Journal: eLife

Article Title: Formation of a giant unilocular vacuole via macropinocytosis-like process confers anoikis resistance

doi: 10.7554/eLife.96178

Figure Lengend Snippet: ( A ) Representative differential interference contrast (DIC) microscopy images of suspended MCF-10A cells showing entotic vacuole or GUVac formation after culture for 24 hr. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm (main panels) or 10 μm (inset). ( B ) Percentage of suspended MCF-10A cells showing entotic vacuole or GUVac formation after 24 hr (n = 772). ( C ) Representative bright-field microscopy images of MCF-10A cells suspended with F-actin cytoskeleton inhibitors. Scale bar: 15 μm ( D ) Percentage of suspended MCF-10A cells showing GUVac formation after exposure to the indicated drugs for the indicated times. dimethyl sulfoxide (DMSO): 0 hr (n = 512), 6 hr (n = 723), 12 hr (n = 690), and 24 hr (n = 690). latrunculin B (LatB): 6 hr (n = 634), 12 hr (n = 693), and 24 hr (n = 428). Cytochalasin D: 6 hr (n = 613), 12 hr (n = 618), and 24 hr (n = 464). Y-27632: 6 hr (n = 448), 12 hr (n = 601), and 24 hr (n = 660). ( E ) Percentage of suspended human primary mammary epithelial cells (HMEpiCs) showing GUVac formation after incubation with DMSO or LatB for the indicated times. DMSO: 6 hr (n = 722), 12 hr (n = 647), 24 hr (n = 417). LatB: 6 hr (n = 1225), 12 hr (n = 1505), and 24 hr (n = 1335). ( F ) Percentage of suspended MCF-10A cells showing GUVac formation after incubation with the indicated drugs for 18 hr. DMSO (n = 1087), LatB (n = 1634), jasplakinolide (n = 2578), Rho inhibitor (n = 839), Y-27632 (n = 1322), EHT 1864 (n = 905), ML 141 (n = 997), nocodazole (n = 1342), and blebbistatin (n = 520). ( G ) Representative DIC microscopy images with phalloidin staining show disruption of the actin cytoskeleton and the GUVac formation in SpvB-expressing cells after suspension culture for 24 hr. Scale bar: 10 μm ( H ) Percentage of GUVac formation in control or SpvB-expressing MCF-10A cells. ( I ) Representative DIC microscopy images after suspension culture of indicated cell lines for 24 hr with LatB. Scale bar: 10 μm ( J ) Representative DIC images of suspended MCF-10A cells treated with DMSO or LatB for the indicated times. White arrowheads indicate the accumulation of vacuoles. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. ( K ) Representative fluorescence and DIC time-lapse images of MCF-10A cells expressing mCherry-H2B and membrane-targeted EGFP obtained at the indicated times after the onset of LatB treatment. Times are presented as hour:minute:second (h:m:s). Scale bars, 10 μm. All quantitative data are means ± SD. The n values represent the total number of cells quantified for two ( D ) or three ( B, E, F, H ) independent experiments. **p<0.01 (two-tailed unpaired t -test) for the indicated comparison ( F, H ) or versus the corresponding value for DMSO ( E ). Figure 1—source data 1. Quantification data corresponding to . Figure 1—source data 2. Quantification data corresponding to . Figure 1—source data 3. Quantification data corresponding to . Figure 1—source data 4. Quantification data corresponding to .

