Journal: bioRxiv

Article Title: Autophagic stress activates distinct compensatory secretory pathways in neurons

doi: 10.1101/2024.11.07.621551

Figure Lengend Snippet: A-D) Representative western blots of isolated plasma from one year old control and LRRK2 G2019S mice against A) Class III β-tubulin, B) TSG101, C) CD81, and D) Synapsin-1. E-H) Quantification of relative band intensity of isolated plasma from one year old control and LRRK2 G2019S mice for E) Class III β-tubulin, F) TSG101, G) CD81, and H) Synapsin-1. N=11, two-tailed t-test, error bars represent SEM. I) Example images of control and LRRK2 G2019S neurons treated with 5 µM of the exosome inhibitor, GW4869. DIC images with overlay of activated CellEvent Caspase3/7 ready probe which is an early indicator of apoptosis. J) Example images of control and LRRK2 G2019S neurons treated with 40 µM of the microvesicle inhibitor, Y27632. DIC images with overlay of activated CellEvent Caspase3/7 ready probe which is an early indicator of apoptosis. Scale bar = 20µM K) Quantification of percent of neurons with activated Caspase 3/7 following two hours of treatment with varying levels of GW4869. N=3, 2way ANOVA with Šídák’s multiple comparison test. Error bars indicate SEM. L) Quantification of percent of neurons with activated Caspase 3/7 following two hours of treatment with varying levels of Y27632. N=3, 2way ANOVA with Šídák’s multiple comparison test. Error bars indicate SEM.

Article Snippet: Primary cortical DIV7 neurons were treated with varying concentrations of either GW4869 (Tocris #6741) or Y27632 (Tocris #1254) for two hours.

Techniques: Western Blot, Isolation, Control, Two Tailed Test, Comparison

Journal: Autoimmunity

Article Title: CAFs-released exosomal CREB1 promotes cell progression and immune evasion in thyroid cancer via the positive regulation of CCL20.

doi: 10.1080/08916934.2025.2458324

Figure Lengend Snippet: Figure 4. CAFs-secreted exosomes carrying CREB1 enhanced CCL20 expression. (A-B) Exosomes isolated from NFs and CAFs were verified by morphology obser vation under TEM (A) and exosomal markers (Alix, CD63, and Tsg101) using western blot (B). (C) NFs and CAFs isolated exosomes were labeled with PKH67 and co-cultured with TC cells, followed by detection of the uptake of exosomes via fluorescence confocal microscopy. (D) CREB1 mRNA expression was quantified by RT-qPCR after TC cells were cultured in NF and CAF media treated with GW4869 or not. (E) Western blot was performed for protein analysis of CREB1 in TC cells incubated with exosomes from CAFs of sh-NC or sh-CREB1 transfection. (F) CCL20 was determined via western blot after TC cells were incubated with exosomes from CAFs of sh-NC, sh-CREB1, sh-CREB1 + pcDNA, sh-CREB1 + CCL20 group. *p < 0.05.

Article Snippet: In addition, TC cells were cultured in NFs and CAFs medium treated with GW4869 (20 μM; Selleck, Houston, TX, USA), and CREB1 mRNA expression was examined by western blot.

Techniques: Expressing, Isolation, Western Blot, Labeling, Cell Culture, Fluorescence, Confocal Microscopy, Quantitative RT-PCR, Incubation, Transfection

Journal: Science Advances

Article Title: Nutrient availability regulates the secretion and function of immune cell–derived extracellular vesicles through metabolic rewiring

doi: 10.1126/sciadv.adj1290

Figure Lengend Snippet: ( A ) PCA scatterplot based on RNA-seq data of M1-Mφs treated with GLN + (2 mM) or without GLN (GLN − ) for 24 hours. ( B ) Heatmap showing the different gene expression profiles in the different groups. ( C ) Volcano plot showing DEGs ( P -adjusted < 0.05) between groups. ( D ) GO enrichment analysis of DEGs (FC > 1.5, top 10) in response to GLN − conditions. ( E ) Schematic illustrating the ESCRT machinery and heatmap showing ESCRT gene expression in the different groups. ( F to H ) Representative images and quantification of TSG101 + early endosomes, CD63 + MVBs, and Lamp2b + late endosomes in M1-Mφs under GLN − conditions (scale bar, 50 μm). ( I ) qPCR analysis of TSG101 and VPS4B gene expression in M1-Mφ under GLN − conditions cotreated with GW4869 for 24 hours. ( J ) EV yield determined by the EV/cell protein ratio after GW4869 treatment. ( K ) qPCR analysis of cytokine gene expression (e.g., IL-6 and IL-1 β) in EV GLN− after GW4869 treatment ( n = 3; **P < 0.01, *P < 0.05 versus the GLN − group).

