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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: miRNAs differentially expressed between A375R and A375 cells and KEGG pathway analysis of putative targets. a Heatmap showing miRNA up-regulated (red = expression above the mean) and down-regulated (blue = expression below the mean) in dabrafenib-resistant A375R cells as compared with dabrafenib-sensitive A375 cells (SAM analysis; FC ≥ 2, FDR = 0%). miRNAs are indicated according to annotation provided by Affymetrix. b Putative target genes of differentially expressed miRNAs were obtained from TargetScan and used for KEGG pathway enrichment analysis. Only pathways with an adjusted P value <0.01 were considered and listed according to a decreasing value of the combined score. c, d Expression of miRNAs down-regulated (c) or up-regulated (d) in A375R cells according to microarray results was validated using specific TaqMan® MicroRNA Assays. The data were normalized to the level of RNU44 in each sample and expressed as 2 -ΔCt x10 5 values. Each value represents the arithmetic mean of at least three independent experiments performed with duplicate samples. Bars, standard error of the mean (SEM). ** P <0.01 and * P <0.05, A375R versus ( vs ) A375.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Expressing, Microarray

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: miR-126-3p is up-regulated by dabrafenib only in drug-sensitive cells and inhibits their proliferation. a, b Melanoma cells were incubated with 100 nM dabrafenib (DAB) or with DMSO alone and after 48 h of culture miR-126-3p expression was evaluated by qRT-PCR. 2 -ΔCt x10 5 values calculated relative to RNU44 as the internal reference are shown. Each value represents the arithmetic mean of three independent experiments performed with duplicate samples. Bars, SEM. ** P <0.01 and * P <0.05, DAB vs matched DMSO; ## P <0.01 and # P <0.05, resistant cells/DMSO vs parental cells/DMSO. c A375 and SK-Mel28 were transiently transfected with 50 nM pre-miR-126-3p or pre-miR-CTRL, cultured for six days and then assayed for proliferation by the MTT assay. Data are expressed in terms of absorbance at 595 nM. Each value represents the arithmetic mean of three (A375) or four (SK-Mel28) independent experiments performed with triplicate samples. Bars, SEM. * P <0.05, pre-miR-126-3p vs pre-miR-CTRL.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Transfection, Cell Culture, MTT Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miR-126-3p down-regulation contributes to dabrafenib acquired resistance in melanoma by up-regulating ADAM9 and VEGF-A

doi: 10.1186/s13046-019-1238-4

Figure Lengend Snippet: ADAM9 silencing delays the development of resistance to dabrafenib. A375 cells were seeded into 96-well plates and every eight days transfected with 50 nM siADAM9 or siCTRL and treated with 100 nM dabrafenib (DAB) or DMSO. Cell cultures were photographed and processed for quantitative analysis of proliferation on day 0 (i.e after the first transfection), 8, 16 and 24. Images from a representative experiment are shown. b Quantitative analysis of proliferation of cell cultures described in (a). Crystal violet was solubilized and absorbance was read at 595 nm. Each value represents the arithmetic mean of three independent experiments performed with triplicate cultures. Bars, SEM. ** P <0.01, siADAM9 vs matched siCTRL; §§ P <0.01, siCTRL/DAB/Day 8 vs siCTRL/DMSO/Day 8; †† P <0.01, siADAM9/DAB/Day 8 vs siADAM9/DMSO/Day8; ## P <0.01, siCTRL/DAB/Day 16 vs siCTRL/DAB/Day 8; ⁋⁋ P <0.01, siCTRL/DAB/Day 24 vs siCTRL/DAB/Day 16.

Article Snippet: The BRAF-mutant human melanoma cell lines A375 and SK-Mel28 were purchased from the European Collection of Cell Cultures (Salisbury, UK) and American Type Culture Collection (ATCC, Manassas, VA), respectively.