Article Snippet: Reagents were obtained from the following sources and used at the indicated concentrations: LatB (Sigma, 5288, used at 5 μM), cytochalasin D (Sigma, C8273, used at 0.5 μM), jasplakinolide (Tocris, 2792, used at 100 nM), Rho inhibitor I (Cytoskeleton, CT04-A, used at 2 μg/ml), Y-27632 (Sigma, Y0503, used at 20 μM), EHT 1864 (Tocris, 3872, used at 2 μM), ML 141 (Tocris, 4266, used at 10 μM), blebbistatin (Sigma, B0506, used at 50 μM), nocodazole (Sigma, M1404, used at 1 μg/ml), EIPA (Sigma, A3085, used at 50 μM), FITC–dextran (70 kDa) (Sigma, 46945, used at 1 mg/ml), tetramethylrhodamine-dextran (70 kDa) (Invitrogen, D1818, used at 0.1 mg/ml), LysoTracker Red (Invitrogen, L7528, used at 50 nM), CellMask Plasma Membrane Stains Deep Red (Invitrogen, C10046), LY294002 (Sigma, L9908, used at 50 μM), VPS34-IN1 (Cayman, 17392, used at 5 μM), TGX-221 (Cayman, 10007349, used at 50 nM), GDC-0941 (Selleckchem, S1065, used at 100 nM), Dynasore (Sigma, D7693, used at 100 μM), Dynole 34-2 (Tocris, 4222, used at 10 μM), OctMAB (Tocris, 4225, used at 20 μM), MitMAB (Abcam, ab120466, used at 10 μM), FCF (Sigma, C2791, used at 100 μM), Matrigel (Corning, 354230), and recombinant human sFasL (Peprotech, 310-03H).

Techniques: Microscopy, Staining, Incubation, Disruption, Expressing, Suspension, Control, Fluorescence, Membrane, Two Tailed Test, Comparison

