 pre-treated CIAR-293 cells were probed on the same western blot, allowing relative levels to be compared. The values shown are from the experiments described in . Bars represent the mean of n=3 replicates and error bars represent standard error of the mean (s.e.m.).

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Expressing, Mutagenesis, Phospho-proteomics, Immunoprecipitation, Incubation

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blots ( top ) of ARaf, BRaf, and CRaf levels in size exclusion chromatography fractions from the lysates of Mia PaCa-2 cells treated with 10 μM LXH-254 or DMSO (n=1). Quantification indicates the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions ( bottom ). b, Representative western blots ( top ) and quantification ( bottom ) of ARaf levels co-immunoprecipitated with CRaf from Mia PaCa-2 treated with a range of LXH-254 doses for 3 h. The percent CRaf levels (normalized to immunoprecipitated BRaf) shown at each LXH-254 concentration are represent singlicate data. DC 50 curves were fit to percent ARaf levels obtained from all three replicates and ARaf:CRaf dimerization DC 50 values and 95% CIs are listed in Table S1 . The Mia PaCa-2 pErk IC 50 and BRaf:CRaf dimerization DC 50 curves from are overlayed to demonstrate at which LXH-254 concentrations pErk IC 50 s and dimerization DC 50 values intersect. Error bars represent standard error of the mean (s.e.m.). c, Representative western blot ( top ) of pErk levels in HCT-116 cells treated with range of encorafenib doses for 1 h followed by a 1 h washout. Quantification (bottom) of normalized pErk percentages and determination of pErk PA 50 value. The percent pErk value shown at each LXH-254 concentration relative to DMSO is the mean of n=3 replicates. PA 50 curves were fit to percent pErk values obtained from all replicates. pErk PA 50 values and 95% confidence intervals (CIs) are listed in Table S1 . d, Representative western blot ( top ) and quantification (bottom) of pErk levels in HCT-116 cells treated with a range of LXH-254 doses for 4 h (same data as shown in ). e, In-cell western assay ( top ) of pErk levels in HCT-116 cells treated with a range of LXH-254 doses for various treatment times. Quantification ( bottom ) of normalized pErk percentages (relative to DMSO-treated cells) observed at each LXH-254 concentration from the in-cell western assay (n=1).

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Size-exclusion Chromatography, Immunoprecipitation, Concentration Assay, In-Cell ELISA

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a and b, Representative western blots ( a ) and quantification ( b ) of CRaf levels co-immunoprecipitated with BRaf from Mia PaCa-2 cells pre-treated with DMSO or a partial (∼85%) inhibitory dose of a Mek inhibitor for 1 h, followed by treatment with a range of LXH-254 doses for 3 h. The BRaf-normalized percent CRaf levels shown at each LXH-254 concentration relative to maximum CRaf levels are the mean of n=3 replicates and error bars represent standard error of the mean (s.e.m.). DC 50 curves were fit to percent CRaf levels obtained from all three replicates. BRaf:CRaf dimerization DC 50 values and 95% confidence intervals (CIs) for each cell line are listed in Table S1 . BRaf:CRaf DC 50 curves shown for LXH-254 only treated cells are from . Mek inhibitor pre-treatment concentrations used: cobimetinib = 200 nM, Ro 5126766 = 30 nM, binimetinib = 80 nM, trametinib = 10 nM, GDC-0623 = 10 nM. c, Correlation plot of LXH-254 BRaf:CRaf DC 50 s from (b) and pErk IC 50 s calculated from , d. Pearson’s r value = 0.595. d, Schematic depicting workflow for quantifying biotinylation of individual Raf isoforms by KRas-TurboID in CIAR-293 cells. e, Representative western blots ( top left ) showing levels of biotinylation of immunoprecipitated ARaf, BRaf, and CRaf in CIAR-293 cells transfected with a KRas-TurboID construct, treated with DMSO, LXH-254 (10 μM), cobimetinib (500 nM), or GDC-0623 (40 nM) for 4 h, followed by treatment with 100 μM biotin for 15 min. Quantified biotinylation levels of ARaf, BRaf, and CRaf (normalized to total immunoprecipitated ARaf, BRaf, and CRaf, respectively, and represented as a fraction relative to DMSO) were obtained from n=3 replicates. Bars equal the mean of all replicates and error bars represent standard error of the mean (s.e.m.). f, Representative western blots ( top ) of pErk levels in Mia PaCa-2 cells pre-treated with 40 nM cobimetinib or DMSO for 1 h, followed by incubation with a range of encorafenib doses for 3 h. The percent pErk values shown at each encorafenib concentration relative to DMSO are the mean of n=3 replicates. Error bars represent s.e.m. PA 50 curves were fit to percent pErk values obtained from all three replicates. pErk PA 50 values and 95% CIs for each cell line are listed in Table S1 .

