braf v600e  (Roche)

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: Journal for Immunotherapy of Cancer

Article Title: HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAFV600E-mutant microsatellite stable colorectal cancer

doi: 10.1136/jitc-2024-010460

Figure Lengend Snippet: Drug screening results in BRAF V600E MSS CRC based on PDOs and cell lines. ( A ) Graphical overview of the study. ( B ) Heatmap of drug inhibition for the two PDOs. ( C ) Venn diagram showing the intersection of drugs with over 60% inhibition on the two PDOs. ( D ) Bar graph detailing specific inhibition rates of common drugs for the two PDOs. ( E ) Heatmap of drug responses on the three cell lines. ( F ) Venn diagram showing the intersection of drugs with over 60% inhibition on the three cells. ( G ) Bar graph showing specific inhibition rates of common drugs across the three cells. ( H ) Venn diagram showing the intersection of common drugs between the two PDOs and the three cell lines. BRAF, B-Raf proto-oncogene, serine/threonine kinase; CRC, colorectal cancer; HDAC, histone deacetylase; IHC, immunohistochemistry; MEK, mitogen-activated protein kinase kinase; MSS, microsatellite stable; PDO, patient-derived organoid; PD-1, programmed death 1; RNA-seq, RNA sequencing.

Article Snippet: To identify potential therapeutic agents for BRAF V600E MSS CRC, we intersected the Anti-cancer Compound Library (L3000, Selleck) and the Food and Drug Administration-approved Drug Library (L1300, Selleck), resulting in a custom subset of 768 drugs for screening.

Techniques: Drug discovery, Inhibition, Histone Deacetylase Assay, Immunohistochemistry, Derivative Assay, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAFV600E-mutant microsatellite stable colorectal cancer

doi: 10.1136/jitc-2024-010460

Figure Lengend Snippet: HOXC6 contributes to treatment resistance in BRAF V600E MSS CRC and regulates the MAPK and AKT pathways and the MYC gene. ( A ) Left: HOXC6 expression in BRAF V600E and BRAF wild-type groups in TCGA COAD. ****p<0.0001 (Mann-Whitney U test). Right: HOXC6 expression in BRAF V600E dMMR, BRAF wild-type dMMR, BRAF V600E pMMR, and BRAF wild-type pMMR groups in TCGA COAD. ns, not significant; ***p<0.001 (Mann-Whitney U test). ( B ) HOXC6 expression in BRAF V600E MSS and BRAF wild-type MSS CRC cell lines from the CCLE database. **p<0.01 (Student’s t-test). ( C ) HOXC6 mRNA expression in HT-29 and HT-29_EnR cells. *p<0.05 (Student’s t-test). ( D ) IC50 values of encorafenib in HOXC6-knockdown HT-29_EnR cells and control cells. ***p<0.001 (Student’s t-test). ( E ) IC50 values of dabrafenib, encorafenib, and trametinib in HOXC6-overexpressing HT29 cells (HT29-HOXC6) and control cells (HT29-Control), respectively. Results represent means±SD (n=4). *p<0.05 (Student’s t-test). ( F ) Top 10 enriched pathways in HOXC6-overexpressing cells revealed by KEGG analysis. ( G ) Western blotting for HOXC6-overexpressing cells and control cells. ( H ) Western blotting for HOXC6-knockdown cells and control cells. ( I ) Cell viability profiles of four BRAF V600E MSS CRC cell lines treated with dabrafenib, encorafenib, and trametinib, respectively. Results represent means±SD (n=4). ( J ) Western blotting for the four BRAF V600E MSS CRC cell lines. ( K ) Peak annotation analysis indicating the distribution of binding sites. ( L ) Chromatin immunoprecipitation-quantitative results on the percentage of Input for MYC promoter. **p<0.01 (Student’s t-test). ( M ) Relative luciferase activity analyzed in the dual luciferase reporter assay to examine HOXC6 binding to the MYC promoter. MYC_WT, MYC_wild-type; MYC_Mut, MYC_mutant. Results represent means±SD (n=3). **p<0.01; ns, not significant (Student’s t-test). ( N ) Schematic of HOXC6 binding to the MYC gene. AKT, protein kinase B; BRAF, B-Raf proto-oncogene, serine/threonine kinase; COAD, colon adenocarcinoma; CRC, colorectal cancer; dMMR, deficient mismatch repair; ERK, extracellular signal-regulated kinase; HOXC6, homeobox C6; IC50, half-maximal inhibitory concentration; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; MSS, mismatch repair; p-AKT, phosphorylated-AKT; p-ERK, phosphorylated-ERK; PI3K, phosphoinositide 3-kinase; pMMR, proficient mismatch repair; TCGA,The Cancer Genome Atlas; TPM, transcripts per kilobase million.

