Journal: British Journal of Cancer

Article Title: The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC

doi: 10.1038/bjc.2014.560

Figure Lengend Snippet: Activation of BMP signalling differentially modulates Wnt signalling dependent on the SMAD4 and p53 status. ( A ) CRC cell lines HCT116, LS174T, HT-29, SW480, RKO and DLD-1 and the embryonic kidney cell line HEK-293 (used as non-cancer cell line) were transfected with WRE-luc and MRE-luc. The next day, the cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, the cells were lysed and luciferase activity was measured. The control (pcDNA) condition was set to 1. ( B ) An overview of APC/ β -catenin, KRAS/BRAF, p53 and SMAD4 mutation status in the cell lines. ( C ) HCT116, HCT116 SMAD4−/−, HCT116 R248 (p53 mutated) and HCT116 p53−/− cells were transfected with WRE-luc and MRE-luc. The next day, the cells were transfected with either BMPR2 (to activate BMP signalling) or the empty control vector pcDNA4/TO. After 24 h, the cells were lysed and luciferase activity was measured. ( D ) DLD-1 (harbouring a p53 mutation) and DLD-1 SIL/+ (one WT copy p53) were transfected with WRE-luc/MRE-luc. The next day, the cells were treated with either 100 ng ml −1 BMP2 or the control vehicle (both diluted in medium with 0,5% FCS). After 24 h, the cells were lysed and luciferase activity was measured. ( E ) SMAD4 was stably knocked down in LS174T cell using lentiviral shRNA transduction. The empty shRNA SHC002 was used to create a control cell line. The cells were transfected with WRE-luc/MRE-luc. The next day the cells were treated with either 100 ng ml −1 BMP2 or the control vehicle (both diluted in medium with 1% FCS). After 24 h, the cells were lysed and luciferase activity was measured. (All the experiments were performed three times. Mean±s.e.m. is shown, * P <0.05, ** P <0.01, *** P <0.001)

Article Snippet: Stock solutions of recombinant human BMP2 ligands (R&D systems, Minneapolis, MN, USA) were prepared in phosphate-buffered saline (PBS) and subsequently dissolved in culture medium (100 ng ml −1 ) containing 0.5% FCS.

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Stable Transfection, shRNA, Transduction

Journal: Cell & Bioscience

Article Title: Bone morphogenetic protein signaling regulation of AMPK and PI3K in lung cancer cells and C. elegans

doi: 10.1186/s13578-022-00817-3

Figure Lengend Snippet: BMP activation of PI3k requires BMPR2. A , B Immunoblot of ( A ) A549 and ( B ) H1299 cells treated with BMP2 ligand with pAkt expression normalized to Akt, n = 2. C Immunoblot of A549 cell treated with BMP2 following starvation with DPBS or in regular media without FCS. D Immunoblot for BMPR2 expression of normal human bronchial epithelial cells (BEC) and lung cancer cell lines. E Immunoblot of BEC treated with BMP2 for 20 min. F , G Immunoblot of H1299 cells treated with BMP inhibitors JL5 2.5 μM or DMH1 2.5 μM for 24 h. H Fluorescence quantification of DAF-16::GFP positive nuclei in the indicated in insulin/DAF-2 daf-2 (1370) and BMPR2/DAF-4 daf-4 (e1364) at L4 stage grown at 20 °C

Article Snippet: For studies examining response to BMP2 ligand (R&D Systems # 355-BM), lung cancer cells were incubated overnight with media without FCS.

Techniques: Activation Assay, Western Blot, Expressing, Fluorescence

Journal: Cell & Bioscience

Article Title: Bone morphogenetic protein signaling regulation of AMPK and PI3K in lung cancer cells and C. elegans

doi: 10.1186/s13578-022-00817-3

Figure Lengend Snippet: BMP suppresses LKB1 in lung cancer cells. A , B Western blot analysis of H1299 cells treated with JL5 or DMH1 demonstrating an increase in pACC and LKB1 at 3 h. Graph represents the mean pLKB1 expression normalized to Actin, n = 3. B Representative immunoblot. Mean percent increase in pLKB1/Actin ratio compared to control at 3 h following treatment with DMH1, n = 3. C Immunoblot of H1299 cells treated with DMH1 for 24 h. Mean percent increase in normalized pAMPK expression compared to control at 24 h following treatment with DMH1, n = 3 D Immunoblot of A549 cells treated with DMH1 for 3 and 24 h, (n = 3) demonstrating no significant change in pACC or pAMPK. The pLKB1, pAMPK, and Actin bands scanned were the 2.5 µM concentration except for one 1.25 µM peak value band obtained from H1299 cells treated with DMH1 for 3 h. E Western blot analysis of A549 cells stably expressing LKB1 and A549 control transfected cells treated with JL5 for 24 h. Both Actin and spectrin were used as loading controls. Graph represents mean pACC/spectin ratio, n = 2. F Immunoblot of H1299 cells following siRNA knockdown of BMPR2. Graph represents the mean of 4 independent studies. G Western blot analysis of starved H1299 cells treated with and without BMP2 ligand. Graph represent the mean change in pLKB1/pLKB1 ratio compared to control over time, n = 3

Article Snippet: For studies examining response to BMP2 ligand (R&D Systems # 355-BM), lung cancer cells were incubated overnight with media without FCS.

Techniques: Western Blot, Expressing, Control, Concentration Assay, Stable Transfection, Transfection, Knockdown

Journal: Cell & Bioscience

Article Title: Bone morphogenetic protein signaling regulation of AMPK and PI3K in lung cancer cells and C. elegans

doi: 10.1186/s13578-022-00817-3

Figure Lengend Snippet: BMP2 ligand decreases expression of TCA cycle intermediates and non-essential amino acids. A549 and H1299 cells were treated with BMP2 ligand for 40 min and metabolomic analysis performed by LC-MC. Data represents mean of 3 experiments for A TCA cycle intermediates, B ATP intermediates, and C , D amino acids

Article Snippet: For studies examining response to BMP2 ligand (R&D Systems # 355-BM), lung cancer cells were incubated overnight with media without FCS.

Techniques: Expressing