Article Snippet: M1-Mφs under GLN depletion conditions were treated with GW4869 (20 μM, HY-19363, MedChemExpress, Monmouth Junction, NJ, USA) or DHMEQ (2.5 μg/ml, HY-14645, MedChemExpress) for 24 hours.

Techniques: RNA Sequencing Assay, Expressing

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: Efficiency of LGTV dissemination within I. scapularis nymphal ticks is affected upon GW4869 treatment (A–C) LGTV loads in midgut (MG), salivary glands (SGs) or carcass (C) of nymphs that were simultaneously exposed to LGTV (1 MOI) and either mock (1.5% DMSO) or GW4869 (150 μM) and incubated for 0 (A), 3 (B) or 17 (C) days post infection (DPI), respectively. (D and E) LGTV loads in MG, C and SGs of nymphs that were pretreated with either mock/GW4869 followed by LGTV infection and incubated for 3 (C) or 17 (D) days p.i., respectively. LGTV loads were normalized to total RNA. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents a pool of either two (A–C) or three (D and E) ticks in the simultaneous or pretreatment groups, respectively. p value less than 0.05 is considered statistically significant and ns indicates not significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Incubation, Infection

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: GW4869 treatment affects viral dissemination within ticks that fed on LGTV-infected mice Photographs (A) showing reduced body sizes of ticks treated with GW4869 (150 μM) in comparison to mock controls (1.5% DMSO-treated) and fully fed on LGTV-infected mice to acquire the viral loads. (B) Quantification of body weights of GW4869 or mock-treated ticks is shown. QRT-PCR analysis showing LGTV loads in midguts (MG) or salivary glands (SGs) of fully engorged repleted ticks (C) or ticks collected during feeding (D) on LGTV-infected mice. In both post fed or during feeing (DF) groups, ticks were first treated with either GW4869 (150 μM) or mock control (1.5% DMSO) followed by feeding on LGTV-infected mice to acquire the viral loads. LGTV loads were normalized to total RNA. Circles/triangles denote the mock-treated groups, whereas squares/inverted triangles represent the GW4869-treated groups. Each circle/square/triangle/inverted triangle represents one MG or a pair of SGs dissected from post-fed ticks (C) or during feeding ticks (D). p value less than 0.05 is considered statistically significant and ns indicates not significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Infection, Comparison, Quantitative RT-PCR, Control

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: LGTV loads and EVs released from salivary glands isolated from I. scapularis female ticks are reduced upon GW4869 treatment (A) QRT-PCR showing LGTV loads in midgut (MG), carcass (C), and salivary glands (SGs) of adult ticks that were simultaneously exposed to LGTV (1 MOI) and to either mock (1.5% DMSO) or GW4869 (150 μM) and incubated for 3 days post-infection. (B) LGTV loads shown from SGs-derived EVs isolated from female ticks incubated at 3 days post LGTV infection with simultaneous treatment with mock/GW4869. (C) Concentration of EVs-derived from SGs isolated from female ticks incubated for 3 days post LGTV infection and mock/GW4869 treatment is shown. (D and E) Graphical representation showing the quantification of EVs-derived from SGs isolated from female ticks after 3 days post LGTV infection and mock/GW4869 treatment. Four-five independent replicates were considered for QRT-PCR or for determining EVs concentration. LGTV loads were normalized to total RNA. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents tissues from one adult tick (in A and B) and salivary glands collected from two female ticks (C), respectively. p value less than 0.05 is considered statistically significant and ns indicates not significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Isolation, Quantitative RT-PCR, Incubation, Infection, Derivative Assay, Concentration Assay