Techniques: Transfection

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: MC1R expression is upregulated in human melanoma cells upon treatment with BRAFi and HDACi. (A) qRT-PCR analysis of MC1R mRNA expression in BRAFV600E A2058 cells after 24 h of incubation with BRAFi dabrafenib (Dabr) and HDACi 4-phenylbutyrate (PBA) and in BRAFWT MEWO cells after 24 h treatment with HDACi PBA and vorinostat (Vor) (n = 3). Data are presented as normalized mean MC1R mRNA ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs controls; (B) flow cytometry histograms and protein expression of MC1R in A2058 cells after 24 h treatment with BRAFi: Vem (5 μM) and Dabr (2 μM); HDACi: PBA (2 mM) and Vor (2 μM); and in MEWO cells after incubation with PBA (2 mM) and Vor (2 μM). Experiments were conducted in triplicate. Data are expressed as relative expression of MC1R vs isotype control (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs controls); (C) MC1R binding with [125I]NDP-α-MSH in A2058 and MEWO cells following incubation with Dabr (1−10 μM), Vor (0.5−10 μM), and PBA (0.5−10 mM) for 12−24 h (n = 4). Data are expressed as MC1R-ligand binding vs dimethyl sulfoxide (DMSO)-treated cells (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs controls); all experiments were performed in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry, Control, Binding Assay, Ligand Binding Assay

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Upregulation of MC1R in melanoma cells is mediated by the transcription factor MITF. Immunoblotting analysis of MC1R and MITF expressions in (A) BRAFV600E cells (A2058) following 24 h incubation with dabrafenib (Dabr) (1−10 μM) and vorinostat (Vor) (1−10 μM) and in (B) BRAFWT cells (MEWO) following 24 h exposure to HDACi vorinostat (Vor) (1−10 μM) and 4-phenylbutyrate (PBA) (1−10 mM); immunoblotting analysis of MC1R in (C) A2058 and (D) MEWO cells in response to 24 h incubation with Dabr (1 μM), Vor (1 μM), or PBA (1 mM), with normal MITF expression (negative scrambled Dsi-RNA) and attenuated MITF expression (MITF Dsi-RNA). All experiments were conducted in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Western Blot, Incubation, Expressing

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Combination of BRAF, HDAC inhibitors, and MC1R-targeted 212Pb α-particle therapy significantly impairs BRAFV600E melanoma A2058 tumor growth and improves survival. (A) Representative IHC staining of MC1R in A2058 melanoma-bearing animals that were treated with HDACi PBA (90 mg kg−1, i.p., q.d.), BRAFi vemubrafenib (10 mg kg−1, p.o., b.i.d.) (n = 2); (B) 2 h postinjection SPECT/CT imaging of A2058 melanoma in athymic nu/nu mice treated with vemurafenib (Vem 10 mg kg−1, p.o.) and 4-phenylbutyrate (PBA 90 mg kg−1, i.p.) using [203Pb]DOTA-MC1L as the imaging tracer. Organs of interest are indicated as T (tumor), K (kidney), and B (bladder); (C) average tumor volume for each group of animals after treatments were initiated; data are expressed as mean ± SD; statistical analysis: *p < 0.05, **p < 0.01; (D) overall fractional survival in each treatment cohort over 100 days (n = 9−10 per group): vemurafenib (Vem) (10 mg kg−1, p.o., b.i.d.), 4-phenylbutyrate (PBA) (90 mg kg−1, i.p. q.d.), and 212Pb α-particle therapy (212Pb) (single dose of 5.2 MBq [212Pb]DOTA-MC1L). Statistical analysis: ****p <0.0001 212Pb versus control; ****p < 0.0001 212Pb versus Vem; **p < 0.01 212Pb/Vem vs 212Pb; **p < 0.01 212Pb/Vem/PBA vs 212Pb/Vem.

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Immunohistochemistry, Single Photon Emission Computed Tomography, Imaging, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Detection of MKRN1-BRAF fusion by next-generation sequencing. ( A ) Representative part of NGS sequencing of the MKRN1–BRAF fusion in THJ-16T cells. Green part, BRAF gene; blue part, MKRN1 gene; red part, the MKRN1-BRAF fusion. ( B ) Schematics of wild-type BRAF (green), wild-type MKRN1 (blue), BRAFV600E (yellow), and the fused MKRN1-BRAF proteins. The zinc finger domains of MKRN1 and the serine-threonine (S/T) kinase domain of BRAF remain intact in the fused protein. WT, wild-type; ex, exon; RBD, Ras-binding domain; CRD, cysteine-rich domain.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 °C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Next-Generation Sequencing, Sequencing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Identification of mutant BRAF expression by reverse transcription–polymerase chain reaction and Sanger sequencing. Reverse transcription–polymerase chain reaction (RT-PCR) detected BRAF exon 15 and MKRN1-BRAF transcripts in ATC cells. Sanger sequencing chromatograph of the BRAF exon 15 and MKRN1–BRAF fusion in THJ-11T ( A ), THJ-16T ( B ), THJ-21T ( C ), and Nthyori 3-1 cells ( A , C ). The arrow shows the breakpoint of the fusion between MKRN1 (NM_001145125, end of exon 3) and BRAF (NM_004333, start of exon 10).

