Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression leads to neuronal cell death and glial cell proliferation in primary mouse cortical mixed culture. Primary cortex mix cultures were prepared from C57BL/6J embryos. Cells were cultured in NB-A for 5 days, then transduced with viral vector, or BRAF WT , or BRAF V600E for 24 hours and then cultured in NB-A medium for 96 hours. (A) BRAF immunoblot and quantified BRAF expression level normalized to GAPDH. (B) Immunostaining for MAP2 and Iba1, and MAP2 + and Iba1 + cell counts in cultures transduced with lentiviral BRAF vectors. MAP2 (red, neurons), Iba1 (green, microglia), DAPI (blue, nuclei). (C) BRAF immunoblot and quantified BRAF expression level normalized to GAPDH. (D) Immunostaining for MAP2 and Iba1, and MAP2 + and Iba1 + cell counts in cultures transduced with retroviral (RV) BRAF vectors. MAP2 (red, neurons), Iba1 (green, microglia), DAPI (blue, nuclei). (E) Immunostaining for MAP2 and GFAP, and MAP2 + and GFAP + cell counting in cultures transduced with lentiviral BRAF vectors. MAP2 (green, neurons), GFAP (red, astrocytes), DAPI (blue, nuclei). (F) Immunostaining for MAP2 and GFAP, and MAP2 + cells and GFAP + cell counting in cultures transduced with RV BRAF vectors. The nuclei were counterstained by DAPI. MAP2 (green, neurons), GFAP (red, astrocytes), DAPI (blue, nuclei). Bars: 100 μm. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates under each condition. DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP2: microtubule-associated protein 2; NB-A: neurobasal media.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Western Blot, Immunostaining, Retroviral, Cell Counting

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in astrocytes promotes proliferation. Primary astrocytes were prepared from C57BL/6J embryos. Cells were transduced with lentiviral vector, or BRAF WT , or BRAF V600E for 24 hours and then cultured in DMEM/F-12 medium for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B) Flow cytometry analysis of cell cycle. (C) MTS cell viability assay at 48, 72, 96 and 120 hours after viral transduction. (D) Immunostaining for GFAP and Ki67, GFAP + cell counting, and % of Ki67 + astrocytes (scale bar: 100 μm). GFAP (red, astrocytes), DAPI (blue, nuclei), Ki-67 (green, proliferative cells). (E, F) qPCR analysis of inflammatory and antioxidant markers normalized to GAPDH. ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; qPCR: quantitative polymerase chain reaction.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Western Blot, Flow Cytometry, Viability Assay, Immunostaining, Cell Counting, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in microglia induces cell proliferation and activation through ERK. Primary microglia cells were prepared from C57BL/6J embryos. Cells were transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours and then cultured in DMEM/F12 for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B, C) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for ERK and JNK 48 hours after viral transduction and quantified expression levels normalized to GAPDH. (D) Cells were transfected with control-siRNA, or si-JNK, or si-ERK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF-related signaling proteins and quantified expression levels normalized to GAPDH. (E, F) Immunostaining for Iba1 and Ki67, Iba1 + cell counts, and percentage of Ki67 + microglia (scale bar: 100 μm), and quantitative morphological analyses (percentage of ameboid-like microglia cells, length, area, length to area ratio in cells without and with siRNA transfection (scale bar: 50 μm). Iba1 (green, astrocytes), DAPI (blue, nuclei), Ki67 (red, proliferative cells). (G, H) Flow cytometry analysis of cell cycle. (I) MTS cell viability assay at 48, 72, 96 and 120 hours following viral transduction. (J) NO release in culture media by Griess reaction. (K) qPCR analysis of inflammatory and antioxidant markers in cells normalized to GAPDH. (L) IL-1β, IL-6 and TNF-α levels in culture medium measured by ELISA. Data are represented as mean ± SEM, n = 9. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates under each condition. ERK: Extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; NO: nitric oxide; qPCR: quantitative polymerase chain reaction.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Activation Assay, Transduction, Plasmid Preparation, Cell Culture, Western Blot, Transfection, Control, Immunostaining, Flow Cytometry, Viability Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: Conditioned medium from BRAF V600E -expressing microglial cells but not astrocytes induces neuronal cell death. Primary cortex neurons were prepared from C57BL/6J embryos and cultured for 5 days, and the original medium was then replaced with conditional medium for 72 hours. (A, B) Immunostaining for MAP2 (scale bar: 50 μm) and MAP2 + cell counting. (C) LDH release in neurons treated with conditioned medium from primary astrocytes transduced with lentiviral BRAF vectors. (D–F) Immunostaining for MAP2 (scale bar: 50 μm; D) and MAP2 + cell counting in neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection (E, F). (G, H) LDH release from neurons treated with conditioned medium from primary microglia transduced with lentiviral BRAF vectors with and without siRNA transfection. * P < 0.05, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in A and D. DAPI: 4′,6-Diamidino-2-phenylindole; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Immunostaining, Cell Counting, Transduction, Transfection