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: Treatment with low dose of GW4869 reduces feeding efficiency and LGTV acquisition in nymphal ticks (A) Micrographs showing sizes of post-fed ticks collected (after repletion) from either control (no treatment) or GW4869 (50 μM) treated nymphs. (B) Nymphal body weights are shown (in milligrams, mg) from either control (no treatment) or GW4869- treated ticks collected at post-repletion. Each circle/square represents the weight of a single nymph per group. (C) LGTV loads in control/GW4869-treated post fed nymphs is shown from acquisition. (D) Is SMase transcript levels from control/GW4869-treated post fed nymphs is shown. (E) Agarose gel image showing LGTV transcripts in infected blood isolated from mice used for tick feeding studies. Mice used for feeding of untreated nymphs were labeled as control and mice that were used for feeding GW4869-treated (50 μM) ticks are labeled as GW4869 group. M represents DNA marker, PC indicate positive control, and NC denotes negative control. The expected band size for LGTV transcript (140 bp) is shown with black arrow. LGTV and Is SMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered statistically significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Control, Agarose Gel Electrophoresis, Infection, Isolation, Labeling, Marker, Positive Control, Negative Control

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: Treatment with higher dose of GW4869 reduces feeding efficiency and LGTV acquisition in nymphal ticks (A) Micrograph showing sizes of mock (1.5% DMSO) or GW4869 (150 μM) treated-nymphs collected post repletion. (B) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869- treated ticks collected at post-repletion. Each circle/square represents one nymphal tick. (C) LGTV loads in mock/GW4869- treated post fed nymphs is shown. (D) Is SMase transcript levels are shown from either mock/GW4869 treated post-fed nymphs. (E) Agarose gel image showing LGTV transcript (of 140 bp, indicated with arrow) in mice that were used for tick feeding studies. Mice used for feeding of untreated nymphs were labeled as control and mice that were used for feeding GW4869-treated (150 μM) ticks were labeled as GW4869. M represents DNA marker, PC indicate positive control, and NC denotes negative control. LGTV and Is SMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered statistically significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Agarose Gel Electrophoresis, Labeling, Control, Marker, Positive Control, Negative Control

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor A, Charles River Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (1.5% DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion. Each circle/square represents the weight of one nymph. (B) LGTV loads from mock/GW4869-treated post fed nymphs is shown. (C) Is SMase transcript levels in mock/GW4869 treated post fed nymphs is shown. (D) Viral loads in tissues are shown from four independent mice that were used for mock/GW4869-treated LGTV-infected nymphs feeding. LGTV and Is SMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered satistically significant and ns indicates not significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Transmission Assay, Infection

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion. Each circle/square represents the weight of one nymph. (B) LGTV loads from mock/GW4869-treated post-fed nymphs are shown. (C) Is SMase transcript levels in mock/GW4869 treated post fed nymphs are shown. (D) LGTV loads in tissues are shown from four independent mice that were used for mock/GW4869-treated LGTV-infected nymphs feeding. LGTV and Is SMase transcripts were normalized to total RNA or tick beta-actin levels, respectively. Circles denote the mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one fed nymphal tick. p value less than 0.05 is considered satistically significant and ns indicates not significant.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Transmission Assay, Infection

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet: GW4869 treatment reduces molting efficiency and survival rates of LGTV-infected ticks (A) Molting rates of untreated uninfected or LGTV-infected or GW4869 (50 μM)-treated and LGTV-infected I. scapularis larvae into nymphs is shown. (B) Total survival rates of untreated uninfected or LGTV-infected or GW4869 (50 μM)-treated and LGTV-infected larvae molting into nymphs are shown. (C) Total survival rates of mock (1.5% DMSO) or GW4869 (150 μM)- treated and LGTV-infected nymphs molting into adults are shown. (D) LGTV loads in adult ticks molted from mock/GW4869-treated and LGTV-infected nymphs is shown. LGTV loads were normalized to total RNA. Circles denote mock-treated group, whereas squares represent the GW4869-treated group. Each circle/square represents one tick. p value less than 0.05 is considered statistically significant and ns indicates not significant. (E) Model showing the effects of the GW4869 treatment of ticks on LGTV transmission to the vertebrate host. Graphical representation shows that LGTV-infected and GW4869-treated (150 μM) ticks are hampered in blood feeding (with reduced tick body weights). GW4869-treated and LGTV-infected ticks fed partially showing reduced feeding efficiency and decreased viral transmission into the vertebrate host. Treatment of GW4869 affected EVs release that corelated with significant reduction in LGTV loads from these ticks. The GW4869 treatment resulted in inefficient tick feeding, lower EVs biogenesis, decreased viral loads during acquisition and transmission, and reduced molting/survival rates. The combination of all these factors eventually affected vector fitness and competence.