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Del-1 interferes with binding of α v integrin to LAP. (A) Binding of α v β 6 integrin (100 nM) to immobilized BSA, Del-1 (100 nM), and LAP (100 nM), and binding of α v β 6 integrin to immobilized LAP in the presence of Del-1, as assessed in a solid-phase binding assay. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD ( n = 4 per group). *** p < 0.001; Student's t -test. (B) Adhesion of α v β 6 -overexpressing HEK293T cells or human small airway alveolar epithelial primary cells (HSAEpCs) onto increasing concentrations of immobilized LAP protein was assessed. Alternatively, adhesion of cells to immobilized inactive TGF-β derived from conditioned medium was assessed. Data are expressed as the mean ± SD ( n = 3). * p < 0.05; Mann-Whitney U -test. (C) Adhesion of α v β 6 integrin-overexpressing HEK 293T cells or HSAEpC to immobilized LAP is impaired by Del-1 (1, 5, and 10 μg/mL), EGF 1−3 (5 μg/mL), or an α v β 6 -blocking antibody (5 μg/mL). Data are expressed as the mean ± SD ( n = 3). * p < 0.05; Mann-Whitney U -test. (D) Representative co-immunoprecipitation assay to validate the interaction between α v integrin and Del-1 and the interaction between α v integrin and LAP in the absence or presence of Del-1. HEK293T cells overexpressing α v β 6 integrin after lentiviral transduction were incubated for 6 h with Del-1 (20 nM) or LAP (20 nM) and then processed for immunoprecipitation and western blotting. Data are representative of three independent experiments, each with similar results.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: Binding Assay, Derivative Assay, MANN-WHITNEY, Blocking Assay, Co-Immunoprecipitation Assay, Transduction, Incubation, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Del-1 inhibits α v integrin-mediated activation of TGF-β in vitro . (A) Activation of inactive TGF-β by α v β 6 integrin lentivirally expressed by HEK293T cells in the absence or presence of Del-1 (100 nM) or an α v -blocking antibody (2.2 μg/mL). Integrin α v β 6 -overexpressing HEK293T cells (1 × 10 5 /well) were plated and incubated overnight at 37°C. Conditioned medium from RAW264.7 cells overexpressing inactive TGF-β was then added in the presence of Del-1 or an α v -blocking antibody. EGF 1−3 (10 μg/mL) was added to some wells instead of recombinant Del-1. After 12 h, active TGF-β levels were measured in an ELISA. Active TGF-β-containing supernatant was used as a positive control. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD ( n = 3 per group). * p < 0.05; n.s., not significant; Mann-Whitney U -test. (B) Relative inhibition of α v β 6 integrin-mediated TGF-β activation by increasing concentrations of Del-1. α v β 6 integrin-overexpressing HEK293T cells (1 × 10 5 /well) were plated and incubated overnight at 37°C. Conditioned medium from RAW264.7 cells overexpressing inactive TGF-β was added in the presence of Del-1 (0, 1, 5, 10, or 20 μg/mL). After 12 h of incubation, active TGF-β levels were measured in an ELISA. Data are presented as a percentage relative to the concentration of active TGF-β in the absence of Del-1, which was set to 100%. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD ( n = 3 per group). Linear regression analysis revealed a significant inverse correlation between TGF-β activation and Del-1 concentration ( p = 0.0002, R 2 = 0.6301). (C) Inhibition of TGF-β activation in co-cultures of cells expressing either α v β 6 integrin or inactive TGF-β by increasing concentrations of Del-1. α v β 6 integrin-overexpressing HEK293T cells (2 × 10 4 /well) and inactive TGF-β-expressing HEK293T cells (2 × 10 4 /well) were plated and then incubated overnight at 37°C in the presence of Del-1 (0, 1, 5, or 10 μg/mL) or an α v β 6 -blocking antibody (5 μg/mL). The supernatants were analyzed in an ELISA to measure active TGF-β. Data are expressed as the mean ± SD ( n = 3 per group). * p < 0.05; Mann-Whitney U -test. (D) Inhibition of TGF-β activation by Del-1 in α v β 6 integrin-overexpressing HEK293T cells or primary epithelial cells exposed to immobilized inactive TGF-β. Conditioned medium from RAW264.7 cells overexpressing inactive TGF-β were coated onto the plate, and then α v β 6 integrin-overexpressing HEK293T cells or HSAEpCs were plated in the presence of Del-1, EGF 1−3 , or an α v β 6 -blocking antibody. After an overnight incubation at 37°C, active TGF-β levels were measured in an ELISA. Data are representative of three independent experiments, each with similar results, and are expressed as the mean ± SD ( n = 3 per group). * p < 0.05; Mann-Whitney U -test. (E) Del-1-mediated reduction in the amount of active TGF-β to weaken TGF-β signaling in cells. Representative western blots showing phospho-Smad2/3 in HEK 293T cells overexpressing α v β 6 integrin in response to inactive TGF-β. Cells were incubated at 37°C with a RAW 264.7 cell-derived conditioned medium containing inactive TGF-β in the presence of Del-1 for 1–3 h, or in the presence of an α v β 6 -blocking antibody for 1 h. Data are derived from three independent experiments.