Journal: bioRxiv

Article Title: Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

doi: 10.1101/2023.09.26.557083

Figure Lengend Snippet: (A) Silencing efficiency of four perfect-match, BRAF-targeting crRNAs at 48h post-knock-in of WT (grey) or V600E (purple BRAF variants, normalised against crNT. (B) Systematic mutagenesis of non-selective crBRAF-1 was used to engineer V600E-selective crRNAs. Sequences for each crRNA are shown below the barplot; the “U” nucleotide (in red) indicates the uracil in the spacer sequence that basepairs with the target V600E but not with the WT BRAF RNA, and the coloured nucleotides show the position of an additional mismatch introduced at various spacer locations through mutagenesis. Barplot shows silencing efficiency of crBRAF-1 and its mutagenesis derivatives against BRAF-WT (grey) vs BRAF-V600E (purple), normalised against crNT at 48h post-transfection. crE 5 -MM 8,15 and crE 5 -MM 11,15 (orange arrows) show the highest selectivity against BRAF V600E. (C) Delta silencing efficiencies between WT (grey) and V600E (purple) variants for the top-performing crRNAs, indicating the degree of SNV-specificity. Schematics depicting the sequence and basepairing configuration of crE5-MM 8,15 and crE5-MM 11,15 with WT vs V600E-mutant BRAF mRNA targets are shown adjacent. (D) Dose-response curves derived from titration of parental crBRAF-1, crE 5 -MM 8,15 and crE5-MM 11,15 against WT (grey) or V600E (purple) BRAF at 48h post-transfection. For all graphs in A-C, individual data points show averaged fluorescence intensity from eight representative fields of view (n=3 independent experiments), and error bars show mean ± SD. (E) Silencing efficiency assessed by WB in HEK293T cells transfected with PspCas13b and full-length WT or SNV constructs. (F) Silencing efficiency assessed by RT-qPCR in cancer cell lines (A375 melanoma and HCT116 colorectal cancer) which endogenously express the SNV-containing oncogene (mean ± SD from n=3 independent experiments). Statistical significance was determined using unpaired t-tests, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cloning into MSCV-IRES reporter backbones encoding GFP (for BRAF-WT) or mCherry (for BRAF V600E and KRAS) was achieved via plasmid digestion, gel purification, and T4 ligation as previously described • Plasmids encoding full-length BRAF wildtype (p3xFLAG-CMV-BRAF, Addgene #131710) and V600E mutant (p3xFLAG-CMV-BRAF-V600E, Addgene #131723) were gifts from Jacques De Greve. (29)

Techniques: Knock-In, Mutagenesis, Sequencing, Transfection, Derivative Assay, Titration, Fluorescence, Construct, Quantitative RT-PCR