Journal: Neural Regeneration Research

Article Title: Oncogenic BRAF V600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase

doi: 10.4103/1673-5374.361516

Figure Lengend Snippet: BRAF V600E expression in neurons promotes cell death through the JNK pathway. Primary cortex neurons were prepared from C57BL/6J embryos. Cells were cultured for 5 days, transduced with lentiviral vector, BRAF WT and BRAF V600E for 24 hours, and then cultured in NB-A for 96 hours. (A) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (B) qPCR analysis c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (C–E) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. (F) Primary cortex neurons were transfected with control-siRNA or si-JNK for 24 hours before transduction with BRAF viral vectors. Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (G) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (H) Immunostaining for MAP2 (sale bar: 50 μm), (I) MAP2 + cell counting, (J) and LDH release. Primary cortex neurons were transfected with control-siRNA or si-ERK for 24 hours before transduction with BRAF viral vectors. (K) Immunoblotting for BRAF and related signaling proteins and quantified expression levels normalized to GAPDH. (L) qPCR analysis of c-Jun, Bax, Bcl-2, p53, Fasl and TNF-α normalized to GAPDH. (M–O) Immunostaining for MAP2 (scale bar: 50 μm), MAP2 + cell counting, and LDH release. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance and Tukey’s post hoc test). All experiments were repeated at least three times with at least three replicates within each condition. MAP2 (green, neurons), DAPI (blue, nuclei) in C, H, and M. DAPI: 4′,6-Diamidino-2-phenylindole; ERK: extracellular signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; JNK: c-Jun N-terminal kinase; LDH: lactate dehydrogenase; MAP2: microtubule-associated protein 2; NB-A: neurobasal media; qPCR: quantitative polymerase chain reaction; TNF-α: tumor necrosis factor-alpha; WT: wide type.

Article Snippet: Constructs used were: pMD2.G (Addgene, Cat# 12259, RRID: Addgene_12259), psPAX2 (Addgene, Cat# 12260, RRID: Addgene_12260), pHAGE- BRAF V600E plasmid (Addgene, Cat# 116204, RRID: Addgene_116204), pHAGE- BRAF WT plasmid (Addgene, Cat# 116719, RRID: Addgene_116719), pBabe-Puro- BRAF V600E plasmid (Addgene, Cat# 15269, RRID: Addgene_15269), gag/pol-Retroviral plasmid (Addgene, Cat# 14887, RRID: Addgene_14887), Control siRNA (Cell Signaling Technology, Boston, MA, USA, Cat# 6568S), SAPK/JNK siRNA (Cell Signaling Technology, Cat# 6232S), ERK1 siRNA (Cell Signaling Technology, Cat# 6436S), ERK2 siRNA (Cell Signaling Technology, Cat# 6578S).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Western Blot, Immunostaining, Cell Counting, Transfection, Control, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: BRAF V600E -induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT