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Infection, Transmission Assay, Plasmid Preparation

Journal: iScience

Article Title: GW4869 inhibitor affects vector competence and tick-borne flavivirus acquisition and transmission by blocking exosome secretion

doi: 10.1016/j.isci.2024.110391

Figure Lengend Snippet:

Article Snippet: Figure 7 Treatment with GW4869 reduces feeding efficiency and LGTV transmission from infected nymphs to mice (from Vendor B, Jackson Laboratories) (A) Nymphal body weights are shown (in milligrams, mg) from either mock (DMSO) or GW4869 (150 μM)- treated and infected ticks collected at post-repletion.

Techniques: Virus, Saline, SYBR Green Assay, Transfection, cDNA Synthesis, Gel Extraction, Software, Imaging

Journal: Frontiers in Cell and Developmental Biology

Article Title: Novel Roles of Small Extracellular Vesicles in Regulating the Quiescence and Proliferation of Neural Stem Cells

doi: 10.3389/fcell.2021.762293

Figure Lengend Snippet: Exosomes involved in cell cycle regulation of qNSCs. (A) Exosomal functions in quiescence entrance and maintenance. (B) Exosome quantification of aNSCs after treatment with GW4869 (orange) and DMSO as a control (blue). Horizontal axis: vesicle size. Vertical axis: vesicle number. (C,D) Quantification of CycD1 levels in NSCs after inducing (C) and maintaining quiescence (D) in presence of GW4869 for 12 h (C) and 24 h (D) . All values were divided by that for DMSO control. Each plot indicates independent experiments. CycD1 band intensities were divided by those of actin bands. Ratios with respect to DMSO control are plotted. Middle lines in plots are medians. (* p < 0.05, **p < 0.01; two-tailed paired t-test, n = 4; For q-keep, one outlier identified by outlier test of Grubbs was removed). (E,F) Representative results of WB of quiescence induction (E) and maintenance (F) . CycD1 were three bands, and actin is loading control. (G,H) Cell counts of Ki-67- and MCM2-positive cells after inducing (G) and maintaining quiescence (H) after incubation with GW4869 for 24 h. Ki-67- and MCM2-positive cell numbers were divided by the cell number based on DAPI staining and shown as the percentage. (** p = 0.013, ****p < 0.0001; one-way ANOVA, Turkey’s multiple comparison test, n = 8). (I,J) Representative photos of Ki-67- (green) and MCM2-positive cells (red) with DAPI staining (blue) in panel G (I) and H (J) . (K) Protein synthesis activity in the quiescence-induced (q induction) and maintained cells (q keep). The intensity of OP-puro labeling (OPP) is represented by a thermal scale.

Article Snippet: Quiescence was induced with quiescent medium [50 ng/ml BMP4 (R&D Systems) in proliferation medium minus EGF] after two washings with phosphate-buffered saline (PBS; Nacalai Tesque) and achieved in 3 d. To prepare reNSCs, qNSCs were washed twice with PBS and cultured in proliferating medium for 2 d. For the exosome inhibition, aNSCs were cultured for 1 d and the medium was supplemented either with 10 μM GW4869 (Cayman Chemical Co., Ann Arbor, MI, United States) or dimethyl sulfoxide (DMSO; Nacalai Tesque) as the control.

Techniques: Control, Two Tailed Test, Incubation, Staining, Comparison, Activity Assay, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Helicobacter pylori induced miR-362-5p upregulation drives gastric cancer progression and links hepatocellular carcinoma through an exosome-dependent pathway

doi: 10.3389/fcimb.2025.1582131

Figure Lengend Snippet: Promotion of HCC progression by Hp-GES-EVs through exosome-enclosed miR-362-5p. (A) miR-362-5p levels in mice serum with or without H. pylori infection. (B) RT-qPCR analysis of miR-362-5p levels in Hep G2 and Hep 3B cells co-incubated with Hp-GES-EVs. (C) Western blot analysis of TLE4 expression in Hep G2 and Hep 3B cells. (D) EdU assay showing the proliferation of Hep G2 and Hep 3B cells treated with Hp-GES-EVs and an additional miR-362-5p inhibitors. (E) Wound healing assay showing migration of Hep G2 and Hep 3B cells treated with Hp-GES-EVs and an additional miR-362-5p inhibitors. (F) serum miR-362-5p in H. pylori -infected mice with GW4869 treatment. (G) Western blot analysis of TLE4 expression in liver tissues with GW4869 treatment. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The scale bar represents 50 μm in (D) and 100 μm in (E) .

