Journal: Medicine International

Article Title: Chemoprophylaxis of precancerous lesions in patients who are at a high risk of developing colorectal cancer (Review)

doi: 10.3892/mi.2024.149

Figure Lengend Snippet: Genes, proteins and inhibitors.

Article Snippet: BRAF , BRAF , ERK, PI3K/AKT/mTOR, RAS, WNT signaling pathways , Uplarafenib , MedChemExpress.

Techniques: Protein-Protein interactions, Mutagenesis, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A,B) Western blots showing the effect of ATP for 30 minutes on AKT and PTEN phosphorylation in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), and the iCCA cell line HUCTT1. Bar graph shows densitometry expressed as % of phosphorylated/total ratios in control conditions (*p<0.05, n=3) (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: Nucleotides are detected by P2Y11 receptor localized in the primary cilium activating AC5, which induces increases in the levels of cAMP and activation of PKA. Once activated PKA phosphorylates and activates LKB1, LKB1 phosphorylates and stabilizes PTEN, leading to AKT inhibition. Inhibited AKT is unable to phosphorylate F-actin that is required to reorganize the cytoplasm and protrusions in migrating cells. HMC can activate LKB1 via a ciliary-independent mechanism emulating the chemosensory function of primary cilia.

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of ARAF, BRAF, or CRAF expression in iM27 cells using corresponding siRNAs. (B) Representative fluorescence microscopy images showing β-catenin expression in iM27 cells transfected with scramble siRNA or siRNAs that deplete ARAF, BRAF, or CRAF expression under PLX4032 treatment. iM27 transfected with scramble siRNA without PLX4032 treatment were used as a control. (C) Quantification of nuclear β-catenin intensity in iM27 and genetically modified iM27 cells as shown in (B) with or without PLX4032 treatment using ImageJ. The data are presented as the means ± SDs (n = 11 randomly selected 20 X fields per group). (D) Representative PLA images showing BRAF-CRAF heterodimerization in DMSO- and PLX4032-treated iM27 cells. Red dots indicate BRAF-CRAF dimers. (E) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (F) Representative PLA images showing BRAF-BRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate BRAF-BRAF dimers. (G) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (H) Representative PLA images showing CRAF-CRAF homodimerization in DMSO- and PLX4032-treated iM27 cells. The red dots indicate CRAF-CRAF dimers. (I) Quantification of PLA signals comparing PLX4032-treated iM27 cells with DMSO-treated iM27 cells. The data are presented as the means ± SDs (n = 9-10 randomly selected 40X fields per group). (J) Western blot analysis showing increased CRAF-CRAF and CRAF-BRAF interactions in PLX4032-treated iM27 cells compared with those in DMSO-treated iM27 cells. Co-IP was performed via the use of an anti-Myc antibody to pull down interacting proteins in iM27 cells coexpressing Myc-tagged and Flag-tagged CRAF. (K-M) Western blot analysis of phosphorylated BRAF (Ser445) and CRAF (Ser338) expression in iM27 (K), BRAF-deficient iM27 overexpressing wild-type BRAF or BRAF (T529N) (L), and CRAF-deficient iM27 overexpressing wild-type CRAF or CRAF (T421N) (M) treated with either DMSO or PLX4032.

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Transfection, Control, Genetically Modified, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot confirming effective silencing of KRAS, HRAS, and NRAS expression in iM27 using siRNAs. (B) PLA results showing BRAF and CRAF heterodimerization in iM27 transfected with scramble siRNA (Scr), scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (C) Western blot showing the phosphorylation of BRAF (Ser445) and CRAF (Ser338) in iM27 transfected with scramble siRNA and RAS-deficient iM27 with and without PLX4032 treatment. (D) Confocal images of nuclei visualized by Hoechst staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The nuclear boundaries are outlined with yellow circles. (E-F) Quantification of nuclear morphology based on the confocal images shown in (D), including the nuclear aspect ratio (E) and circularity (F), was performed via ImageJ. The data are presented as the means ± SDs (n = 30-67 nuclei per group). (G) Confocal images of F-actin expression and organization in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032 (scramble), and RAS-deficient iM27 (siRASs) treated with PLX4032. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate actin caps. (H-I) Quantification of F-actin intensity (H) and actin cap intensity (I) using ImageJ. The data are presented as the means ± SDs (n = 6 randomly selected 20 X fields per group for H; n = 25-52 nuclei per group for I). (J) Confocal images showing Nesprin-2 distribution in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. The yellow arrow indicates abnormal cytosolic localization of Nesprin-2. (K) Representative fluorescence images showing nuclear β-catenin staining in iM27 transfected with scramble siRNA, scramble iM27 treaded with PLX4032, and RAS-deficient iM27 (siRASs) treated with PLX4032. (L) Quantification of nuclear β-catenin intensity under the indicated conditions shown in (K). The data are presented as the means ± SDs (n = 9 randomly selected 20X fields per group).

