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: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 1. gRNA Design for Cpf1, Cas9, and Cas9-EQR Systems and Dual-Fluorescence Reporter Plasmids for Evaluation of Editing Efficacy and Selectivity (A) Design of single-guide RNAs (sgRNAs) for Cpf1, Cas9, and Cas9-EQR systems targeting mutant BRAF allele, with a hypothesis that they cannot bind and cleave wild-type BRAF allele. Two gRNAs (gRNA-1 and gRNA-2) were designed for both AsCpf1 and LbCpf1. gRNA-3 was used for spCas9. Mutant BRAF forms a PAM of spCas9 EQR variant, which is absent from the wild-type BRAF, and thus gRNA-4 was designed for Cas9-EQR. (B) Schematic illustration of the GFP-RFP reporter plasmids for evaluation of the editing efficacy and selectivity. The reporter vector contains a CMV promoter and sequences encoding EGFP and RFP, which are separated by multiple cloning sites. gRNA binding regions and PAM motifs were inserted between EcoRI and XhoI sites, and this insertion caused RFP coding region to be shifted out of frame. Target cleavage and subsequent non-homologous end-joining repair can lead to sequence indels/frameshifts and bring the downstream RFP back in frame.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Fluorescence, Mutagenesis, Variant Assay, Plasmid Preparation, Cloning, Binding Assay, Non-Homologous End Joining, Sequencing

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 2. Evaluation of Editing Efficacy and Selectivity in HEK293T Cells (A) Transient transfection of GFP-vehicle-RFP control, GFP-WT-RFP reporter, and GFP-Mut-RFP reporter in HEK293T cells for 48 hr. (B–E) Evaluation of the editing efficacy and selectivity of the indicated CRISPR systems. All gRNAs were designed for targeting mutant BRAF allele. HEK293T cells constitutively expressing AsCpf1, LbCpf1, Cas9, or Cas9-EQR were established respectively by lentiviral transduction and puromycin selection. Images were acquired 60 hr after co-transfection with indicated GFP-RFP reporter vectors and gRNAs. (F) Statistical analysis of editing efficiency and selectivity by FACS. Error bars represent SD from experiments performed in triplicates. One-way ANOVA: *p < 0.05, **p < 0.01. WT, wild-type; Mut, mutant. The scale bar represents 200 mm.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Transfection, Control, CRISPR, Mutagenesis, Expressing, Transduction, Selection, Cotransfection

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 3. Representative Sequencing Results of Corresponding DNA Samples in HEK293T Cells Sixty hours after co-transfection as described above, genome DNA was extracted and subjected to PCR amplification. PCR products were sequenced directly. All gRNAs were designed for targeting mutant BRAF allele as indicated. (A, C, and E) Sequencing results of gene editing of wild-type allele. (B, D, and F) Results of mutant allele. Regions with continuous overlapping peaks, caused by cleavage and subsequent non-homologous end-joining repair, are indicated with dashed boxes. Alignment to the mutant BRAF regions inserted in EGFP-Mut-RFP reporter is shown in (F).

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Sequencing, Cotransfection, Mutagenesis, Non-Homologous End Joining

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 4. Evaluation of Editing Efficacy and Selectivity in Cpf1-gRNA-Treated A375, a Melanoma Cell Line with a Homozygous BRAF V600E Mutation (A) Schematic illustration of the lentiviral vectors used for gRNA expressing and fluorescently labeling. (B) Highly efficient transduction of A375 stable cells with indicated lentiviral vectors. Images were acquired on day 2 following transduction. (C) Cpf1 stable cells were transducted with vehicle or gRNA-expression lentiviral vectors. Images were acquired on day 7 following transduction. A375 cells constitutively expressing AsCpf1 or LbCpf1 were established respectively by lentiviral transduction and puromycin selection. (D) Statistical analysis of cell viability by trypan blue exclusion test. Error bars represent SD from experiments performed in triplicate. One-way ANOVA analysis: NS, not significant; **p < 0.01. (E) Expression status of BRAF, ERK1/2, and pERK1/2 in A375 stable cells following Cpf1-gRNA treatment for 72 hr by western blotting. AsCpf1 positive stable A375 cells transducted with lenti-mCherry-vehicle were set as control group. GAPDH serves as a loading control. AsgRNA, gRNA for AsCpf1; LbgRNA, gRNA for LbCpf1. The scale bar represents 100 mm.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Mutagenesis, Expressing, Labeling, Transduction, Selection, Western Blot, Control

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 5. Mutant Sequences Induced at the Cpf1-gRNA-1 Target Site in A375 Cells Genome DNA was extracted after lentiviral transduction of A375 stable cells as described above. All gRNAs were designed for targeting mutant BRAF allele as indicated. Data are representative of three independent experiments. Mutant sequence induced by AsCpf1-gRNA-1 and LbCpf1-gRNA-1 are shown on the left and right, respectively. (A–D) PCR products harboring target region were sequenced directly 72 hr after transduction. Regions with continuous overlapping peaks, caused by cleavage and subsequent non-homologous end-joining repair, are indicated with dashed boxes. (E–J) PCR products harboring target region were subjected to TA cloning 7 days after transduction. In total, 60 clones from three independent samples (20 clones per independent sample) were analyzed by sequencing. (E and F) Representative mutant sequences are shown. The deleted bases are marked with dashes, and the inserted or substituted bases are shown in red. Mutation types and the occurrence of each sequence are indicated to the left of the alignment. Unmodified reads are marked as “WT.” (G and H) Size distribution of deletions identified at the target site. (I and J) Mutation profiling of the sequenced clones.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Mutagenesis, Transduction, Sequencing, Non-Homologous End Joining, TA Cloning, Clone Assay