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Immunoprecipitation, Concentration Assay, Transfection, Construct, Incubation

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blots ( top ) of ARaf, BRaf, CRaf, and Mek levels in size exclusion chromatography fractions from the lysates of Mia PaCa-2 cells treated with cobimetinib or GDC-0623 (n=1). Quantification indicates the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions ( bottom ). b, Quantification shows the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions.

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Size-exclusion Chromatography

Journal: International journal of cancer

Article Title: The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl-2 genes in non-small cell lung cancer.

doi: 10.1002/ijc.28062

Figure Lengend Snippet: Figure 2. Western blot analysis of phospho-BRAF (Ser445), BRAF and Bcl-2 in MCPyV-infected and noninfected NSCLC specimens. (a) Compared to MCPyV noninfected cells (lanes 1–3), both activa- tion of BRAF at Ser445 and overexpression of BRAF protein were evident in MCPyV-infected tissues (lanes 4–6). (b) Bcl-2 protein levels were significantly decreased in MCPyV-positive samples (lanes 4–6) compared to noninfected tissue specimens. Actin was used as a stable endogenous control. 2ve: negative samples; 1ve: positive samples.

Article Snippet: Primary antibodies were then added at the appropriate dilution [phosphor-BRAF (Ser445), BRAF and Bcl-2 1/ 1,000; beta-actin 1/2,000) with overnight incubation at 4 C. All primary antibodies were purchased from Cell Signaling Technology, MA.

Techniques: Western Blot, Infection, Over Expression, Control

Journal: PLoS ONE

Article Title: Functional Consequences of the Macrophage Stimulating Protein 689C Inflammatory Bowel Disease Risk Allele

doi: 10.1371/journal.pone.0083958

Figure Lengend Snippet: (A) Single cell suspensions of the indicated cell populations or tissues were analyzed by flow cytometry for RON expression using a monoclonal antibody specific for murine RON (blue histogram) or an isotype control antibody (shaded histogram). Macrophage populations were gated as CD45 + F4/80 + CD11b + MHC class II + for ex vivo -derived cells and F4/80 + CD11b + for bone marrow cultured cells. Colonic epithelial cells were gated as CD45 − ECadherin + . B) Representative image from IHC analysis of RON expression (brown) on tissue sections from normal mouse colon. Scale bars = 100 µm. Data are representative of at least three similar experiments.

Article Snippet: Murine cells were stained with fluorochrome-labeled antibodies against F4/80 (clone BM8), CD11b (clone M1/70), MHC class II (clone M5/114.15.2), and either mouse RON (clone PH4, Genentech, Inc.) or a murine IgG2a isotype control antibody (Genentech, Inc).