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: 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: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DNA content of normal and tumor tissue from Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); p53(lf); alb(lf) zebrafish. B) Scales from wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. Melanin pigment is dispersed throughout the cytoplasm of zebrafish melanocytes, revealing markedly different cell sizes. Scale bar = 250μm, insets are at same scale as one another. C) Quantification of melanocyte densities of wild-type, Tg(mitfa:BRAF V600E );p53(lf), Tg(mitfa:BRAF V600E ) and p53(lf) strains. One-way ANOVA with Tukey’s multiple comparisons test, ***p<0.001, **p<0.01, ns = not significant. Error bars represent mean ± SEM. D) Images from brightfield (left), anti-Mitfa (middle) and DAPI (right) staining of a single wildtype (top) or Tg(mitfa:BRAF V600E ) (bottom) epidermal melanocyte. Only the melanocyte nuclei stain positively for Mitfa. White arrowheads indicate nuclei within a single melanocyte. Scale bar = 5μm. E) Percent binucleate cells as determined by anti-Mitfa staining of pigmented melanocytes. One-way ANOVA with Tukey’s multiple comparisons test, ****p<0.0001, ns = not significant. Error bars represent mean ± SEM. F) Flow cytometry and DNA content analysis of control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) melanocytes with brightfield, EGFP and DAPI images of single melanocytes. G) Quantification of percent mononucleate and binucleate melanocytes from Tg(mitfa:EGFP);alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) strains. Chi Square test, p=0.000009. H) DNA content analysis of wild-type and Tg(mitfa:BRAF V600E ) melanocyte nuclei by confocal densitometry.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Generation of BRAF V600E -expressing RPE-1 FUCCI cell lines with a lentiviral-based doxycycline-inducible vector. B) Western blot showing inducible expression of BRAF V600E (+Dox) using a BRAF and BRAF V600E -specific antibody. Expression of the Tet repressor protein is shown. Tubulin is used as the loading control. C) Flow cytometry plots of control (-Dox) and BRAF V600E -expressing (+Dox) cells. Tetraploid cells accumulating in G1 were quantified based on Cdt1-mCherry positivity and Hoechst incorporation. Percentages of G1 tetraploid cells in control and BRAF V600E -expressing cultures are indicated. D) Fold change in G1 tetraploids relative to the control (-Dox) are shown for BRAF V600E , BRAF WT and BRAF K483W (kinase-dead) -expressing cell lines. Fold change from 3 independent experiments is shown; unpaired Student’s t test, ** p < 0.01, ns = not significant. Error bars represent mean ± SEM. E) Merged phase contrast and GFP photomicrographs of H2B-GFP expressing control (-Dox) and BRAF V600E -expressing (+Dox) cells that have recently undergone mitosis. White dotted lines indicate 2 cells with 1 nucleus each that have separated following a successful cytokinesis (-Dox) and 1 cell with 2 nuclei that has failed cytokinesis (+Dox). F) Quantification of cytokinesis failure in H2B-GFP RPE-1 control (-Dox) and BRAF V600E -expressing (+Dox) cells. Percent cells with cytokinesis failure from 3 independent experiments is shown (total cells are n= 934 for -Dox and n=568 for +Dox). Unpaired Student’s t test, **** p< 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Expressing, Plasmid Preparation, Western Blot, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI and anti-Anillin staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Mean Anillin fluorescence intensity at the equator of control (n=80) and BRAF V600E -expressing (n=61) anaphase cells. Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. Unpaired Student’s t test, ****p < 0.0001. Error bars represent mean ± SEM. C) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with MEKi or ERKi. Drugs were added coincident with Dox administration. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. D) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=41), BRAF V600E -expressing (+Dox) cells (n=68), and BRAF V600E -expressing (+Dox) cells treated with MEKi or ERKi (n=21, n=21, respectively). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM. E) Western blot analysis of immunoprecipitated RhoA-GTP from control (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Total RhoA protein and alpha tubulin were used as a controls. F) Western blot quantification of immunoprecipitated RhoA-GTP levels from (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Samples were normalized to the -Dox condition. Measurements from 3 independent experiments are shown. Unpaired Student’s t test, * p< 0.05, ** p < 0.01, *** p < 0.001. Error bars represent mean ± SEM. G) Fold change in G1 tetraploid cells following addition of RhoA activators. LPA (1μM) and S1P (1μM) were added coincident with DOX administration. Fold change in G1 tetraploids relative to the control (+Dox no drug) are shown. Measurements from 3 independent experiments are shown. Unpaired Student’s t test, *** p<0.001. Error bars represent mean ± SEM. H) G1 tetraploid generation following expression of HA-tagged-BRAF V600E in RHOA Q61L -inducible cells. Experimental design (top): RPE-1 FUCCI cells with Dox inducible RHOA Q61L were transiently transfected with an HA-tagged-BRAF V600E -expressing construct and selected accordingly. G1 tetraploids were quantified (left) by gating HA-positive, Cdt-1:mCherry-positive cells with increased Hoechst incorporation. Fold change in G1 tetraploids (right), normalized to control (-Dox) cells. Unpaired Student’s t test, *** p < 0.001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing, Fluorescence, Western Blot, Immunoprecipitation, Transfection, Construct