Article Snippet: To assess the role of exosome-associated miRNAs in mediating liver disease due to H. pylori infection, GW4869 (Macklin, 6823-69-4, China), an inhibitor targeting exosome biogenesis or release, was utilized during mouse infection assays.

Techniques: Infection, Quantitative RT-PCR, Incubation, Western Blot, Expressing, EdU Assay, Wound Healing Assay, Migration

Journal: Cancer Drug Resistance

Article Title: Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

doi: 10.20517/cdr.2022.26

Figure Lengend Snippet: Characterization of EV concentration and size distribution following GW4869 treatment in PC3-R cells. EVs were isolated from the conditioned media of paclitaxel-resistant PC3-R cells following 24 h (upper panel) and 48 h (lower panel) of treatment with GW4869 (10-20 µM) and characterized for concentration and size distribution by NTA. (A) Concentration and size distribution of EVs isolated from DMSO (VC) or GW4869 (10 and 20 µM) treated cells are represented with green, orange, and red colors, respectively ( n = 3). Each line represents the mean of three samples, and an average data of 5 videos of 30 sec each was used for each sample. (B-C) Total EV concentration per million cells and mean size are plotted. (D) Size distribution of EVs is presented as percent particles in the mentioned size range. Each bar represents mean ± SEM ( n = 3). VC: Vehicle control; * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.0001.

Article Snippet: Overall, it appears that the dose and treatment duration of GW4869, as well as the cancer-type or study model, could also impact its effect on EV biogenesis and secretion in biofluids.

Techniques: Concentration Assay, Isolation, Control

Journal: Cancer Drug Resistance

Article Title: Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

doi: 10.20517/cdr.2022.26

Figure Lengend Snippet: Effect of GW4869 treatment on the survival and clonogenicity of PC-R cells. (A) Paclitaxel-resistant PC3-R cells were treated with DMSO (VC) or GW4869 (5-20 µM), and cell viability was measured in MTT assay after 24, 48, and 72 h. Data are presented as mean ± SEM ( n = 5 replicates per group). * P < 0.05, ** P < 0.005. (B) Colony formation was measured in PC3-R cells after GW4869 treatment (10 and 20 μM) as described in the methods. Representative images are shown (left panel), and the colony number is presented as mean ± SEM ( n = 3 replicates per group). **** P < 0.0001. VC: Vehicle control.

Article Snippet: Overall, it appears that the dose and treatment duration of GW4869, as well as the cancer-type or study model, could also impact its effect on EV biogenesis and secretion in biofluids.

Techniques: MTT Assay, Control

Journal: Cancer Drug Resistance

Article Title: Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

doi: 10.20517/cdr.2022.26

Figure Lengend Snippet: In vivo effect of GW4869 treatment on the growth of paclitaxel-resistant PC3-R cells’ xenografts. Male athymic nude mice were treated with vehicle or GW4869, and various study parameters were assessed as described in the methods. (A) Average body weight (mean ± SEM) at the start and day 40 of the study for control mice ( n = 3) and GW4869 treated mice ( n = 4) mice. (B) Average xenograft volume (mean ± SEM) in control and GW4869 treated mice survived at measured time points is presented. (C) In the end, mice were sacrificed, xenografts were excised, and their weight was measured. Average tumor weight in the control group ( n = 6 xenografts) and the GW4869 group ( n = 8 xenografts) is presented as mean ± SEM. Unpaired t test was used to calculate the statistical significance, * P = 0.017. (D) CD63 expression in xenograft tissues from the vehicle control (VC) group and GW4869 group ( n = 6 each) was measured by IHC. For each image, ten random areas were analyzed for IHC scoring as described in the methods. Mean IHC scores are presented as mean ± SEM in the bar diagram. Representative images are shown. (E) EVs isolated from the plasma of control ( n = 3) and GW4869 treated mice ( n = 3) were analyzed by NTA, and the size distribution (mean ± SEM) of EVs was plotted as a percentage of total EVs. (F) EVs from VC ( n = 3) and GW4869 treated ( n = 4) mice were used for the analysis of syntenin, CD63, and GOLGA2 by Western blotting. Representative immunoblots are shown. (G) Densitometry analysis of syntenin and CD63 expression was performed and normalized with corresponding band intensity in Ponceau-stained membrane. The relative band intensity is presented as mean ± SEM.