Techniques: Flow Cytometry, Expressing, Ex Vivo, Derivative Assay, Cell Culture

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: Schematic illustration of the therapeutic system based on EVs used for the delivery of nucleic acid drugs targeting gene mutation sites for the precise treatment of CRC with the BRAF V600E mutation. siBRAF V600E was transfected into HEK293T cells via a lentiviral vector to stably produce EVs carrying siBRAF V600E . After treatment with these EVs- siBRAF V600E , the BRAF-MEK1/2-ERK1/2 pathway was inhibited in CRC cells. Subcutaneous xenograft tumor models and metastasis models of CRC cell lines were used to validate the therapeutic effects and toxicity of the EVs-siBRAF V600E . The efficacy of the EVs-siBRAF V600E was further confirmed via PDX models

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Stable Transfection

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: BRAF mutation is a poor prognostic factor in CRC patients. ( A ) K-M survival curves of patients with BRAF mutation and wild-type CRC. ( B ) Multivariate COX regression model analysis of gender, stage and BRAF mutation status in CRC patients. ( C ) K-M survival curve of BRAF expression level in BRAF MT CRC. ( D ) Representative IHC images of BRAF V600E and KI-67 in CRC tissues. The scale bar represents 10 μm for the 10x objective and 5 μm for the 20x objective. ( E ) KI67 positivity rate in CRC tissues with BRAF V600E or BRAF WT . The data are reported as the means ± SDs of the experiments ( n = 3). Two-tailed Student’s t-tests, ** p < 0.01

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Mutagenesis, Expressing, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: Construction and characterization of EVs-siBRAF V600E . ( A ) BRAF mRNA levels were analyzed by qRT-PCR. BRAF mRNA levels were measured after treating HEK293T, COLO320, and RKO cells with 50 µg/mL of EVs-siBRAF V600E for 24 h. ( B ) Construction of the therapeutic agent. ( C ) NTA analysis of EVs-NC (blue) and EVs-siBRAF V600E (red). ( D )( E ) TEM images of eVs-NC ( D ) and EVs-siBRAF V600E ( E ); scale bar: 200 nm. ( F ) WB analysis of the extracellular vesicle marker proteins, TSG101, CD81 and CD63. ( G ) Fluorescence microscopy images showing the uptake of EVs-NC and EVs-siBRAF V600E by RKO cells. The cell nuclei were stained with DAPI, and the EVs were stained with DiD; scale bar: 20 μm. ( H ) qRT-PCR analysis of siBRAF V600E levels in EVs-NC and EVs-siBRAF V600E . ( I ) WB analysis of BRAF protein levels. After COLO320 and RKO cells were treated with PBS, or 50 µg/mL of EVs-NC or EVs-siBRAF V600E for 24 h, cell proteins were extracted to measure BRAF protein expression. The data are reported as the means ± SDs of the experiments ( n = 3). Two-tailed Student’s t-tests for ( a ) and ( h ), and one-way ANOVA followed by Tukey test multiple comparisons for ( I ), * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Marker, Fluorescence, Microscopy, Staining, Expressing, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: EVs-siBRAF V600E can inhibit BRAF V600E CRC cells. RKO ( A ) and HT29 ( B ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 48 h, followed by EdU assays to measure cell proliferation activity and imaging via fluorescence microscopy. Scale bar: 20 μm. RKO( C ) and HT29 ( D ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 5 days, and cell viability was measured via CCK-8 assays. ( E ) Colony formation. Images of single-cell-derived colonies formed by RKO and HT29 cells after treatment with PBS, EVs-NC or EVs-siBRAF V600E for 2 weeks. ( F ) Flow cytometry analysis of cell apoptosis. RKO and HT29 cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, stained with Annexin V-FITC and PI, and apoptosis was detected via flow cytometry. ( G ) Cell migration was assessed by wound healing assays. RKO cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h. The concentration of EVs-NC or EVs-siBRAF V600E in all the treatments was 50 µg/mL. The data are reported as the means ± SDs of the experiments ( n = 3). Two-way ANOVA for ( A ) and ( B ), and one-way ANOVA followed by Tukey test multiple comparisons for the others, * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Activity Assay, Imaging, Fluorescence, Microscopy, CCK-8 Assay, Derivative Assay, Flow Cytometry, Staining, Migration, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: EVs-siBRAF V600E cannot inhibit BRAF WT CRC cells. COLO320( A ) and HCT116 ( B ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 5 days, and cell viability was measured via CCK-8 assays. COLO320( C ) and HCT116 ( D ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 48 h, followed by EdU assays to measure cell proliferation activity and imaging using fluorescence microscopy. Scale bar: 20 μm. ( E ) Colony formation. Images of single-cell-derived colonies formed by COLO320 and HCT116 cells after treatment with PBS, EVs-NC or EVs-siBRAF V600E for 2 weeks. ( F ) Flow cytometry analysis of cell apoptosis. COLO320 and HCT116 cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, stained with Annexin V-FITC and PI, and apoptosis was detected via flow cytometry. ( G ) Cell migration was assessed by wound healing assays. COLO320 and HCT116 cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h. The concentration of EVs-NC and EVs-siBRAF V600E in all treatments was 50 µg/mL. The data are reported as the means ± SDs of the experiments ( n = 3). Two-way ANOVA for ( A ) and ( B ), and one-way ANOVA for the others