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: Activation Assay, In Vitro, Blocking Assay, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, MANN-WHITNEY, Inhibition, Concentration Assay, Expressing, Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Endogenous and exogenous Del-1 inhibit the interaction between α v integrin and LAP in vivo . (A) Representative lung sections showing colocalization of α v integrin with LAP in WT and Del-1 −/− mice with BLM-induced PF supplemented with control Fc or Del-1-Fc (intravenous injection; 50 μg/dose Fc or Del-1-Fc on Days 5, 9, and 13 post-BLM instillation; n = 3 mice/group). Lung samples were collected at 21 dpa. Green, α v integrin; red, LAP. Scale bar = 200 μm. Quantification of the colocalization is shown in the right panel. In each section, the number of double-positive cells per 200 μm 2 was counted. Data are expressed as the mean ± SEM. * p < 0.05; Mann-Whitney U -test. (B) Representative images showing colocalization of α v integrin with LAP in fibroblasts or epithelial cells in lungs from Del-1 −/− mice with BLM-induced PF ( n = 3 mice/group). Green, α v integrin; red, LAP; blue, activated fibroblasts (α-SMA); blue, epithelial cells (E-cadherin). Scale bar = 50 μm.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: In Vivo, Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Endogenous Del-1 inhibits activation of TGF-β and subsequent collagen production. (A) Active TGF-β was measured in the BALF of WT and Del-1 −/− mice with PF induced by an adenovirus expressing inactive TGF-β. Mice received adenovirus intratracheally, and BALF was collected at 7 days post administration. Data are expressed as the mean ± SEM ( n = 3 mice per group). * p < 0.05; n.s., not significant; Mann-Whitney U -test. (B) Hydroxyproline analysis of lung tissue from WT and Del-1 −/− mice with PF induced by adenovirus expressing inactive TGF-β. Lungs were collected at Day 14 post-adenovirus administration. The concentrations of hydroxyproline are shown as a percentage of the mean absolute concentration in WT mice treated with a mock adenovirus, which was set to 100%. Data are expressed as the mean ± SEM ( n = 6 mice/group). * p < 0.05, ** p < 0.01; n.s., not significant; Student's t -test.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: Activation Assay, Expressing, MANN-WHITNEY, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Supplementation with Del-1 attenuates the pathological characteristics of PF in mice. (A) Representative ex vivo micro-computed tomography (μ-CT) scans of lung sections from WT and Del-1 −/− mice with BLM (1 U/kg)-induced PF, which were supplemented intravenously with bovine serum albumin (BSA; 50 μg/dose) or soluble Del-1 (sDel-1; 50 μg/dose) at 5, 9, and 13 days post-BLM administration (dpa). Lung sections were taken at 21 dpa. Dashed lines indicate fibrotic regions. Quantification of fibrosis in the μ-CT images is shown in the right panel. Data are represented as the mean ± SEM ( n = 3 mice per group). * p < 0.05; n.s., not significant; Mann-Whitney U -test. (B) Survival analysis of WT and Del-1 −/− mice with BLM-induced PF (1.5 U/kg BLM) that were supplemented with BSA or sDel-1 (intravenous injection, 50 μg/dose sDel-1 at 5, 9, and 13 dpa [ n = 12 mice per group]). * p < 0.0001; log-rank test. (C) The levels of active TGF-β and LAP in the lungs of mice with BLM-induced PF that were supplemented with Fc or Del-1-Fc (intravenous injection; 50 μg/dose at 5, 9, and 13 dpa). The lung lysates were collected at 14 dpa and subject to ELISA. Data are expressed as mean ± SEM ( n = 3 mice per group). * p < 0.05, *** p < 0.001; Mann-Whitney U -test. (D) Representative western blots to detect the expression of α-SMA and the level of phosphorylated Smad3 in WT mice with BLM-induced PF (2 U/kg BLM) supplemented with Fc or Del-1-Fc (intravenous injection; 50 μg/dose Fc or Del-1-Fc at 14, and 18 dpa). Lung lysates were collected at 21 dpa. Densitometric data are shown in the right panels. Data are expressed as mean ± SEM ( n = 3–4 mice per group). ** p < 0.01, *** p < 0.001; Student's t -test. (E) Hydroxyproline analysis of lung tissues from WT mice with BLM-induced PF (2 U/kg BLM) supplemented with Fc or Del-1-Fc, as in (D) . Data are expressed as the mean ± SEM ( n = 3–4 mice per group). * p < 0.05, *** p < 0.001; Student's t -test. (F) Hydroxyproline analysis of lung and skin tissues from WT mice with systemic fibrosis (8 U/kg/dose BLM, daily subcutaneous injection for 15 days) supplemented with Fc or Del-1-Fc (intravenous injection; 50 μg/dose Del-1-Fc at 7, 11, 15, and 19 dpa after the first BLM instillation). Tissues were harvested at 23 days after the first BLM instillation. Data are expressed as the mean ± SEM ( n = 3 mice per group). * p < 0.05; Mann-Whitney U -test.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: Ex Vivo, Micro-CT, MANN-WHITNEY, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Del-1, an Endogenous Inhibitor of TGF-β Activation, Attenuates Fibrosis

