Journal: Journal of enzyme inhibition and medicinal chemistry

Article Title: Identification of novel inhibitors of dengue viral NS5 RNA-dependent RNA polymerase through molecular docking, biological activity evaluation and molecular dynamics simulations.

doi: 10.1080/14756366.2025.2463006

Figure Lengend Snippet: Figure 1. Crystal structure of DENV-NS5 (PDB ID: 4V0R) and crystal structure of DENV-NS5 RdRp domain in complex with Compound 27 (PDB ID: 5K5M).

Article Snippet: Firstly, the docking protocol has been validated by redocking original ligand (Compound 27) of crystal structure of DeNV-Ns5 to its binding domain. the docked pose is in agreement with the original pose, and the RsMD value of the two is 0.346 Å (Figure s1). after systematic molecular docking process, 200 candidates were retained from the 1.6 million compounds in the chemDiv and targetMol (Usa) database. their performance was evaluated using three key metrics: docking score, Uni-score Rescore, and MM/GBsa binding free energy.

Techniques:

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: Cellular and Molecular Life Sciences: CMLS

Article Title: LncRNA SNHG15 promotes angiogenesis and improves cardiac repair after myocardial infarction through MiR-665-mediated KDR expression

doi: 10.1007/s00018-025-05737-2

Figure Lengend Snippet: SNHG15 regulates HCAECs proliferation, migration, and tube formation by upregulating KDR. ( A) , Efficiency of knocking down and overexpressing SNHG15 by RT-qPCR, n = 3. ( B) , Representative and quantitative KDR protein levels in HCAECs after the knockdown and overexpression of SNHG15 by Western blotting detection, n = 3. ( C) , Representative and quantification of AKT/eNOS/ERK/Raf1/p38 expressions and phosphorylation in HCAECs treated with the knockdown and overexpression of SNHG15 following western blotting detection, n = 3. ( D and E ), Cell proliferation was measured through EdU staining and quantification of EdU-positive cells at 6 h after the knockdown and overexpression of SNHG15. Scale bar = 20 μm, n = 3. ( F and G ), Migration ability was measured by the transwell assay and quantification of transwell cells at 24 h after the knockdown and overexpression of SNHG15. Scale bar = 200 μm, n = 3. ( H and I) , Migration ability was measured through wound healing assays and migration rate quantification at the indicated time points. Scale bar = 200 μm, n = 3. ( J and K ), The angiogenic response was measured by conducting tube formation assay and quantification analysis of the total tube length and tube branches number. Scale bar = 50 μm, n = 3. * P < 0.05 versus siRNA ctrl group; P < 0.05 versus SNHG15 OE ctrl group. Data is the mean ± SEM. Two-way ANOVA with Bonferroni multiple comparisons test ( B , C , E , G , I , K ). HCAECs, human coronary artery endothelial cells; KDR, kinase domain-containing receptor

Article Snippet: Membranes were blocked with 5% skim milk for 1 h at 37 °C before incubated with primary antibodies against KDR (Cat. No. 26415-1-AP, Proteintech), AKT (Cat. No. 10176-2-AP, Proteintech), eNOS (Cat. No. A20985, Abclonal), ERK (Cat. No. A4782, Abclonal), Raf1 (Cat. No. A19638, Abclonal), p38 (Cat. No. A14401, Abclonal), p-AKT (Cat. No. 66444-1-lg, Proteintech), p-eNOS (Cat. No. AP1404, Abclonal), p-ERK (Cat. No. AP0974, Abclonal), p-Raf1 (Cat. No. AP0498, Abclonal), and p- p38 (Cat. No. AP0526, Abclonal), followed by at 4 °C for overnight.