Article Snippet: To identify potential therapeutic agents for BRAF V600E MSS CRC, we intersected the Anti-cancer Compound Library (L3000, Selleck) and the Food and Drug Administration-approved Drug Library (L1300, Selleck), resulting in a custom subset of 768 drugs for screening.

Techniques: Expressing, MANN-WHITNEY, Knockdown, Control, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Luciferase, Activity Assay, Reporter Assay, Mutagenesis, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAFV600E-mutant microsatellite stable colorectal cancer

doi: 10.1136/jitc-2024-010460

Figure Lengend Snippet: Immune microenvironment characteristics and immune cell infiltration differ between BRAF V600E MSS CRC and BRAF wild-type MSS CRC. ( A ) Volcano plot demonstrating differentially expressed genes between patients with BRAF V600E MSS CRC and patients with BRAF wild-type MSS CRC. ( B ) Top 10 significantly enriched pathways in BRAF V600E MSS CRC identified by GO enrichment analysis. ( C )-( F ) GSEA analysis revealing significant enrichment in pathways such as immunoregulatory interactions between a lymphoid and a non-lymphoid cell ( C ), MHC class II antigen presentation ( D ), Hallmark_IL-2_STAT5_signaling ( E ), and Hallmark_IL-6_JAK_STAT3_signaling ( F ) in BRAF V600E MSS CRC. ( G )-( H ) GSEA analysis illustrating significant enrichment of IL-17 signaling pathway ( G ) and IL-10 signaling pathway ( H ) in BRAF V600E MSS CRC cell lines. ( I )- ( J ) Representative images of multiplex fluorescent immunohistochemistry for CD8 + cells, PD-1 + cells, and DAPI in BRAF wild ( I ) and BRAF V600E ( J ) MSS CRC tissues. Scale bars, 200 µm. ( K ) Quantification of PD-1 + cells in BRAF V600E MSS CRC and BRAF wild-type MSS CRC groups. *p<0.05 (Student’s t-test). ( L ) Quantification of CD8 + cells in BRAF V600E MSS CRC and BRAF wild-type MSS CRC groups. ns, not significant (Student’s t-test). ( M ) ssGSEA analysis showing the scores of immune cell infiltration in BRAF V600E MSS CRC and BRAF wild-type MSS CRC groups. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 (Mann-Whitney U test). BRAF, B-Raf proto-oncogene, serine/threonine kinase; CRC, colorectal cancer; GO, Gene Ontology; GSEA, gene set enrichment analysis; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes; MHC, major histocompatibility complex; MSS, microsatellite stable; PD-1, programmed death 1; ssGSEA, single sample gene set enrichment analysis.

Article Snippet: To identify potential therapeutic agents for BRAF V600E MSS CRC, we intersected the Anti-cancer Compound Library (L3000, Selleck) and the Food and Drug Administration-approved Drug Library (L1300, Selleck), resulting in a custom subset of 768 drugs for screening.