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Expressing, Transfection, Phospho-proteomics, Staining, Fluorescence

Journal: bioRxiv

Article Title: Actin-modulated nuclear shape controls adaptive reprogramming in cancer-associated fibroblasts

doi: 10.1101/2025.07.19.665630

Figure Lengend Snippet: (A) Western blot showing increased ERK phosphorylation in response to increasing concentrations of PLX4032 in iM27 cells. (B) Western blot showing the phosphorylation of ERK in iM27 transfected with scramble siRNA and RAS-deficient iM27 (siRASs) with and without PLX4032 treatment. (C-D) Western blots showing ERK phosphorylation in BRAF-deficient iM27 overexpressing wild-type BRAF and BRAF(T529N) (C) and in CRAF-deficient iM27 overexpressing wild-type CRAF and CRAF(T421N) (D) with and without PLX4032 treatment. (E) Western blot showing increased phosphorylation of MYPT1 in PLX4032-treated iM27. (F) Western blot showing the phosphorylation of MYPT1 in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ERK inhibitor SCH772984 (SCH). (G) Representative confocal images of nuclear morphology visualized by Hoechst staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (H-I) Quantification of nuclear morphology based on the confocal images shown in (G), including the nuclear aspect ratio (H) and circularity (I), was performed via ImageJ. The data are presented as the means ± SDs (n = 27-38 nuclei per group). (J) Confocal images of F-actin expression and organization in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. Insets display enlarged views of representative individual cells (highlighted by yellow boxes). Yellow arrows indicate the actin caps. (K) Quantification of the actin cap intensity via ImageJ shown in (J). The data are presented as the means ± SDs (n = 20 nuclei per group). (L) Confocal images showing SUN2 distribution in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. The yellow arrow indicates abnormal nuclear localization of SUN2 in PLX4032-treated cells. (M) Representative fluorescence images showing nuclear β-catenin staining in iM27 treated with DMSO, PLX4032, and a combination of PLX4032 and the ROCK inhibitor Y27632. (N) Quantification of the nuclear β-catenin intensity under the indicated conditions shown in (M). The data are presented as the means ± SDs (n = 40 nuclei per group).

Article Snippet: The membranes were blocked with 5% fat-free milk or 5% BSA in TBST for one hour at room temperature and then incubated with the following primary antibodies: ARAF (Cell Signaling Technology, 4432, 1:1000), BRAF (Proteintech, 20899-1-AP, 1:1000), CRAF (Proteintech, clone 1D6A1, 1:1000), CRAF (Proteintech, 26863-1-AP, 1:1000), phospho-BRAF (Cell Signaling Technology, 2696, 1:1000), phospho-CRAF (Cell Signaling Technology, 9127, 1:1000), Flag (Proteintech, 20543-1-AP, 1:1000), MYC (Proteintech, clone 1A5A2, 1:1000), MYC (Cell Signaling Technology, clone 71D10, 1:1000), KRAS (Proteintech, 12063-1-AP, 1:1000), HRAS (Proteintech, 18295-1-AP, 1:1000), NRAS (Proteintech, 10724-1-AP, 1:1000), ERK1/2 (Cell Signaling Technology, clone 3A7, 1:1000), and phospho-ERK (Cell Signaling Technology, clone D13.14) in blocking buffer.

Techniques: Western Blot, Phospho-proteomics, Transfection, Staining, Expressing, Fluorescence

Journal: Molecules

Article Title: Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAF V600E Protein

doi: 10.3390/molecules27238513

Figure Lengend Snippet: Concentration–response curves for isolated BRAF V600E inhibition for compounds 5 – 15 , 54 and encorafenib ( 1 ). ( a ) Comparison of compounds with flexible PEG-type linkers of different length; ( b ) Comparison of compounds with triazole ring-type linker of different length; ( c ) comparison of compounds with different E3 ligase ligands of similar length or lacking the E3-ligase binder ( 54 ). Data are the results of three different experiments run in duplicate.

Article Snippet: Inhibition of B-RAF (V600E) Kinase activity by synthesized compounds was measured with a B-RAF (V600E) Kinase Assay Kit purchased from BPS Bioscience using Kinase-Glo MAX ® (Promega Corporation, Madison, Wisconsin, USA) as detection reagent.

Techniques: Concentration Assay, Isolation, Inhibition

Journal: Molecules

Article Title: Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAF V600E Protein

doi: 10.3390/molecules27238513

Figure Lengend Snippet: Self-docking of dabrafenib superposed to dabrafenib crystal pose ( a ) and docking poses of ( b ) BI 882730 and ( c ) encorafenib in BRAF V600E . In ( a ), the dabrafenib docking pose is colored lilac, while X-ray binding pose (PDB ID: 5CSW) is colored orange. In ( b ), BI 882730 (green) and in ( c ) encorafenib (pink) ligands are depicted as sticks; the linker attachment point for obtaining P5B and 10 is encircled and labelled. Waters retained in docking are shown as red spheres and labelled as ‘w1’ and ‘w2’, hydrogen bonds are shown as dashed black lines and non-polar hydrogens are omitted for clarity. Protein is represented as cartoon and colored in beige, binding site residues are shown as sticks and labelled with one letter code and the DFG loop is highlighted in yellow.