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Fold change in G1 tetraploids following inhibitor treatment. Fold changes are expressed relative to control (+ BRAF V600E , no drug) cells. Fold change from 3 independent experiments is shown; unpaired Student’s t test, * p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Error bars represent mean ± SEM. B) ELISA-based quantification of RAC1-GTP levels in control (-Dox) and BRAF V600E -expressing (+Dox) RPE-1 cells. Cells were measured at the indicated timepoints post thymidine release. RAC-1 GTP signal was measured using a colorimetric assay at 490nM absorbance. Unpaired Student’s t test, **** p < 0.0001. Error bars represent mean ± SEM. C) Fold change in G1 tetraploids following addition of RAC1 inhibitors. NSC2366 and EHT1864 were added coincident with BRAF V600E induction. Fold changes are expressed relative to control (+ BRAF V600E , no drug) cells. Fold change from 3 independent experiments is shown; unpaired Student’s t test, **** p<0.0001. Error bars represent mean ± SEM. D) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with NSC2366 or EHT1864. Drugs were added coincident with Dox administration. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. E) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=40), BRAF V600E -expressing (+Dox) cells (n=38), and BRAF V600E -expressing (+Dox) cells treated with NSC2366 or EHT1864 (n=38, n=36, respectively). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Colorimetric Assay, Staining, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI and anti-CENTRIN-2 staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Insets show centrioles at one pole. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Quantification of cells in mitosis with supernumerary (>4) centrioles. -Dox (n=131); +Dox (n=169); +Dox MEKi (n=85) and +Dox ERKi (n=91). Drugs were added coincident with Dox administration. Percent cells from 3 independent experiments is shown; one-way ANOVA with Tukey’s multiple comparisons test, ** p < 0.05. Error bars represent mean ± SEM. C) DAPI, anti-Mitfa and anti-CENTRIN-2 staining of control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) non-cycling zebrafish melanocytes. Scale bar = 7.5μM. Insets show centrioles. D) Percent cells with normal (2) and extra (>2) centrioles in control Tg(mitfa:EGFP); alb(lf) and Tg(mitfa:EGFP); Tg(mitfa:BRAF V600E ); alb(lf) zebrafish melanocytes; chi squared test p = 0.000843.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) DAPI, anti-CENTRIN-2 and anti-RhoA staining in control (-Dox) and BRAF V600E -expressing (+Dox) anaphase cells. Insets show centrioles at one pole. Images are maximum intensity projections of z-stacks. Scale bar = 7.5μM. B) Mean RhoA fluorescence intensity at the equator of control (n=30) and BRAF V600E -expressing anaphase cells with normal (n=54) and supernumerary (n=38) centrosomes. Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, ** p < 0.01, **** p < 0.0001. Error bars represent mean ± SEM. C) Quantification of cells in mitosis with supernumerary (>4) centrioles. -Dox (n=119); +Dox (n=124) and +Dox +Centrinone (n=122). Centrinone was added coincident with Dox administration. Percent cells from 3 independent experiments is shown; one-way ANOVA with Tukey’s multiple comparisons test, *** p < 0.001, ns = not significant. Error bars represent mean ± SEM. D) DAPI and anti-RhoA staining in - BRAF V600E (-Dox) cells, BRAF V600E -expressing (+Dox) cells, and BRAF V600E -expressing (+Dox) cells treated with Centrinone. Centrinone was added coincident with Dox administration (G1/S/G2/M) or only during G1/S. Images are maximum intensity projections of z-stacks (0.20μM). Scale bar = 7.5μM. E) Mean RhoA fluorescence intensity at the equator of - BRAF V600E (-Dox) (n=32), BRAF V600E -expressing (+Dox) cells (n=40), and BRAF V600E -expressing (+Dox) cells treated with Centrinone coincident with Dox administration (G1/S/G2/M; n=44) or only during G1/S (n=41). Fluorescence intensities (mean gray values) of the equator were measured by sum intensity projections of z-stacks. One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM. F) Fold change in G1 tetraploids in control cells (-Dox), BRAF V600E -expressing cells (+Dox) and BRAF V600E -expressing cells treated with Centrinone (+Dox +cent.). Fold changes are expressed relative to the control cells. Fold change from 3 independent experiments is shown; One-way ANOVA with Tukey’s multiple comparisons test, **** p < 0.0001. Error bars represent mean ± SEM.