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: CCK-8 Assay, Activity Assay, Imaging, Fluorescence, Microscopy, Derivative Assay, Flow Cytometry, Staining, Migration, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: EVs-siBRAF V600E Inhibit BRAF-MEK1/2-ERK1/2 Signaling Pathway in BRAF V600E CRC cells. ( A ) and ( B ) qRT-PCR analysis of BRAF transcription levels. RKO ( A ) and HT29 ( B ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, followed by detection of BRAF mRNA levels. ( C ) and ( D ) WB analysis of BRAF expression levels and MEK1/2-ERK1/2 phosphorylation levels. RKO ( C ) and HT29 ( D ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, and the BRAF protein levels and MEK1/2-ERK1/2 phosphorylation levels were measured. ( E ) and ( F ) qRT-PCR analysis of BRAF transcription levels. COLO320 ( E ) and HCT116 ( F ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, followed by the detection of BRAF mRNA levels. ( G ) and (H) WB analysis of BRAF expression levels and MEK1/2-ERK1/2 phosphorylation levels. COLO320 ( G ) and HT29 ( H ) cells were treated with PBS, EVs-NC or EVs-siBRAF V600E for 24 h, and the BRAF protein levels and MEK1/2-ERK1/2 phosphorylation levels were measured. The concentration of EVs-NC and EVs-siBRAF V600E in all the treatments was 50 µg/mL. ( I ) Schematic illustration of the mechanism by which EVs-siBRAF V600E treat CRC cells. The data are reported as the means ± SDs of the experiments ( n = 3). One-way ANOVA followed by Tukey test multiple comparisons, * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Expressing, Phospho-proteomics, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: EVs-siBRAF V600E inhibit BRAF V600E CRC in vivo. ( A ) Animal model handling protocol. ( B ) Subcutaneous tumor volume changes. Tumor volume measurements were performed every 3 days. ( C ) Images of the subcutaneous tumors in the three groups. ( D ) The weights of the subcutaneous tumors. ( E ) Immunohistochemistry images of BRAF and p-ERK1/2. Scale bar: 50 μm. ( F )~( I ) WB detection of BRAF expression and MEK1/2-ERK1/2 phosphorylation levels in subcutaneous tumor tissues. ( J ) Bioluminescence images of lung metastases. ( K ) Bioluminescence images of liver metastases. Each group of animals was treated with PBS, EVs-NC or EVs-siBRAF V600E . Each mouse was injected with 20 µg of extracellular vesicles per injection. Subcutaneous tumors: n = 5 per group. Lung metastasis and liver metastasis: n = 4 per group. The data are reported as the means ± SDs of the experiments. Two-way ANOVA for ( B ), and one-way ANOVA followed by Tukey test multiple comparisons for the others, * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: In Vivo, Animal Model, Immunohistochemistry, Expressing, Phospho-proteomics, Injection

