Journal: Cancer Research

Article Title: Resistance to Selective BRAF Inhibition Can Be Mediated by Modest Upstream Pathway Activation

doi: 10.1158/0008-5472.can-11-1875

Figure Lengend Snippet: Figure 2. CRAF knockdown restores vemurafenib sensitivity in resistant clones, and CRAF overexpression confers resistance to vemurafenib in the parental sensitive A375 cells. A, Western blot analysis conducted with cell lysates collected 72 hours posttransfection show that CRAF protein expression is downregulated following transfection of a CRAF-directed siRNA construct in sensitive and resistant cell lines. B, effect of CRAF protein knockdown on cellular proliferation is shown in a bar graph of IC50. Sensitive or resistant cells transfected with scrambled (ctrl) or CRAF siRNA were treated with various concentrations of vemurafenib for 4 days, and cell viability was measured to plot growth curves. C, Western blot analysis confirms overexpression of tagged CRAF protein in the sensitive parental A375 cells transfected with a CRAF expression plasmid. D, the sensitive A375 cells were transfected with vector plasmid or plasmid carrying CRAF. Sixteen hours after transfection, cells were treated with various concentrations of vemurafenib for 3 days, and cell viability was measured to plot growth curves.

Article Snippet: The CRAF expression plasmid (OriGene Technologies, Inc.; RC201983) and KRAS wild-type plasmid (Biomyx Technology; p1025) were transfected with FuGENE 6 (Roche Diagnostics Corporation) according to the manufacturer's protocol.

Techniques: Knockdown, Clone Assay, Over Expression, Western Blot, Expressing, Transfection, Construct, Plasmid Preparation

Journal: European journal of medicinal chemistry

Article Title: Design, synthesis, and biological evaluation of a series of new anthraquinone derivatives as anti-ZIKV agents.

doi: 10.1016/j.ejmech.2023.115620

Figure Lengend Snippet: Fig. 7. Compound 22 decreases ZIKV NS5 RdRp ac tivities. The effect of 22 on ZIKV NS5 activity. HEK293T cells were transfected with Gluc reporter plasmid or co- transfected Gluc reporter and ZIKV NS5 plasmid and then treated with different concentrations (1, 5 and 10 μM) of 22 for 24h. Gluc activity in supernatants was quantified. 20 μM of fidaxomicin was treated as a positive control which is a potent inhibitor of ZIKV. 1 % DMSO was treated as a negative control. Western Blot analysis of the effect of the expression level of NS5 (upper panel). GAPDH was used as a loading control (lower panel). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: A potent ZIKV NS5 inhibitor fidaxomicin (MedChemExpress LLC, Shanghai, China) at a concentration of 20 μM was used as the positive control.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Positive Control, Negative Control, Western Blot, Expressing, Control

Journal: European journal of medicinal chemistry

Article Title: Design, synthesis, and biological evaluation of a series of new anthraquinone derivatives as anti-ZIKV agents.

doi: 10.1016/j.ejmech.2023.115620

Figure Lengend Snippet: Fig. 6. 22 binds directly to the ZIKV NS5 protein. (A) The overview of ZIKV NS5 bound to 22 and 22 shown as green sticks. (B) Two-dimensional ligand- interaction maps of co-crystals of ZIKV NS5 domains bound with 22 were generated using Molecular Operating Environment. Polar residues are colored light purple, charged residues have an additional blue ring, and lipophilic residues are green. The degree of solvent exposure is shown by the blue halos. H-bond interactions to the amino acid side chain or main chain are shown as dashed green arrows, respectively, pointing towards the H-bond acceptor. (C) and (D) SPR assay to examine and characterize the binding of 22 or ribavirin to ZIKV RdRp, using a BIAcore T100 system. ZIKV RdRp protein was immobilized on a CM5 chip, respectively. The analytes were consisting of serial dilutions of 22 or ribavirin, as indicated. The Kd values presented in (C) and (D) were calculated via the BIAcore T100 analysis software (BIAevaluation Version 3.1).

Article Snippet: A potent ZIKV NS5 inhibitor fidaxomicin (MedChemExpress LLC, Shanghai, China) at a concentration of 20 μM was used as the positive control.

Techniques: Generated, Solvent, SPR Assay, Binding Assay, Software

Journal: Cancer genetics

Article Title: A new NFIA:RAF1 fusion activating the MAPK pathway in pilocytic astrocytoma.

doi: 10.1016/j.cancergen.2016.09.002

Figure Lengend Snippet: Figure 1 Identification of a novel NFIA:RAF1 fusion in pilocytic astrocytoma. (a) Graphic illustration of translocation between the chromosomal regions 1q31.3 and 3p25.2, resulting in fusion between NFIA (NM_001134673.3) exons 1–6 and RAF1 (NM_002880.3) exons 9–17, including the Raf-1 kinase domain. (b) RT-PCR with primers in NFIA exon 6 and RAF1 exon 9 re- sulted in a 406 bp PCR product. An in-house non-CNS tumor was used as negative control. (c) The junction was verified by Sanger sequencing of the RT-PCR product.

Article Snippet: Cells were transfected with empty vector, EV (pCMV6Entry-FLAG, Origene), wild type NFIA (pCMV6-NFIA-FLAG, Origene), wild type RAF1 (pCMV6-RAF1-FLAG, Origene), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG) (Primer sequences are available on request).

Techniques: Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing

Journal: Cancer genetics

Article Title: A new NFIA:RAF1 fusion activating the MAPK pathway in pilocytic astrocytoma.

doi: 10.1016/j.cancergen.2016.09.002

Figure Lengend Snippet: Figure 2 Functional characterization and subcellular localization of NFIA:RAF1 fusion. (a) HEK293 cells were transfected with empty vector, EV (pCMV6-Entry-FLAG), wild type NFIA (pCMV6-NFIA-FLAG), wild type RAF1 (pCMV6-RAF1-FLAG), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG). Western blot analysis was performed using antibodies against Raf-1 and GAPDH. The molecular weight of the fusion protein was calculated to 80 kDa, while the endogenous Raf-1 is 75 kDa as depicted. (b) HEK293 cells were transfected as described above, or stimulated for 10 minutes with 10 ng/ml human recombinant epidermal growth factor (EGF) as positive control for activated MAPK pathway. Antibodies against phospho-Ser221-MEK1/2, MEK1/2 were used. (c) Cell growth of trans- fected HeLa cells was measured 3 days after transfection. Cell growth is calculated relative to EV transfected cells. Error bars shown are standard error of the mean (SEM) and the asterisk denotes statistical significance (P = 0.049) compared to EV. (d) HeLa cells were transfected with EV, wild type RAF1 or NFIA:RAF1 constructs and fractionation into cytoplasmic and membrane fractions was performed as described in the Materials and Methods section. Western blotting was performed using an antibody against FLAG-tag. (e–g) Transfected HeLa cells were fixed and stained for FLAG-tag (green) indicating the FLAG-tagged constructs; NFIA-FLAG, RAF1- FLAG and NFIA:RAF1-FLAG, F-actin (red), reflecting the localization of the cellular membrane and nucleus (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells were transfected with empty vector, EV (pCMV6Entry-FLAG, Origene), wild type NFIA (pCMV6-NFIA-FLAG, Origene), wild type RAF1 (pCMV6-RAF1-FLAG, Origene), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG) (Primer sequences are available on request).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Clone Assay, Western Blot, Molecular Weight, Recombinant, Positive Control, Construct, Fractionation, Membrane, FLAG-tag, Staining