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Control, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Oncogenic BRAF Induces Whole-Genome Doubling Through Suppression of Cytokinesis

doi: 10.1101/2021.04.08.439023

Figure Lengend Snippet: A) Anti-Mitfa and DAPI staining of nuclei in Tg(mitfa:BRAF V600E ) and Tg(mitfa:BRAF V600E ); p53(lf) melanocytes. Melanin pigment was bleached to more clearly visualize nuclear size. White arrowheads indicate 2 nuclei within a single melanocyte. Scale bar = 10μm. B) DNA content per nucleus as measured by confocal densitometry. Each data point represents a single nucleus. C) Flow cytometry plots of BRAF V600E -expressing (+Dox) p53 wild-type and p53 mutant tetraploid cells. Prior to analysis, G1 tetraploid cells were isolated and plated for 24 hours in the presence of nocodazole. Percentages of Geminin-GFP-positive cells in S/G2/M are indicated. D) Quantification of S/G2/M cells from flow cytometry analysis in (C). Percent cells from 3 independent experiments is shown; unpaired Student’s t test, ** p < 0.01. Error bars represent mean ± SEM. E) Flow cytometry and DNA content analysis of normal tissue (left, top) and nascent melanomas (left, bottom) from Tg(crestin:EGFP); Tg(mitfa:BRAF V600E ); p53(lf) zebrafish. Brightfield, EGFP and DAPI images of single melanocytes are shown. Quantification of percent mononucleate and binucleate cells in the 4N and 8N peaks of nascent melanomas (right).

Article Snippet: Antibodies against the following proteins were used: pMEK1/2 (S217/221), MEK1/2 and p44/42 MAPK (ERK1/2), alpha-Tubulin (Cell Signaling Technology 9154, 8727, 4695, 3873, respectively); pERK (Sigma m8159); BRAF V600E (Spring Bioscience E19290); BRAF (Millipore 10146); IRDye 800CW Donkey anti-Rabbit and IRDye 680RD Goat anti-Mouse (LI-COR 926-32213 and 926-68070, respectively).

Techniques: Staining, Flow Cytometry, Expressing, Mutagenesis, Isolation

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of PFS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for PFS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. (F, G) Kaplan-Meier plots of PFS for CRC patients undergoing exclusive first-line chemotherapy (F) and chemotherapy combined with bevacizumab (G) , stratified by BRAF mutation status. PFS, progression-free survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Prognostic associated somatic mutated genes in FPHYP CRC cohort. (A) Univariable analyses of OS concerning somatic gene mutations in FPHYP CRC tumors. (B) Kaplan-Meier curves for OS between three genes( BRAF , ARID2 , and KMT2C ) combined MT and WT groups. (C–E) Kaplan-Meier curves for PFS based on BRAF (C) , ARID2 (D) , and KMT2C (E) mutation status. OS, overall survival; MT, mutation type; WT, wiled type.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Construction of a four-gene mutation signature prediction disease progression and prognosis in FPHYP cohort. (A, B) Univariate and multivariate analyses were performed to assess the impact of clinicopathological features, individual somatic gene mutations, and the four-gene mutation signature on PFS (A) and OS (B) in CRC. (C) The Kaplan-Meier survival analysis for PFS in CRC patients between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (D) The Kaplan-Meier survival analysis for OS in CRC cases between the four-gene combined MT and WT groups based on BRAF , ARID2 , KMT2C , and GNAQ mutation status. (E, F) ROC curves for PFS (E) and OS (F) that dependent on time were generated to evaluate the prognostic model’s performance, which is based on the gene mutation status within the FPHYP cohort. PFS, progression-free survival; OS, overall survival; MT, mutation type; WT, wiled type; ROC, receiver operating characteristic.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Mutagenesis, Biomarker Discovery, Generated