doi: 10.3389/fimmu.2020.00068

Figure Lengend Snippet: Reduced expression of Del-1 correlates with the development of PF in humans, which accompanies increased active TGF-β in plasma. (A) Del-1 protein levels in the plasma of healthy controls and PF patients: healthy controls ( n = 20); mild-to-moderate PF ( n = 20; FVC > 50%); and severe PF ( n = 8; FVC ≤ 50% and DLCO ≤ 35%). Data are expressed as the mean ± SEM. * p < 0.05; n.s., not significant; Dunnett's test and Student's t -test. (B) Active TGF-β levels in the plasma of healthy controls ( n = 20) and patients with PF ( n = 20; FVC <50%). Data are expressed as mean ± SEM. *** p < 0.001; Student's t -test.

Article Snippet: The plates were coated with 100 nM recombinant human Del-1 (R&D Systems), LAP (R&D Systems), or BSA in PBS at 37°C for 2 h. After washing with washing buffer (0.05% Tween-20 in PBS), the plates were blocked at room temperature (RT) for 1 h with wash buffer containing 0.5% BSA.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition

doi: 10.1074/jbc.RA119.008619

Figure Lengend Snippet: MDAMB231-MV–mediated transfer of miR221 induces EMT in MCF7 via the down-regulation of PTEN and activation of the AKT/NF-κB signaling pathway. A, MV were collected from PAR2-activated MDAMB231 cells and incubated with MCF7 for various time points. MV fusion was analyzed upon observing TF band in MV-incorporated MCF cells and B, accordingly a line diagram was plotted that indicates that MV fusion occurs at ∼2 h post-MV incubation with MCF7. C, the level of PTEN was analyzed in MCF7 cells upon fusion of PAR2-activated MDAMB231-derived MV at various time points and D, accordingly the graph was generated, which suggests that the PTEN level goes down significantly at ∼5 h of MV incubation. E, both control as well as PAR2-activated MDAMB231-derived MV were fused with MCF7 and the PTEN level was monitored at 5 h of MV treatment. F, MDAMB231 cells were treated with PAR2AP beside untreated controls and MV were isolated from the cell supernatant after 24 h. miR221 expression in the MV was monitored by the real-time PCR approach. G, miR221 expression was also analyzed in MDAMB468-derived MV (both control and PAR2-activated). miR221 expression was found to be significantly higher in MV derived from PAR2-activated cells. H, relative miR221 expression was analyzed in both MCF7 and MDAMB231 cells, which depict that miR221 expression is significantly higher in MDAMB231 than MCF7. I, MCF7 cells were treated with both control as well as PAR2-activated MDAMB231-derived MV for 3 h followed by the analysis of miR221 level by real-time PCR. The incorporation of MV significantly up-regulated the miR221 level in MCF7 and maximum effect was monitored in a PAR2-activated MV-treated set. J, MCF7 cells were pre-treated with actinomycin D (5 μg/ml) for 8 h followed by the addition of MV. The miR221 level was analyzed in those cells by real-time PCR. The treatment of actinomycin D does not interfere with the level of miR221 in MV-fused MCF7 cells. K and L, MDAMB231 cells were transfected with anti-miR221 beside Scrambled control miR followed by the addition of PAR2AP or FVIIa. MV were isolated and fused with MCF7. Alternatively, MCF7 cells were pre-treated with anti-miR221 alongside Scrambled miR followed by the addition MDAMB231-derived MV. The analysis of PTEN, phospho-AKT, nuclear phospho-p65, Snail, Slug, E-cadherin, N-cadherin, and vimentin expression in MV-fused MCF7 cells was performed by Western blotting, which indicates that the introduction of anti-miR221 reverses the EMT phenotype induced by MDAMB231-MV.

Article Snippet: MDAMB231 and MDAMB468 cells, grown in 100-mm dishes to 80% confluence were serum starved for 2 h followed by the stimulation with PAR2-activation peptide (PAR2AP; SLIGKV-NH 2 ; 100 μ m ; GL Biochem, China) or human recombinant FVIIa (100 n m ; Novo-Nordisk).