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 °C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Mutagenesis, Expressing, Reverse Transcription, Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Differential expression of pBRAF, BRAF, pMEK, MEK, pERK, ERK in Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells. ( A ) Western blotting analyses of pBRAF, BRAF, pMEK, MEK, pERK, and ERK levels in Nthyori 3-1, THJ-16T, and THJ-21T cells. GAPDH served as a loading control. ( B ) Immunocytochemical staining (scale bar, 5 μm) of pERK performed on Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 °C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Quantitative Proteomics, Western Blot, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Differential pBRAF, BRAF, pMEK, MEK, pERK, ERK expression in THJ-11T, THJ-16T, and THJ-21T cells without and with resveratrol (R), trametinib (T), dabrafenib and trametinib (D+T), dabrafenib (D) and/or trametinib (T) in combination with resveratrol (R) treatment. ( A ) Western blotting was performed on the sample proteins of THJ-11T, THJ-16T, and THJ-21T cells before and after 48h drug treatment, GAPDH served as a loading control. ( B ) pERK immunocytochemical staining (scale bar, 5 μm) performed on THJ-11T, THJ-16T, and THJ-21T cells after 48h drugs treatment.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 °C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot, Control, Staining

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: Blood

Article Title: Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults

doi: 10.1182/blood-2016-12-757823

Figure Lengend Snippet: Patients. Summary of adult patients included in the study. Asterisks indicate the number of patients with BRAFV600E detectable in PBMCs. The BRAF wild-type (WT) ECD group includes 1 patient with RAS mutation. NT, not tested.

Article Snippet: BRAF mutation and reference quantitative PCR was performed with competitive allele-specific Taqman mutation detection assays: Mutation Allele Assay, BRAF _476_mu 4465804 Hs00000111_mu; Gene Reference Assay, BRAF _rf - 4465807 Hs00000172_rf, according to the manufacturer’s instructions (Life Technologies; http://www.lifetechnologies.com ).

Techniques: Mutagenesis

Journal: Blood

Article Title: Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults

doi: 10.1182/blood-2016-12-757823

Figure Lengend Snippet: Detection of BRAFV600E alleles. (A) A standard curve constructed using DNA purified from a dilution series of BRAFV600E-positive melanoma cell line A375 into WT Epstein-Barr virus–transformed lymphoblastoid cell line. The quantitative limit of detection was 0.1%; the absolute limit of detection was 0.01% (positive outside of quantitative range [POQR]). (B) BRAFV600E allele frequency in bulk PBMCs in cases of lesion BRAFV600E + LCH, LCH/ECD, and ECD. Contingency of positive PBMCs upon MS-LCH tested by Fisher’s exact test. (C) Correlation between mutated allele burden in cell-free plasma and PBMC DNA in LCH, ECD, and HCL. (Di) Test of exogenous free DNA uptake by PBMCs. BRAF-mutated DNA (derived from melanoma cell line A375) was spiked into whole blood for 24 hours at room temperature. Plasma and PBMCs were isolated by density centrifugation. DNA was extracted from both fractions and subjected to allele-specific PCR. (Dii) Test of exogenous free DNA uptake by sorted cells incubated at 37°C for 24 hours in medium supplemented with 20% human serum from a patient with HCL. DNA from supernatants and cell pellets, as indicated, was subjected to allele-specific PCR. mDC, myeloid dendritic cell.

Article Snippet: BRAF mutation and reference quantitative PCR was performed with competitive allele-specific Taqman mutation detection assays: Mutation Allele Assay, BRAF _476_mu 4465804 Hs00000111_mu; Gene Reference Assay, BRAF _rf - 4465807 Hs00000172_rf, according to the manufacturer’s instructions (Life Technologies; http://www.lifetechnologies.com ).