Journal: Frontiers in Oncology

Article Title: Comprehensive genomic profiling reveals prognostic signatures and insights into the molecular landscape of colorectal cancer

doi: 10.3389/fonc.2023.1285508

Figure Lengend Snippet: Immunohistochemical analysis of BRAF and ARID2 in CRC. (A, B) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of the BRAF -MT (A) and BRAF -WT (B) groups. (C) Comparison of the IOD/Area value between BRAF -MT and BRAF -WT groups. (D, E) The BRAF expression original field was acquired from tissue sections (magnification, 200x) of stage I (D) and stage IV (E) groups. (F) Comparison of the IOD/Area value between stage I and IV groups. (G, H) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of the ARID2 -MT (G) and ARID2 -WT (H) groups. (I) Comparison of the IOD/Area value between ARID2 -MT and ARID2 -WT groups. (J, K) The ARID2 expression original field was acquired from tissue sections (magnification, 200x) of stage I (J) and stage IV (K) groups. (L) Comparison of the IOD/Area value between stage I and IV groups. MT, mutation type; WT, wiled type; IOD, cumulative optical density.

Article Snippet: Staining was performed with BRAF antibody (Bioss, Beijing, China), ARID2 antibody (Boster, CA, USA), and AR (Bioss, Beijing, China) to demonstrate protein expression, respectively.

Techniques: Immunohistochemical staining, Expressing, Comparison, Mutagenesis

Journal: bioRxiv

Article Title: Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

doi: 10.1101/2021.10.03.462422

Figure Lengend Snippet: (A, E, I, and M) Hub and spoke diagram of four major neoPPI hubs revolved around (A) SMAD4 G386D , (E) SPOP F133L , (I) AKT1 E17K and (M) BRAF V600E . (B, F, J and N) GST pull-down validation results of the selected Go-PPI, comPPI and/or Lo-PPI in HEK293T cell overexpressing the construct as indicated. Confirmation of (B) SMAD4 G386D ’s Lo-PPI with SMAD3, and neoPPIsneoPPIs with GSK3β and AXIN2, (F) SPOP F133L ’s Lo-PPI with BRD4 and NCOA3, and neoPPI with c-JUN (J) AKT1 E17K ’s neoPPIs with ATM, FANCC and FANCE, (N) BRAF V600E ’s comPPIs with 14-3-3β 3β and NRAS, Lo-PPI with MEK1, and neoPPIs with KEAP1 and ACO1. (C, G, K and O) Functional examination of the selected neoPPIs. (C) TCF/LEF transcriptional reporter activity in HEK293T cells transfected with GST-SMAD4 G386D or other mutants comparing to the WT controls. The data are presented as mean ± SD from three independent experiments. *p≤0.05. (G) AP-1 transcriptional reporter activity in HEK293T cells transfected with GST-SPOP F133L or other mutants comparing to the WT controls. The data are presented as mean ± SD from three independent experiments. *p≤0.05. (K) Western blots showing the γH2AX and total H2AX levels in MCF7 WT (PI3KCA WT /AKT1 WT ) and MCF7 E17K (PI3KCA WT /AKT1 E17K ) upon treatment with cisplatin at concentration as indicated. The relative γH2AX was calculated as the fold induction of γH2AX/H2AX comparing to no cisplatin control. The data are presented as mean ± SD from three independent experiments. *p≤0.05. (O) Western blots showing the NRF2 protein levels in HEK293T cells co-transfected with GST-BRAF V600E and flag-tagged NRF2 comparing to the WT control ( upper ), and the ferritin protein levels in HEK293T cells co-expressing GST-BRAF V600E and flag-ACO1 in the presence of 100 μM deferoxamine mesylate (DFOM) ( lower ). (D, H, L and P) Hypothetical model for further interrogation of neoPPI-induced pathway reprograming. (D) Model of SMAD4 G386D /AXIN2 and GSK3β neoPPIs in activating Wnt/β-catenin signaling pathways. (H) Model of SPOP F133L /c-JUN neoPPI in regulating AP-1 transcriptional activities. (L) Model of AKT1 E17K /ATM and FANCC/E neoPPIs in regulating cisplatin-induced DNA damage response pathway. (P) Model of BRAF V600E /ACO1 and KEAP1 neoPPIs in reprogramming iron and ROS homeostasis pathways.