Techniques: Immunopeptidomics, Multiplex Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: HDAC and MEK inhibition synergistically suppresses HOXC6 and enhances PD-1 blockade efficacy in BRAFV600E-mutant microsatellite stable colorectal cancer

doi: 10.1136/jitc-2024-010460

Figure Lengend Snippet: The characteristics of CT26 BRAF V637E cell line. ( A ) Western blotting for CT26 cell, CT26-Control cell, and CT26 BRAF V637E cell. ( B ) Tumor formation rates of subcutaneous inoculation of CT26-Control cells and CT26 BRAF V637E cells. ( C ) Inhibition rates of CT26 BRAF V637E cells under different treatments (trametinib, 0.1 µM; panobinostat, 0.3 µM). Results represent means±SD (n=3). **p<0.01 (Student’s t-test). ( D )-( F ) Gene expression changes of Ifng ( D ), Cxcl9 ( E ) and H2-T3 ( F ) in CT26 BRAF V637E cells under different treatments (trametinib, 0.1 µM; panobinostat, 0.3 µM), respectively. Results represent means±SD (n=3). *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test). ( G ) Western blotting for the cGAS/STING pathway in CT26 BRAF V637E cells with different treatments (trametinib, 0.1 µM; panobinostat, 0.3 µM). ( H ) Western blotting for MMR proteins in CT26 BRAF V637E cells with different treatments (trametinib, 0.1 µM; panobinostat, 0.3 µM). ( I ) Cytokine arrays evaluating changes in the levels of cytokines secreted by CT26 BRAF V637E cells before and after the combination treatment (trametinib, 0.1 µM; panobinostat, 0.3 µM). BRAF, B-Raf proto-oncogene, serine/threonine kinase; cGAS/STING, cyclic guanosine monophosphate–adenosine monophosphate synthase/stimulator of interferon genes; ERK, extracellular signal-regulated kinase; Pano, panobinostat; p-ERK, phosphorylated-ERK; Tra, trametinib.

Article Snippet: To identify potential therapeutic agents for BRAF V600E MSS CRC, we intersected the Anti-cancer Compound Library (L3000, Selleck) and the Food and Drug Administration-approved Drug Library (L1300, Selleck), resulting in a custom subset of 768 drugs for screening.

Techniques: Western Blot, Control, Inhibition, Gene Expression

Journal: bioRxiv

Article Title: Deleting PTEN, but not SOCS3 or myelin inhibitors, robustly boosts BRAF-elicited intraspinal regeneration of peripheral sensory axons

doi: 10.1101/2024.09.18.613685

Figure Lengend Snippet: (A) A schematic diagram of the experimental procedure for 3 week analysis of all transgenic lines. Boxed area specifies region of interest in the spinal cord. (B) tdTomato expression indicates high transgene activation within sensory neurons and afferent projections of the dorsal horn and dorsal columns. Scale bar: 200 μm , 100 μm . (C) Western blot analysis confirms expression of BRAF V600E in kaBRAF iTg but not WT DRG lysates. Total B-RAF protein levels remain unchanged. Molecular mass is noted in kilodaltons. Cross sections of WT (D-D”) or kaBRAF iTg (E-E”’) groups show GFP-labeled axon regeneration at 3 weeks after DR crush injury. WT show spontaneous regeneration along the DR but no regeneration past the DREZ. (E”’, inset) kaBRAF expression promotes axon regeneration across the DREZ and into the superficial laminae of the spinal cord. GFAP staining denotes the astrocytic boundary of the CNS. Dotted line indicates the relative area of the DREZ. (F) Quantification of axon growth shows significantly more axons penetrate the DREZ in kaBRAF iTg than in WT controls. Scale bar: D-E” =100 μm, E”’ = 50 μm. n=4-5 mice per group, at least 5-6 sections per mouse, **** P < 0.0001, ** P < 0.01, two-way ANOVA with Sidak’s multiple comparisons test. Values represent mean ±SEM.

Article Snippet: Antibodies used were mouse anti-BRAF V600E (1:1000, Spring Bioscience), rabbit anti-BRAF (1:1000, Cell Signaling #9433), rabbit anti-PTEN (1:1000, Cell Signaling, #9559), rabbit anti-pS6 (1:1000, Cell Signaling #2215), rabbit anti-survivin (1:1000, CST #2808), and mouse anti-β-actin (1:5000, Sigma, A5441).

Techniques: Transgenic Assay, Expressing, Activation Assay, Western Blot, Labeling, Staining