Article Snippet: Inhibition of B-RAF (V600E) Kinase activity by synthesized compounds was measured with a B-RAF (V600E) Kinase Assay Kit purchased from BPS Bioscience using Kinase-Glo MAX ® (Promega Corporation, Madison, Wisconsin, USA) as detection reagent.

Techniques: Binding Assay

Journal: Molecules

Article Title: Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAF V600E Protein

doi: 10.3390/molecules27238513

Figure Lengend Snippet: Occupancy of ligand–protein contacts along MD productions, mapped on the surface of BRAF. Occupancy is defined as the normalized number of MD frames in which atoms of the ligand are at a distance equal or lower than 4 Å from protein atoms. Occupancy is shown as a range of colors spanning from blue (low occupancy) to orange (high occupancy) areas. Black arrows highlight areas of BRAF V600E which were accessed by P5B but not by 10 . Protein is represented as surface, ligand is depicted as sticks and labelled.

Article Snippet: Inhibition of B-RAF (V600E) Kinase activity by synthesized compounds was measured with a B-RAF (V600E) Kinase Assay Kit purchased from BPS Bioscience using Kinase-Glo MAX ® (Promega Corporation, Madison, Wisconsin, USA) as detection reagent.

Techniques:

Journal: Oncogene

Article Title: Growth suppression by dual BRAF(V600E) and NRAS(Q61) oncogene expression is mediated by SPRY4 in melanoma

doi: 10.1038/s41388-018-0632-2

Figure Lengend Snippet: Differential cell growth response upon rival oncogene expression. The rival oncogene was induced with doxycycline (50–100 ng/ml) and subjected for 6 days cell viability assays using cell-titer-glow reagent. a A panel of four isogeneic stable NRAS(Q61)/Tet-On BRAF(V600E) and five BRAF(V600E)/Tet-On-NRAS(Q61R) mutant cell lines and an immortalized primary human melanocyte line (Pmel) were assayed for cell viability at fifth day following rival oncogene induction with doxycycline. Cell lines showing antagonism such as b two NRAS(Q61R)/Tet-On-BRAF(V600E) and c two BRAF(V600E)/Tet-On-NRAS(Q61R). Cell lines showing cooperativity/neutral such as d one BRAF(V600E)/Tet-On-NRAS(Q61R) and one NRAS(Q61R)/Tet-On-BRAF(V600E). The protein expression was confirmed by western blotting. For each cell line, cell viability was performed independently more than three times in triplicates. Student’s t test, doxycycline vs. no-doxycycline, ◇ p ≤ 0.05

Article Snippet: Full-length BRAF(WT) and BRAF(V600E) were PCR amplified from pBABE-B-Raf, pBABE-B-Raf-V600E vector (Addgene) using primers (Forward: 5′- ATATGGCCCCCGGGGACGCGTGCCATGGCGGCGCTGAGC-3′ and Reverse: 5′- TCCCCTACCCGGTAGAATTCTCAGTGGACAGGAAACGCAC-3′) with amplicon possessing the t1799a/v600e mutation.

Techniques: Expressing, Mutagenesis, Western Blot

Journal: Oncogene

Article Title: Growth suppression by dual BRAF(V600E) and NRAS(Q61) oncogene expression is mediated by SPRY4 in melanoma

doi: 10.1038/s41388-018-0632-2

Figure Lengend Snippet: Rival oncogene-induced growth suppression is rescued by SPRY4 silencing in vitro. a Western blot analysis and quantitative densitometry of the protein expression in SK- MEL-119 NRAS* + iBRAF*cells that ectopically express rival oncogenes and siRNA SPRY4 constructs. Total cell lysate extracts at 48 h were probed with antibodies for BRAF(V600E), BRAF(WT), NRAS(Q61R), NRAS(WT), SPRY4 and internal loading control GAPDH. b Silencing of SPRY4 abrogates the growth inhibition effect of rival oncogene expression in SK-MEL-119 NRAS* cells as compared to siRNA nontarget control (siNTC) vector. c Differential regulation of SPRY4 and p21 proteins upon second oncogene induction. d SPRY4 overexpression and ( e ) SPRY4 silencing effect on p21 protein expression in the presence of rival oncogene, among suppressive (Red) and nonsuppressive (Green) cell lines was confirmed by western blotting. Student’s t test, doxycycline vs. no-doxycycline, ◇ p ≤ 0.05. The data presented are representative of three independent experiments

Article Snippet: Full-length BRAF(WT) and BRAF(V600E) were PCR amplified from pBABE-B-Raf, pBABE-B-Raf-V600E vector (Addgene) using primers (Forward: 5′- ATATGGCCCCCGGGGACGCGTGCCATGGCGGCGCTGAGC-3′ and Reverse: 5′- TCCCCTACCCGGTAGAATTCTCAGTGGACAGGAAACGCAC-3′) with amplicon possessing the t1799a/v600e mutation.

Techniques: In Vitro, Western Blot, Expressing, Construct, Inhibition, Plasmid Preparation, Over Expression