doi: 10.3892/ol.2019.10360

Figure Lengend Snippet: The TCGA_THCA dataset was used for the following analysis. (A) Compared with the BRAF wild type group, the expression of KRT19 was increased in BRAF V600E group, however, not in BRAF wild type group. The (B) wild type BRAF or (C) V600E mutated BRAF overexpression plasmids were transfected into 8505C cells, and may be overexpressed significantly compared with their empty vectors, BRAF WT /BRAF V600E . (D) mRNA and (E) protein expression levels of the BRAF V600E mutation group exhibited higher expression levels of KRT19 in 8505C cells when compared with BRAF wild type group. BRAF, B-Raf Proto-Oncogene, Serine/Threonine Kinase; TCGA, The Cancer Genome Atlas Network; THCA, thyroid cancer; KRT19, keratin 19; WT, wild type.

Article Snippet: The wild type (WT) BRAF WT (pcDNA3.1, cat. no. 40775; Addgene, Inc.) with its empty vector were transfected to the cells followed by Lipofectamine 2000 instruction and mutant BRAF V600E (pBabe, cat. no. 15269; Addgene, Inc.) with its empty vector were transfected into retrovirus packaging phoenix cells (with an VSV.G envelope plasmid; cat. no. 14888; Addgene, Inc.), after 48 h culturing the virus supernatant was harvested and filtered.

Techniques: Expressing, Over Expression, Transfection, 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 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: Oncology Letters

Article Title: Analysis of the clonal origin and differences in the biological behavior of multifocal papillary thyroid carcinoma

doi: 10.3892/ol.2024.14677

Figure Lengend Snippet: Histology of individual multifocal papillary thyroid carcinoma samples. Representative images of (A) BRAF protein-positive, (B) BRAF protein-negative, (C) TERT protein-positive and (D) TERT protein-negative samples. Magnification, ×40. BRAF, v-raf murine sarcoma viral oncogene homolog B1; TERT, telomerase reverse transcriptase.

Article Snippet: The primary antibodies used were mouse anti-human BRAF monoclonal antibody (cat no. TA500431) and rabbit anti-human TERT polyclonal antibody (cat. no. TA324097), both from OriGene Technologies, Inc, a dilution of 1:200.

Techniques: Reverse Transcription

Journal: Oncology Letters

Article Title: Analysis of the clonal origin and differences in the biological behavior of multifocal papillary thyroid carcinoma

doi: 10.3892/ol.2024.14677

Figure Lengend Snippet: BRAF V600E mutation in multifocal papillary thyroid carcinoma. Representative (A) BRAF V600E mutant and (B) wild-type amplification plots generated using quantitative PCR. The red marking represents the threshold line. BRAF, v-raf murine sarcoma viral oncogene homolog B1; BRAF-IC, BRAF internal reference gene detection curve; BRAF-W, BRAF V600E wild-type gene detection curve; BRAF-M, BRAF V600E mutation gene detection curve; DRn, change in relative fluorescence normalized.

Article Snippet: The primary antibodies used were mouse anti-human BRAF monoclonal antibody (cat no. TA500431) and rabbit anti-human TERT polyclonal antibody (cat. no. TA324097), both from OriGene Technologies, Inc, a dilution of 1:200.

Techniques: Mutagenesis, Amplification, Generated, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Oncology Letters

Article Title: Analysis of the clonal origin and differences in the biological behavior of multifocal papillary thyroid carcinoma

doi: 10.3892/ol.2024.14677

Figure Lengend Snippet: Gene mutation and protein expression in cases of multifocal papillary thyroid carcinoma with different clonal origins.

Article Snippet: The primary antibodies used were mouse anti-human BRAF monoclonal antibody (cat no. TA500431) and rabbit anti-human TERT polyclonal antibody (cat. no. TA324097), both from OriGene Technologies, Inc, a dilution of 1:200.

Techniques: Mutagenesis, Expressing