Techniques: Migration, Quantitative RT-PCR, Knockdown, Over Expression, Western Blot, Phospho-proteomics, Staining, Transwell Assay, Tube Formation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: LncRNA SNHG15 promotes angiogenesis and improves cardiac repair after myocardial infarction through MiR-665-mediated KDR expression

doi: 10.1007/s00018-025-05737-2

Figure Lengend Snippet: SNHG15 in ECs promotes therapeutic angiogenesis to against cardiac remodeling and dysfunction post-MI. ( A ), Schematic diagram depicting the time course of MI-induced cardiac remodeling and dysfunction in mice receiving AAV-SNHG15 or AAV-Scr. ( B ), Kaplan–Meier survival analysis of mice treated with sham ( n = 8), MI ( n = 22), MI + AAV- Scr ( n = 23) and MI-AAV-SNHG15 ( n = 23) post-MI. ( C and D ), Echocardiography and measured LVEF, FS, LVEDD and LVSDD in mice, n = 6. ( E and F ), Hematoxylin-eosin staining, Masson’s trichrome, and quantification of cardiac fibrosis, n = 6. ( G) , Representative and quantitative of KDR protein levels in the ECs of infarct border zone after MI through western blot detection, n = 6. ( H) , Representative and quantification of AKT/eNOS/ERK/Raf1/p38 expressions and phosphorylation in the border zone were analyzed using western blotting, n = 6. ( I and J ), Representative images and quantification of immunofluorescence staining for CD31 + /Ki67 + cells in the border zone of infarcted hearts at 7 days post-MI. Scale bar = 20 μm, n = 6. ( K and L) , Representative images and quantification of immunofluorescence staining for CD31 + cells in the border zone of infarcted hearts at 28 days post-MI. Scale bar = 20 μm, n = 6. Data are the mean ± SEM. HRF, high resolution field. The survival rate was analyzed by Kaplan-Meier method and compared by log-rank test ( B ). Two-way ANOVA with Bonferroni multiple comparisons test ( D , F , G , H , J , L ). ECs, endothelial cells; MI, myocardial infarction; LVEDD, Left ventricular end-diastolic diameter; LVESD, Left ventricular end-systolic diameter; LVEF, Left ventricular ejection fraction; FS, fractional shortening; KDR, kinase domain-containing receptor; Scr, scramble

Article Snippet: Membranes were blocked with 5% skim milk for 1 h at 37 °C before incubated with primary antibodies against KDR (Cat. No. 26415-1-AP, Proteintech), AKT (Cat. No. 10176-2-AP, Proteintech), eNOS (Cat. No. A20985, Abclonal), ERK (Cat. No. A4782, Abclonal), Raf1 (Cat. No. A19638, Abclonal), p38 (Cat. No. A14401, Abclonal), p-AKT (Cat. No. 66444-1-lg, Proteintech), p-eNOS (Cat. No. AP1404, Abclonal), p-ERK (Cat. No. AP0974, Abclonal), p-Raf1 (Cat. No. AP0498, Abclonal), and p- p38 (Cat. No. AP0526, Abclonal), followed by at 4 °C for overnight.

Techniques: Staining, Western Blot, Phospho-proteomics, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Nordihydroguaiaretic acid inhibits bladder cancer metastasis through suppression of α1,3-mannosyltransferase expression and LRFN4 N-glycosylation

doi: 10.1186/s12967-025-06571-7

Figure Lengend Snippet: ALG3 activated the Ras signaling pathway. A RNA-seq analysis identified differentially expressed genes between ALG3-knockdown and scramble control groups in UMUC3 cells. Thresholds: |log2 fold change|≧0.59 and P < 0.05. Red/green dots represented significantly up-/down-regulated genes. B GO enrichment analysis of ALG3-regulated DEGs, showing the top 20 significantly enriched biological processes (BP), molecular functions (MF), and cellular components (CC). Bubble size indicated gene count; color represented P-value. C KEGG pathway analysis of ALG3-associated DEGs to identify the altered signaling pathways, revealed Ras signaling pathway as one of the most significantly enriched pathways. Bubble size indicated gene count; color represented P-value. D , E Western blot validation of Ras signaling pathway activation (Ras-GTP, p-Raf1, p-MEK1/2, p-ERK1/2) following transfection with ALG3 siRNA or cDNA. GAPDH served as the loading control. Band intensities were quantified using ImageJ and normalized to GAPDH. ** P < 0.01, *** P < 0.001