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: Efficacy of EVs-siBRAF V600E in human CRC PDX models. ( A ) PDX model handling protocol. ( B ) Subcutaneous tumor volume changes in the BRAF V600E PDX models. Tumor volume measurements were performed every 3 days. ( C )Images of subcutaneous tumors from the BRAF V600E PDX models. ( D ) Subcutaneous tumor weights of the BRAF V600E PDX models. ( E ) Immunohistochemistry images of BRAF and p-ERK1/2 in the BRAF V600E PDX models. Scale bar: 50 μm. ( F ) and ( G ) Subcutaneous tumor volume changes in the BRAF WT PDX models. Tumor volumes of BRAF WT PDX1( F ) and PDX2( G ) were measured every 3 days. ( H ) Images of subcutaneous tumors from the BRAF WT PDX1 models. ( I ) Subcutaneous tumor weights of the BRAF WT PDX1 models. ( J ) Images of subcutaneous tumors from the BRAF WT PDX2 models. ( K ) Subcutaneous tumor weights of the BRAF WT PDX2 models. Each group of animals was treated with PBS, EVs-NC or EVs-siBRAF V600E . Each mouse was injected with 20 µg of extracellular vesicles per injection. N = 6 for each group of BRAF V600E PDX models. N = 5 for each group in the BRAF WT PDX1 and PDX2 models. The data are reported as the means ± SDs of the experiments. Two-way ANOVA for ( B ), ( F ) and ( G ), and one-way ANOVA followed by Tukey test multiple comparisons for the others, * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: Immunohistochemistry, Injection

Journal: Journal of Nanobiotechnology

Article Title: Extracellular vesicle-mediated gene therapy targets BRAF V600E -mutant colorectal cancer by inhibiting the MEK1/2-ERK1/2 pathway

doi: 10.1186/s12951-025-03205-4

Figure Lengend Snippet: Tumor targeting capability and safety of EVs-siBRAF V600E in vivo. ( A ) Biodistribution images of EVs-NC or EVs-siBRAF V600E in major organs and tumors. ( B ) Semi-quantitative analysis of the distribution in tumors. ( C )~( D ) After treatment with PBS, EVs-NC or EVs-siBRAF V600E , related serum biochemistry indicators were analyzed, including hepatic function enzymes, such as ALT ( C ) and AST ( D ); renal function enzymes, such as BUN ( E ) and CREA ( F ); and cardiac function indicators, such as CK ( G ) and CK-MB ( H ), were analyzed in RKO xenograft models. ( I ) ~( L ) Representative images of histological assessments of major organs. After treatment with PBS, EVs-NC and EVs-siBRAF V600E , the lungs, kidneys, spleens and livers of RKO xenograft models from each group were collected. The data are reported as the means ± SDs of the experiments ( n = 5). One-way ANOVA

Article Snippet: The primary antibodies used were against BRAF V600E (ZA-0668, ZSGB-BO, China), KI67 (27309-1-AP, Proteintech), BRAF (ab33899, Abcam), and phospho-p44/42 MAPK (p-ERK1/2) (4370s, Cell Signaling Technology).

Techniques: In Vivo

Journal: BMJ Case Reports

Article Title: BRAF V600E-mutated combined large cell neuroendocrine carcinoma and adenocarcinoma responding to targeted therapy

doi: 10.1136/bcr-2021-243295

Figure Lengend Snippet: (A) The cerebral tumour is a mixture of adenocarcinoma-like cells that form and proliferate small ducts, and atypical cells with abundant acidophilic cytoplasm and distinct nucleoli. (B) Synaptophysin is diffusely positive in all regions. (C) Immunostaining of BRAF V600E with cerebellar tumours stain both adenocarcinoma and large cell neuroendocrine carcinoma. Both cells showing adenocarcinoma morphology and LCNEC cells stained for synaptophysin are stained with BRAF V600E antibody.

Article Snippet: Immunostaining of BRAF V600E (anti- BRAF V600E rabbit monoclonal antibody clones: RM8, RevMAb Biosciences) with cerebellar tumours tested positive in both adenocarcinoma and LCNEC ( ).

Techniques: Immunostaining, Staining