Techniques: Activation Assay, Incubation, Derivative Assay, Generated, Isolation, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition

doi: 10.1074/jbc.RA119.008619

Figure Lengend Snippet: MDAMB231 cell-derived MV confer resistance against cisplatin-induced apoptosis of MCF7. A, MCF7 cells were exposed to various concentrations of cisplatin for 30 h and cell death was monitored by the MTT assay and the IC50 of cisplatin on MCF7 was shown to be 100 μm. B, the degree of apoptosis induced by various concentrations of cisplatin was also measured by a caspase 7 activity assay, which also indicates that maximum caspase 7 activity was monitored at 100 μm cisplatin. C, MCF7 cells were exposed to cisplatin (100 μm) for various time points and apoptosis was monitored by a FACS analyzer as mentioned briefly under “Experimental procedures,” which indicates that maximum cell death was observed at ∼30 h of cisplatin exposure. D–F, MDAMB231 cell-derived MV (both control, as well as PAR2AP-treated) were fused with MCF7 cells followed by the addition of cisplatin (100 μm). Cell viability, caspase 7 activity, and the degree of apoptosis by FACS were measured thereafter, which demonstrates that MDAMB231-MV rescued MCF7 cells from cisplatin-induced apoptosis. G–I, MDAMB231 cells were pre-transfected with anti-miR221 followed by FVIIa addition and MV isolated were introduced into MCF7 cells. The recipient MCF7 cell death was monitored upon challenging the cells with cisplatin and the data indicates that anti-miR221 introduction diminished the anti-apoptotic effect of MDAMB231-MV on MCF7.

Article Snippet: MDAMB231 and MDAMB468 cells, grown in 100-mm dishes to 80% confluence were serum starved for 2 h followed by the stimulation with PAR2-activation peptide (PAR2AP; SLIGKV-NH 2 ; 100 μ m ; GL Biochem, China) or human recombinant FVIIa (100 n m ; Novo-Nordisk).

Techniques: Derivative Assay, MTT Assay, Activity Assay, Transfection, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition

doi: 10.1074/jbc.RA119.008619

Figure Lengend Snippet: MDAMB231-MV–mediated transfer of miR221 promotes AKT/NF-κB-dependent migration and invasion of nonmetastatic MCF7 cells. A–D, to determine the role of miR221 transported from MDAMB231 to MCF7 cells via MV in inducing MCF7 cell migration, MDAMB231 cells were transfected with anti-miR221 beside Scrambled control miR followed by the addition of PAR2AP or FVIIa. MV were isolated and fused with MCF7. Alternatively, MCF7 cells were pre-transfected with anti-miR221 alongside Scrambled miR followed by the addition MDAMB231-derived MV. The migratory property of these MV-fused MCF7 cells was analyzed thereafter, which indicates that MDAMB231-MV–mediated induction of MCF7 migration was suppressed upon introduction of anti-miR221; scale bar 100 μm. E–H, to understand the contribution of MDAMB231-MV in promoting MCF7 invasion, the donor MDAMB231 cells were transfected with anti-miR221 followed by the addition of PAR2AP or FVIIa. MV were collected and incorporated into MCF7. Alternatively, MCF7 were pre-transfected with anti-miR221 followed by the fusion of MDAMB231-derived MV. MV-fused MCF7 cell invasion was measured by Transwell invasion assay; scale bar 100 μm, as mentioned briefly under “Experimental procedures.” The induction of MCF7 invasion by MDAMB231-MV was repressed upon administration of anti-miR221.

Article Snippet: MDAMB231 and MDAMB468 cells, grown in 100-mm dishes to 80% confluence were serum starved for 2 h followed by the stimulation with PAR2-activation peptide (PAR2AP; SLIGKV-NH 2 ; 100 μ m ; GL Biochem, China) or human recombinant FVIIa (100 n m ; Novo-Nordisk).

Techniques: Migration, Transfection, Isolation, Derivative Assay, Transwell Invasion Assay