Techniques: Construct, Purification, Virus, Transformation Assay, Clinical Proteomics, Derivative Assay, Isolation, Centrifugation, Incubation

Journal: Blood

Article Title: Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults

doi: 10.1182/blood-2016-12-757823

Figure Lengend Snippet: Mononuclear profile and BRAF allele frequency in CD34+ BM progenitors. (A) Relative proportions of progenitor fractions among CD34+ BM mononuclear cells compared with healthy controls (n = 21), expressed as percentage of total live cells. Error bars depict 95% confidence intervals of healthy controls. Populations: B/NK, B/NK cell progenitors; LMPP, lymphoid-primed multipotent progenitors; MEP, megakaryocyte–erythroid progenitor; MPP, multipotent progenitor. (B) The distribution of BRAFV600E among CD34+ BM progenitors. Gray shading indicates positive fractions. BM aspirate was obtained in parallel with the peripheral blood shown previously. *Child; †NRASQ61R Sanger sequencing. NT, not tested owing to insufficient number of cells.

Article Snippet: BRAF mutation and reference quantitative PCR was performed with competitive allele-specific Taqman mutation detection assays: Mutation Allele Assay, BRAF _476_mu 4465804 Hs00000111_mu; Gene Reference Assay, BRAF _rf - 4465807 Hs00000172_rf, according to the manufacturer’s instructions (Life Technologies; http://www.lifetechnologies.com ).

Techniques: Sequencing

Journal: Blood

Article Title: Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults

doi: 10.1182/blood-2016-12-757823

Figure Lengend Snippet: Table summarizing BRAF V600E mutant allele frequency among blood monocytes and myeloid DCs (data from ) and the potential of WT cells to form langerin high LCH-like cells or foamy macrophages in vitro

Article Snippet: BRAF mutation and reference quantitative PCR was performed with competitive allele-specific Taqman mutation detection assays: Mutation Allele Assay, BRAF _476_mu 4465804 Hs00000111_mu; Gene Reference Assay, BRAF _rf - 4465807 Hs00000172_rf, according to the manufacturer’s instructions (Life Technologies; http://www.lifetechnologies.com ).

Techniques: Mutagenesis

Journal: Blood

Article Title: Hematopoietic origin of Langerhans cell histiocytosis and Erdheim-Chester disease in adults

doi: 10.1182/blood-2016-12-757823

Figure Lengend Snippet: Potential precursor pathways in MS-LCH and ECD. Schema summarizing potential precursor pathways in MS-LCH and ECD based on the distribution of BRAFV600E alleles in peripheral blood and the differentiation potentials of healthy control cells in vitro. A minority of cells (filled) in the BM and blood contain BRAF mutation. The principal observation is that <1% clonal hematopoiesis gives rise to <3% mutated blood precursors that appear to be strongly selected for in peripheral tissue environments, resulting in lesional histiocyte mutation levels of up to 100%. Lesions potentially consist of >1 precursor as indicated by blue for CD1c+ DCs, red for classical monocytes, and brown for nonclassical monocytes. The depiction of lesion composition is conjectural and not based on experimental observation. Furthermore, additional contributions from other, more primitive myeloid progenitors cannot be excluded at present.

Article Snippet: BRAF mutation and reference quantitative PCR was performed with competitive allele-specific Taqman mutation detection assays: Mutation Allele Assay, BRAF _476_mu 4465804 Hs00000111_mu; Gene Reference Assay, BRAF _rf - 4465807 Hs00000172_rf, according to the manufacturer’s instructions (Life Technologies; http://www.lifetechnologies.com ).

