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: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 5. Mechanism of PDP1-mediated promotion of MAPK signaling. (A) GSEA results from TCGA-COAD database showing enrichment of KRAS signaling hallmark and MAPK pathway in CRC cases with high PDP1 expression. (B-C) Western blot analysis of KRAS mutant CRC cells, transfected with either (B) PDP1 knockdown or (C) overexpression constructs, probed with specific antibodies targeting MAPK signaling components. (D) Representative IHC images showing MAPK signaling in subcutaneous tumors formed by KRAS mutant CRC cells with indicated transfections. Scale bar = 200 μm. (E) Identification of proteins interacting with PDP1 in KRAS mutant CRC cells, as detected by IP-MS. (F) Detection of the interaction between BRAF and PDP1 via LC-MS/MS. The mass spectrometry image of BRAF is displayed. (G) Western blot analysis of co-immunoprecipitation assays in KRAS mutant CRC cells using antibodies against BRAF, MEK1, and PDP1. (H) Observation of reduced endogenous BRAF and MEK1 interaction in KRAS mutant CRC cells with PDP1 knockdown. (I) HEK293T cells with the indicated plasmids transfection, were treated with MG132 (25 μm), followed by immunoprecipitation and immunoblotting. (J) Schematic of truncated PDP1 expression plasmids. (K) HEK293T cells transfected with various truncated PDP1 plasmids, were treated with MG132 (25 μm) and processed to immunoprecipitation and immunoblotting.

Article Snippet: Following blocked utilizing 5% skim milk, the PVDF membranes were incubated overnight with indicated primary antibodies against phosphoERK1/2 (CST, #4370S, 1:1000), ERK1/2 (CST, #4695S, 1:1000), Vinculin (Abcam, #ab129002, 1:2000), phospho-MEK1/2 (CST, #2338S, 1:1000), MEK1/2 (CST, #4694S, 1:1000), PDP1 (CST, #65575S, 1:1000), KRAS (Proteintech, #1263-1-AP, 1:1000), β-Actin (Proteintech, #81115-1-RR, 1:2000), KLF5 (Proteintech, #21017-1-AP, 1:1000), GAPDH (Proteintech, # 60004-1-Ig, 1:3000), phospho-BRAF (CST, #2696S, 1:1000), BRAF (CST, #14814S, 1:1500), MEK1 (Abcam, #ab32576, 1:1000), Flag (CST, #14793S, 1:1000), HA (CST, #3724S, 1:1000), His (CST, #12698S, 1:1000), phospho-AKT (CST, #4060T, 1:1000), AKT (CST, #4691T, 1:1000).

Techniques: Expressing, Western Blot, Mutagenesis, Transfection, Knockdown, Over Expression, Construct, Protein-Protein interactions, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation

Journal: Cancer letters

Article Title: PDP1 promotes KRAS mutant colorectal cancer progression by serving as a scaffold for BRAF and MEK1.

doi: 10.1016/j.canlet.2024.217007

Figure Lengend Snippet: Fig. 6. PDP1 knockdown enhances therapeutic effect of KRAS inhibitors. (A) Western blot analysis of SW837 cells with control or PDP1 knockdown, treated with sotorasib (100 nM) for varying durations (0, 8, 24, 48, 72 h), probed with specific antibodies. (B-C) Colony formation assays and (D) cell viability assays of SW837 cells with control or PDP1 knockdown, treated with different concentrations of sotorasib. (E) Representative images of SW837 xenograft tumors in mice treated with either vehicle or sotorasib, and with or without PDP1 knockdown (n = 5 per group). (F) Tumor weights and (G) growth curves of SW837 xenograft tumors under the indicated treatments. (H) Representative IHC images showing p-ERK1/2 and Ki-67 protein levels in SW837 xenograft tumors with various treatments. Scale bar = 200 μm. (I) Schematic model illustrating the role of PDP1 in KRAS mutant CRC. Mutant KRAS upregulates KLF5, leading to increased PDP1 expression. Elevated PDP1 then enhances the BRAF-MEK1 interaction, amplifying MAPK signaling and facilitating CRC progression. Values are mean ± SD. **p < 0.01, ***p < 0.001, determined by one-way ANOVA (C, F, G) and two-tailed Student’s t-test (D).

Article Snippet: Following blocked utilizing 5% skim milk, the PVDF membranes were incubated overnight with indicated primary antibodies against phosphoERK1/2 (CST, #4370S, 1:1000), ERK1/2 (CST, #4695S, 1:1000), Vinculin (Abcam, #ab129002, 1:2000), phospho-MEK1/2 (CST, #2338S, 1:1000), MEK1/2 (CST, #4694S, 1:1000), PDP1 (CST, #65575S, 1:1000), KRAS (Proteintech, #1263-1-AP, 1:1000), β-Actin (Proteintech, #81115-1-RR, 1:2000), KLF5 (Proteintech, #21017-1-AP, 1:1000), GAPDH (Proteintech, # 60004-1-Ig, 1:3000), phospho-BRAF (CST, #2696S, 1:1000), BRAF (CST, #14814S, 1:1500), MEK1 (Abcam, #ab32576, 1:1000), Flag (CST, #14793S, 1:1000), HA (CST, #3724S, 1:1000), His (CST, #12698S, 1:1000), phospho-AKT (CST, #4060T, 1:1000), AKT (CST, #4691T, 1:1000).

Techniques: Knockdown, Western Blot, Control, Mutagenesis, Expressing, Two Tailed Test

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: 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: