Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: TGF-β induces cholesterol accumulation to regulate the secretion of tumor-derived extracellular vesicles
doi: 10.1186/s13046-025-03291-0
Figure Lengend Snippet: TGF-β induces EV secretion via activation of the MEK/ERK pathway. ( A , B ) EVs released by the three indicated cell models were quantified by NTA in terms of particle size ( A ) and particle number after normalization to the total cell number ( B ). The cells were stimulated with vehicle (Ctrl), 5 ng/mL TGF-β1 for 48–120 h, 5 µM LY2157299 TGF-β type I receptor inhibitor (TβRi) or combination of TGF-β with TβRi for 120 h. ( C ) Pearson correlation of nanoparticle numbers per cell with corresponding cellular TGFB1 mRNA expression from the same cell. Each data point represents one of the seven cell lines analyzed. ( D ) Protein expression levels of the indicated EV-specific proteins in EV extracts (isolated as VSF or after CD81-specific enrichment) derived from MDA-MB-231 cells stimulated with 5 ng/mL TGF-β1 in the absence or presence of 5 µM TβRi for the indicated time periods, and densitometric values were normalized to the vehicle control. ( E , F ) Representative cryo-TEM ( E ) and SEM ( F ) pictures of EVs isolated as VSF ( E ) or CD81-enriched fraction ( F ) from MDA-MB-231 cells stimulated with 5 ng/mL TGF-β1 for 48–120 h or not (Ctrl). A negative control image of CD81-specific immunobeads alone is also shown ( F ). Scale bars are included and red arrows mark individual or clustered EVs. ( G ) Protein expression levels of SMAD2, SMAD3 and β-ACTIN (as loading control) in MDA-MB-231 protein extracts transiently transfected with the indicated siRNAs, and densitometric values were normalized to the control siRNA. ( H ) EVs released by MDA-MB-231 cells, which were first transiently transfected with SMAD-specific siRNAs, were quantified by NTA in terms of particle size (left) and particle number after normalization to the total cell number (right). The cells were stimulated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for 48 h. ( I ) Protein expression levels of the indicated signaling proteins and β-TUBULIN (as loading control) in MDA-MB-231 cells stimulated with 5 ng/mL TGF-β1 in the absence or presence of 5 µM MEK inhibitor (MEKi; U0126) for the indicated time periods, and densitometric values were normalized to the vehicle control. ( J ) EVs released by MDA-MB-231 cells treated with vehicle (DMSO) or 5 µM MEKi and quantified by NTA in terms of particle size (left) and particle number after normalization to the total cell number (right). The cells were stimulated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for 48 h. ( K ) Expression levels of the indicated proteins and β-ACTIN (as loading control) in A549 cells stimulated with 5 ng/mL TGF-β1 in the absence or presence of 5 µM gefitinib or 0.5 µM lapatinib for 48 h, and densitometric values normalized to the vehicle control. ( L ) EVs released by A549 cells treated with vehicle (DMSO) or 5 µM gefitinib or 0.5 µM lapatinib and quantified by NTA in terms of particle size (left) and particle number after normalization to the total cell number (right). The cells were stimulated with vehicle (Ctrl) or 5 ng/mL TGF-β1 for 48 h. Data in ( A , B , H , J and L ) are presented as mean values of three biological replicates ± SEM, each in technical duplicates and p-values are shown based on two-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s post-test method. p-values: ** p ≤ 0.01; *** p ≤ 0.001. The data in ( D , G , I and K ) show representative immunoblots of three independent biological replicates along with molecular mass markers in kDa
Article Snippet: Cells were treated with inhibitors (i) LY2157299 (TβRi; #15409, Cayman Chemical Co, Ann Arbor, MI, USA), U0126 (MEKi; #19–147 Merck/Millipore, Stockholm, Sweden), AY9944 (DHCR7i; #14611, Cayman Chemical Co, Ann Arbor, MI, USA), simvastatin (HMGCRi; #S6196, Sigma-Aldrich AB, Stockholm, Sweden), heparin (#7980, STEMCELL Technologies UK Ltd, Cambridge, UK), GM6001 (MMPi; #CC1010, Sigma-Aldrich AB, Stockholm, Sweden), monoclonal anti-TGF-β1/2/3 antibody (TGF-β Ab; MAB1835, R&D Systems Inc./Biotechne, Abingdon, UK), gefitinib (EGFRi; #Y0001813, Sigma-Aldrich AB, Stockholm, Sweden), lapatinib (EGFRi# CDS022971, Sigma-Aldrich AB, Stockholm, Sweden), doxorubicin hydrochloride (Dox; #D1515, Sigma-Aldrich AB, Stockholm, Sweden) or paclitaxel (Taxol; #T7402, Sigma-Aldrich AB, Stockholm, Sweden).
Techniques: Activation Assay, Expressing, Isolation, Derivative Assay, Control, Negative Control, Transfection, Western Blot
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