Techniques: Control, In Vitro, Mutagenesis

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 and TG003 chemical structures. ( b ) Pharmacokinetic profile of TG693 after a single 30 mg kg −1 dose administered by subcutaneous injection in imprinting control region (ICR) mice. Data indicate the mean ± SEM (n = 3). ( c ) Recombinant CLK1 was incubated with the substrate peptide in the presence of the indicated concentrations of small molecules. Data represent the means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) TG693 competitive ATP inhibition is shown in Michaelis-Menten (left) and Hanes-Woolf (right) plots. CLK1 kinase activity was measured at the indicated concentrations of TG693 and ATP. Velocity was plotted versus [ATP] and [ATP]/velocity was plotted versus [ATP]. ( e ) Map of the inhibitory activities of TG693 on a kinase dendrogram. Percent inhibition by 1 μM TG693 was measured for a panel of 313 kinases. Red circles indicate the inhibited kinases and are sized according to percent inhibition. The illustration was reproduced courtesy of Cell Signaling Technology, Inc. ( www.cellsignal.com ).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Injection, Recombinant, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) SR protein phosphorylation was assessed in HeLa cells treated with TG693 and TG003 for 1 h. Lamin B served as a loading control. Uncropped images have been provided in . ( b , c ) Effect of TG693 on exon 31 skipping with the reporter plasmid. Transfected HeLa cells were incubated in the presence of TG693, TG003, or DMSO vehicle for 24 h. Reporter and endogenous CLK1 splicing was then analyzed by RT-PCR. GAPDH served as a control. The Splicing ratios were quantified by intensity analysis and normalized to GAPDH expression. Uncropped images have been provided in and , respectively. Data represent the means ± SD (n = 3).

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Plasmid Preparation, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Scientific Reports

Article Title: Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy

doi: 10.1038/srep46126

Figure Lengend Snippet: ( a ) TG693 bioavailability in the tibialis anterior (TA) muscle of ICR mice after oral administration of a single 30 mg kg −1 dose. Data represent the mean ± SEM (n = 3). ( b ) SR protein phosphorylation status in the TA muscle of ICR mice after oral administration. Lamin B served as a loading control. SRSF4 phosphorylation was quantified by densitometry. Uncropped images are provided in . Data represent means ± SD (n = 5). * p < 0.05. ( c,d ) Clk1 expression in the TA muscle, heart and diaphragm were analyzed by RT-PCR with a GAPDH internal control. Uncropped images are provided in and in , respectively. Data represent the means ± SD (n = 3). * p < 0.05.

Article Snippet: The reaction mixture containing serially diluted inhibitors, 10 mM MOPS-KOH (pH 6.5), 10 mM magnesium chloride, 200 μM EDTA, 1 μM ATP, 0.167 μCi of [γ- 32 P] ATP, 0.417 μg of synthetic RS peptide, and recombinant GST-tagged human CLK1 (Cat #04–126, Carna Biosciences, Kobe, Japan) was prepared in a final volume of 25 μL.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits proliferation in BRAF V600E or RAS mutant cell lines. a Chemical structure of erianin. b Cytotoxicity of erianin in normal NHEM and NHDF cell lines using MTT assay. c The left panel shows representative dose–response curves by MTT assay. The SK-MEL-2 (NRAS mut), HCT116 (KRAS mut), A375 (BRAF V600E), and SK-MEL-28 (BRAF V600E) cell lines were exposed to erianin for 72 h. The concentrations are transformed to Log10 values; the Y -axis shows the corresponding relative cell viability. The right panel shows IC50 values of erianin, three BRAF inhibitors (vemurafenib, dabrafenib and encorafenib) and three MEK inhibitors (cobimetinib, trametinib, and binimetinib) calculated in GraphPad Prism 7.0. d The effect of erianin on growth of SK-MEL-2, A375, SK-MEL-28, and HCT 116 cells was estimated by MTT assay at 24, 48, or 72 h. Data were shown as means ± S.D. e The effect of erianin on anchorage-independent growth in above cells was evaluated. Data were shown as means ± S.D. Scale bars: 400 μm. The colonies numbers were calculated in Image-Pro Plus software. * p < 0.05; ** p < 0.01; *** p < 0.001. f Synergism effect of erianin and vemurafenib in SK-MEL-2, A375, SK-MEL-28, and HCT 116. The synergism and antagonism (CI value) were determined and analyzed using CompuSyn 1.0. CI value > 1.1 indicates antagonism, 1.1 ≥ CI value > 0.9 shows addictive effect and CI value ≤ 0.9 indicates synergism