Article Snippet: HEK293T cells were grown in 6-well plates and transfected using FuGene with Venus-flag-BRAF WT, or V600E, or Venus vector along with NRF2-ARE, or TCF/LEF, or AP-1 luciferase reporter plasmids (BPS Bioscience, 60514; Promega, E4661; Qiagen, CCS-011L).

Techniques: Biomarker Discovery, Construct, Functional Assay, Activity Assay, Transfection, Western Blot, Concentration Assay, Control, Expressing, Protein-Protein interactions

Journal: bioRxiv

Article Title: Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

doi: 10.1101/2021.10.03.462422

Figure Lengend Snippet: (A) BRET n saturation curve of Venus-tagged KEAP1 interaction with NLuc-tagged BRAF WT versus V600E from qHT-dS. The data is presented by combining four replicates from the primary qHT-dS. (B) Venus-PCA shows the cytoplasm localization of BRAF V600E /KEAP1 neoPPI using CHL-1 melanoma cell line transfected with N-Venus-tagged BRAF WT or V600E and C-Venus-tagged KEAP1. Green: reconstituted Venus signal. Blue: nuclear stained with Hoechest. Venus-PCA signal was presented as the normalized fluorescence intensity. (C) Endogenous interaction of BRAF V600E with KEAP1. The BRAF V600E /KEAP1 complex was co-immunoprecipitated with KEAP1 antibody from a pair of isogenic colon cancer cells, RKO and RKO (+/-/-), with anti-IgG as control. (D) GST-affinity pull-down assay with BRAF and KEAP1 fragments suggests the interaction domains including KEAP1 KELCH domain (left panel) and BRAF V600E kinase domain (right panel). (E) Bio-layer interferometry assay validation of the direct interaction between KEAP1 KELCH domain and BRAF V600E kinase domain using human recombinant proteins. Interaction between 14-3-3ζ and BRAF WT kinase domain was used as positive control, and 14-3-3ζ K49E and BRAF WT were used as negative control. (F) Effect of BRAF V600E kinase inhibition by vemurafenib on BRAF V600E /KEAP1 neoPPI. HEK293T cells transfected with GST-BRAF V600E and flag-KEAP1 were treated with vemurafenib at indicated concentrations for six hours.

Article Snippet: HEK293T cells were grown in 6-well plates and transfected using FuGene with Venus-flag-BRAF WT, or V600E, or Venus vector along with NRF2-ARE, or TCF/LEF, or AP-1 luciferase reporter plasmids (BPS Bioscience, 60514; Promega, E4661; Qiagen, CCS-011L).

Techniques: Transfection, Staining, Fluorescence, Immunoprecipitation, Control, Pull Down Assay, Biomarker Discovery, Recombinant, Positive Control, Negative Control, Inhibition