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight: ALG3 (DF14333, 1:1000, Affinity Biosciences, Jiangsu, China), E-cadheirn (A22850, 1:1000, ABclonal Technology, Wuhan, China), N-cadherin (A19083, 1:2000, ABclonal Technology, Wuhan, China), VE-cadherin (A22659, 1:1000, ABclonal Technology, Wuhan, China), MMP14 (A2549, 1:500, ABclonal Technology, Wuhan, China), MMP2 (1:1000, ABclonal Technology, Wuhan, China), MMP9 (1:500, ABclonal Technology, Wuhan, China), Ras (A23382, 1:1000, ABclonal Technology, Wuhan, China), Ras-GAP (A4876, 1:500, ABclonal Technology, Wuhan, China), Raf1 (A0223, ABclonal Technology, Wuhan, China), p-Raf1 (AP0498, 1:500, ABclonal Technology, Wuhan, China), MEK1/2 (A4868, 1:2000, ABclonal Technology, Wuhan, China), p-MEK1/2 (AP1349, 1:2000, ABclonal Technology, Wuhan, China), ERK1/2 (A4782, 1:1000, ABclonal Technology, Wuhan, China), p-ERK1/2 (AP0974, 1:1000, ABclonal Technology, Wuhan, China) and GAPDH (AC001, 1:5000, ABclonal Technology, Wuhan, China).

Techniques: RNA Sequencing, Knockdown, Control, Protein-Protein interactions, Western Blot, Biomarker Discovery, Activation Assay, Transfection

Journal: Journal of Translational Medicine

Article Title: Nordihydroguaiaretic acid inhibits bladder cancer metastasis through suppression of α1,3-mannosyltransferase expression and LRFN4 N-glycosylation

doi: 10.1186/s12967-025-06571-7

Figure Lengend Snippet: N-glycosylation of LRFN4 drove malignant progression in bladder cancer cells. A Cell proliferation assessed by CCK-8 assays in T24 and UMUC3 cells transfected with wild-type LRFN4 (LRFN4 WT) or N-glycosites-deficient mutant-type LRFN4 (LRFN4 MUT). Absorbance at 450 nm was measured at 0, 24, 48, 72, and 96 h post-transfection. B Colony formation capability after 14 days culture. Left: Representative crystal violet-stained wells. Right: Quantification of colonies (> 50 cells/colony). C , D Transwell assays assessing metastatic potential of bladder cancer cells after LRFN4 WT and LRFN4 MUT transfection. Left: Representative images (scale bars = 50 μm). Right: Quantified cell counts (10 random fields/well). E Tube formation assays evaluating the vasculogenic mimicry formation ability in LRFN4 WT or LRFN4 MUT transfected cells. Left: Representative images (scale bars = 50 μm). Right: Quantified parameters (total tube length, number of meshes and junctions). F – H Western blot analysis of Ras signaling pathway activation (Ras-GTP, p-Raf1, p-MEK1/2, p-ERK1/2) in T24 ( F ) and UMUC3 ( G ) cells transfected with LRFN4 WT or LRFN4 MUT. Statistical analysis were shown ( H ). * P <0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Membranes were blocked with 5% non-fat milk in TBST for 2 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight: ALG3 (DF14333, 1:1000, Affinity Biosciences, Jiangsu, China), E-cadheirn (A22850, 1:1000, ABclonal Technology, Wuhan, China), N-cadherin (A19083, 1:2000, ABclonal Technology, Wuhan, China), VE-cadherin (A22659, 1:1000, ABclonal Technology, Wuhan, China), MMP14 (A2549, 1:500, ABclonal Technology, Wuhan, China), MMP2 (1:1000, ABclonal Technology, Wuhan, China), MMP9 (1:500, ABclonal Technology, Wuhan, China), Ras (A23382, 1:1000, ABclonal Technology, Wuhan, China), Ras-GAP (A4876, 1:500, ABclonal Technology, Wuhan, China), Raf1 (A0223, ABclonal Technology, Wuhan, China), p-Raf1 (AP0498, 1:500, ABclonal Technology, Wuhan, China), MEK1/2 (A4868, 1:2000, ABclonal Technology, Wuhan, China), p-MEK1/2 (AP1349, 1:2000, ABclonal Technology, Wuhan, China), ERK1/2 (A4782, 1:1000, ABclonal Technology, Wuhan, China), p-ERK1/2 (AP0974, 1:1000, ABclonal Technology, Wuhan, China) and GAPDH (AC001, 1:5000, ABclonal Technology, Wuhan, China).

Techniques: Glycoproteomics, CCK-8 Assay, Transfection, Mutagenesis, Staining, Western Blot, Activation Assay

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