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, MTT Assay, Transformation Assay, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin suppresses either BRAF V600E or RAS mutant cell growth in CDX model. a NOD-SCID mice were injected subcutaneously with SK-MEL-2 (NRAS mut, 5 × 10 6 cell/mouse), A375 (BRAF V600E, 1 × 10 7 cell/mouse), SK-MEL-28 (BRAF V600E, 5 × 10 6 cell/mouse) and HCT116 (KRAS mut, 1 × 10 7 cell/mouse) cells mixed with Matrigel (1:1); erianin (50 mg/kg), vemurafenib (50 mg/kg) or the combine was given through oral gavage and the size of the tumors was monitored twice per week. Tumor volume (mm 3 ) = (length × width × height) × 0.52. SK-MEL-2: n = 10; A375 and SK-MEL-28: n = 8; HCT116: n = 9. b The photographs show tumors from CDX mice treated with vehicle, erianin, vemurafenib, or the combination. c The weight of the tumors was quantified and expressed as the treatment groups compared with the vehicle-treated group. Data were presented as mean ± S.D. One-way ANOVA test. * p < 0.05; ** p < 0.01. d Western blotting shows the expression of phospho-MEK1/2 and phospho-ERK1/2 by erianin in SK-MEL-2, A375, and SK-MEL-28 CDX tumor tissues. The tissue lysates were prepared from CDX tumor tissues in each treatment group. Three samples were randomly prepared for each group and every blot shows one sample. e The quantization (IOD values) of IHC staining in the treatment groups compared with the vehicle-treated group. Each point represents the IOD values of four quantified data from one mouse. Scale bars: 50 μm. One-way ANOVA test. *** p < 0.001. f Kaplan–Meier curve depicting tumors less than 1000 mm 3 in the treatment groups compared with the vehicle-treated group

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, Injection, Western Blot, Expressing, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin exerts antitumor efficacy in melanoma and colorectal cancer in vivo. a Tumor pharmacodynamic assay was performed in tumor-bearing NPG mice (tumor has been passaged from melanoma patient to mice for three generations). The photographs show tumors from melanoma PDX mice treated with vehicle or drugs. b The effect of erianin on the volume of PDX tumors over time (within 78 days) was plotted. Vehicle, erianin (50 mg/kg, once a day), vemurafenib (50 mg/kg, once a day), erianin and vemurafenib combination therapy, cobimetinib (5 mg/kg, twice a week) or vemurafenib and cobimetinib combination therapy (once a day and twice a week, respectively) were administered by oral gavage, n = 8 in each group. Tumor volume was measured once a week. One-way ANOVA test. * p < 0.05; ** p < 0.01. c Tumor weight was measured after treatment on the last day of the study. d The expression of phospho-MEK1/2 and phospho-ERK1/2 were examined by immunofluorescence analysis. Scale bars: 20 μm. One-way ANOVA test. *** p < 0.001. e Antitumor efficacy of erianin with or without immunity using B16F10 cell xenograft in C57BL-6J mouse. f , g Trend of tumor volume over time and tumor weight was measured after treatment on the last day of the study. One-way ANOVA test. * p < 0.05; ** p < 0.01. h The model depicts that erianin suppresses constitutive activation of MAPK signaling pathway in either BRAF V600E or RAS mutant cancers (Created with BioRender.com). Through inhibition of CRAF and MEK1/2 kinases, erianin suppresses phospho-MEK1/2 and phospho-ERK1/2 without paradoxical activation in vitro and in vivo

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: In Vivo, Expressing, Immunofluorescence, Activation Assay, Mutagenesis, Inhibition, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

doi: 10.1074/jbc.RA119.012365

Figure Lengend Snippet: MAP3K19 maintains MEK phosphorylation in the presence of RAF inhibitors. A, 48 h after transfection with EV, MAP3K19-WT, or MLK1-WT HEK293T cells were treated with DMSO vehicle control, 1 μm L779450, 1 μm AZD6244, or a combination of both RAF and MEK inhibitors for 1 h, and Western blotting was performed on cell lysates. B, 48 h after transfection with EV, WT, or KD-MAP3K19, HEK293T cells were treated with DMSO vehicle control or indicated inhibitors: 1 μm vemurafenib (BRAFi), 1 μm selumetinib (S, MEKi), or 500 nm cobimetinib (C, MEKi) for 1 h, and Western blotting was performed on cell lysates. Band density was quantified by ImageJ software. The data are shown as mean phospho:total protein density ± S.D. Dunnett's multiple comparisons test was used for statistical analysis, with EV-DMSO group as control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: In vitro kinase assay Recombinant human GST-tagged MAP3K19 (YSK4) kinase domain (Thermo Fisher Scientific) or human MLK1 kinase domain (Carna Biosciences) was incubated at room temperature with kinase-inactive MEK1, ERK2, MKK7, JNK1, or JNK2 (Carna Biosciences) in the absence or presence of MAPK pathway inhibitors for 5 min.