Journal: bioRxiv

Article Title: Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

doi: 10.1101/2021.10.03.462422

Figure Lengend Snippet: (A) BRAF V600E stabilizes endogenous NRF2. Immunoblot showing NRF2 and actin levels at different time points after inhibition of protein synthesis with cycloheximide in HEK293T cells overexpressing BRAF WT or V600E. (B) Graph of NRF2 protein levels at indicated time points based on densitometric analysis of results in (A). (C) BRAF V600E activates NRF2 transcriptional activity. HEK293T cells were co-transfected with the NRF2-ARE luciferase reporter and either WT or V600E BRAF. Relative luciferase activity was measured, normalized to internal Renilla luciferase control. Representative results of three independent experiments are shown. The error bars show the mean ± SD of three replicates. ***p<0.001. (D) BRAF V600E increases NRF2 and its target gene NQO1 protein levels in HEK293T cells transfected with GST-BRAF V600E versus WT. (E) Effect of BRAF V600E on NRF2 mRNA levels in HEK293T cells transfected with flag-NRF2 and GST-BRAT WT or V600E plasmids. ns p>0.05. (F) Correlation of NRF2 and its target gene NQO1 protein levels with BRAF genetic status in six melanoma cell lines with WT or V600E BRAF. (G) Bar graph of NQO1 protein levels in indicated cell lines based on densitometric analysis of results in (F). **p<0.01. (H) Violin plot of the correlation between NQO1 mRNA levels and BRAF genetic status in 967 cell lines from the Cancer Cell Line Encyclopedia genomic dataset. The lines indicate mean and SD. ***p<0.001. (I-J) Effect of BRAF V600E kinase inhibitor, vemurafenib (I) , or MEK1 kinase inhibitor, selumetinib (J) , on NRF2 and NQO1 protein levels in WM3482 melanoma cell line with BRAF V600E mutation. (K) Competitive binding between BRAF V600E and NRF2 to KEAP1. GST-affinity pull down of GST-KEAP1 complex from lysate of HEK293T cells transfected with equal amounts of GST-KEAP1 and flag-NRF2, and with increasing amounts of flag-BRAF V600E plasmid. (L) Violin plot showing the CERES scores for CRISPR knockout of KEAP1 in 342 cancer cell lines from CCLE dataset. The lines indicate mean and SD. ***p<0.001. (M) Parallel HTS of chemical genomic compound library in a pair of isogenic MCF10A cell lines. The identification of quinone and its derivatives with selective growth inhibition of cell line with BRAF V600E are highlighted in red. Data were presented as percentage of inhibition in parental MCF10A cells with BRAF WT versus its V600E knock-in counterpart for 3200 bioactive compounds. (N) Chemical structure of one of the quinones, deoxynyboquinone (DNQ). (O) AUC analysis of DNQ-induced dose-dependent growth inhibition of twelve cell lines with BRAF WT or V600E. WT: CHL-1, HMCB, MCF10A, MeWO, WM3311 and RKO +/-/- ; V600E: A2058, A375, MCF10A BRAF V600E , WM3482, SK-MEL-5 and RKO. Each dot represents one cell line and the data are presented as mean ± SD. *p<0.05. (P) Representative DNQ-induced dose-dependent growth inhibition of CHL-1 and WM3482 cell lines. The experiments were repeated independently three times. The data are presented as mean ± SEM from triplicates from a representative experiment. (Q) Sequential combination effect of DNQ and vemurafenib in growth inhibition of WM3482 cell lines carrying BRAF V600E mutation. DNQ-induced dose-dependent growth inhibition of WM3482 cell viability were tested in three conditions: (1) DNQ alone, (2) pretreatment with 100nM vemurafenib for 24h followed by DNQ for 3 days (vemurafenib (1st) + DNQ (2nd)), and (3) pretreatment with DNQ for 24h followed by 100nM vemurafenib for 3 days (DNQ (1st) + vemurafenib (2nd)). The experiments were repeated independently three times. Data are presented as mean ± SEM from triplicates from a representative experiment. (R) AUC analysis of the combination effect of DNQ and vemurafenib in three melanoma cell lines, A2058, SK-MEL-5, and WM3482, with BRAF V600E mutation. The experiments were repeated independently three times. Data are presented as mean of triplicates from a representative experiment. Each dot represents a cell line and the lines indicate mean ± SD. *p<0.05 from paired t-test. (S) Proposed working model of BRAF V600E /KEAP1 neoPPI in re-wiring KEAP1/NRF2/NQO1 ROS pathway and generating vulnerability to NQO1 substrate.

Article Snippet: HEK293T cells were grown in 6-well plates and transfected using FuGene with Venus-flag-BRAF WT, or V600E, or Venus vector along with NRF2-ARE, or TCF/LEF, or AP-1 luciferase reporter plasmids (BPS Bioscience, 60514; Promega, E4661; Qiagen, CCS-011L).

Techniques: Western Blot, Inhibition, Activity Assay, Transfection, Luciferase, Control, Mutagenesis, Binding Assay, Plasmid Preparation, CRISPR, Knock-Out, Drug discovery, Knock-In