Techniques: Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

doi: 10.1074/jbc.RA119.012365

Figure Lengend Snippet: MAP3K19 directly phosphorylates MAP2Ks. A, MAP3K19 was immunoprecipitated (IP) from HEK293T cells and subjected to a kinase assay with kinase-inactive MEK1. B and C, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MEK1 or ERK2, respectively. D, kinase-inactive MEK1 and purified GST-MAP3K19 or GST-MLK1 kinase domain were subjected to in vitro kinase assay in the presence or absence of inhibitors: 5 μm L779450, 1 μm PLX4032, 5 μm U0126, or 2 μm AZD6244. E and F, in vitro kinase assay using recombinant MAP3K19 protein and kinase-inactive MKK7 or JNK1/2, respectively. The data are shown as mean phospho:total protein density ± S.D. Dunnett's multiple comparisons test was used for statistical analysis, with samples in the first lane as control. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ♢, kinase-inactive.

Article Snippet: In vitro kinase assay Recombinant human GST-tagged MAP3K19 (YSK4) kinase domain (Thermo Fisher Scientific) or human MLK1 kinase domain (Carna Biosciences) was incubated at room temperature with kinase-inactive MEK1, ERK2, MKK7, JNK1, or JNK2 (Carna Biosciences) in the absence or presence of MAPK pathway inhibitors for 5 min.

Techniques: Immunoprecipitation, Kinase Assay, In Vitro, Recombinant, Purification

Journal: Cancer Discovery

Article Title: A Next-Generation BRAF Inhibitor Overcomes Resistance to BRAF Inhibition in Patients with BRAF -Mutant Cancers Using Pharmacokinetics-Informed Dose Escalation

doi: 10.1158/2159-8290.CD-24-0024

Figure Lengend Snippet: Preclinical characterization of pan-mutant BRAF selective monomer/dimer inhibitor PF-07799933. A, Heatmap of 50% inhibitory concentration (IC 50 ) values for inhibition of pERK in human cancer cell lines for PF-07799933 and comparator RAF inhibitors, as derived from dose–response curves in Supplementary Fig. S1. Each cell line with specific BRAF mutation is summarized in Supplementary Table S1. Note color scaling for BRAF wild-type cell lines is distinct from BRAF -mutant cell lines. Unsupervised clustering of compounds is shown using Euclidean distance as a similarity metric. B, Immunoprecipitation of BRAF and CRAF dimer complexes in the MEL21514 (p61 BRAF splice variant) melanoma cell line. Comparison of dimer-breaking effects of PF-07799933 and encorafenib are shown at indicated drug concentrations for a 1-hour incubation period. C, Efficacy curves of mean tumor volumes in mice ( n = 8–10) bearing subcutaneous xenografts (left) and change in flux measurements of intracranial xenografts (right) of Class I A375 ( BRAF V600E ) melanoma cells following oral treatment with the indicated agents. D, Efficacy curves of mean tumor volumes in mice ( n = 8–10) bearing subcutaneous patient-derived or cell line xenografts of Class II, indel, and Class I acquired resistance models following oral treatment with the indicated agents. IC 50 , 50% inhibitory concentration; nM, nanomolar; INDEL, insertion/deletion; WT, wild-type; IP, immunoprecipitated; FL, full-length; mm 3 , cubic millimeter; SEM, standard error of mean; QD, once daily; BID, twice daily; mpk, milligrams per kilogram; NSCLC, non–small cell lung cancer; PDAC, pancreatic adenocarcinoma.

Article Snippet: Lysates were incubated with either BRAF (CST #53745) or CRAF (CST #14814) rabbit polyclonal antibodies at 1:100 dilution overnight at 4°C followed by incubation with protein-A conjugated magnetic beads (Dynabeads, Thermo Inc.) for 90 minutes at room temperature (22 °C).

Techniques: Mutagenesis, Concentration Assay, Inhibition, Derivative Assay, Immunoprecipitation, Variant Assay, Comparison, Incubation