Article Snippet: Overall, it appears that the dose and treatment duration of GW4869, as well as the cancer-type or study model, could also impact its effect on EV biogenesis and secretion in biofluids.

Techniques: In Vivo, Control, Expressing, Isolation, Clinical Proteomics, Western Blot, Staining, Membrane

Journal: Cancer Drug Resistance

Article Title: Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells

doi: 10.20517/cdr.2022.26

Figure Lengend Snippet: Effect of GW4869 treatment on paclitaxel-resistant DU145-R cells. EVs were isolated from the conditioned media of paclitaxel-resistant DU145-R cells following 24 h (upper panel) and 48 h (lower panel) of treatment with GW4869 (10-20 µM) and characterized for concentration and size distribution by NTA. (A-B) Each colored line in the left panel represents the mean of three samples, and an average data of 5 videos of 30 s each was used for each sample. Concentration (particles/mL)-size distribution, average EV concentration per million cells, average size, and percent particles for various size ranges are presented. Each bar represents mean ± SEM ( n = 3). * P < 0.05, ** P < 0.005 *** P < 0.0005 **** P < 0.0001. (C) Colony formation was measured in DU145-R cells after GW4869 treatment (10 and 20 μM) as described in the methods. Representative images are shown (left panel), and the colony number is presented as mean ± SEM ( n = 3 replicates per group). *** P < 0.0005, **** P < 0.0001.

Article Snippet: Overall, it appears that the dose and treatment duration of GW4869, as well as the cancer-type or study model, could also impact its effect on EV biogenesis and secretion in biofluids.

Techniques: Isolation, Concentration Assay

Journal: Journal of Inflammation Research

Article Title: PM2.5 Promotes Macrophage-Mediated Inflammatory Response Through Airway Epithelial Cell-Derived Exosomal miR-155-5p

doi: 10.2147/JIR.S482509

Figure Lengend Snippet: GW4869 improves inflammation in Mφ. PM2.5-BEAS-2B cells were pretreated with GW4869 and co-cultured with Mφ. ( A ) Cellular supernatant IL-6, IL-1β, TNF-α contents of Mφ; ( B ) mRNA expression levels of IL-6, IL-1β, TNF-α in Mφ; ( C ) miR-155-5p expression level in Mφ; ( D ) mRNA expression levels of SOCS1, NF-κB in Mφ; ( E and F ) Protein expression level of SOCS1, P65, PP65 in Mφ as detected by Western blot analysis as well as statistical analysis of gray values. Experimental group: group A PM2.5 0 -BEAS-2B+Mφ, group C PM2.5 200 -BEAS-2B+Mφ, group I PM2.5 200 -BEAS-2B+GW4869+Mφ. ** P <0.01, * P <0.05 vs group A; ▲▲ P <0.01, ▲ P <0.05 vs group C.

Article Snippet: In Group I, 10 μM of GW4869 (Topscience, Shanghai, China) was added to the upper layer and incubated at 37°C and 5% CO 2 for 48h.

Techniques: Cell Culture, Expressing, Western Blot

Journal: European Journal of Medical Research

Article Title: BMSC-derived exosomes protect against kidney injury through regulating klotho in 5/6 nephrectomy rats

doi: 10.1186/s40001-022-00742-8

Figure Lengend Snippet: Effect of exosome transplantation on klotho activation and expression. A Fluorescent report plasmid containing klotho promoter and pRL-TK were co-transfected into 293 T cells. The transfected cells were randomly divided into five groups: control group (PBS), TGF-β1 group (1 ng/μL TGF-β1), BMSCs group (1 ng/μL TGF-β1+co-cultured with 5 × 10 4 well BMSCs), exosomes (1 ng/μL TGF-β1+0.4 mg exosomes), and exosome inhibitor group (1 ng/μL TGF-β1+co-cultured with 5 × 10 4 well BMSCs+GW4869). Then the firefly and Renilla luciferase activities were measured using the dual luciferase reporter gene assay. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01. The protein expression of klotho was also evaluated using western blotting B , C qRT-PCR were performed to detect the mRNA expression of klotho. mRNA expression levels were normalized to that of GAPDH. Quantitative data ( n = 6–7) are provided as the mean ± SD. * p < 0.05, ** p < 0.01. D Expression of klotho protein in the control, 5/6 Nx, and exosome-treated 5/6 Nx rats was evaluated using western blotting

Article Snippet: The transfected cells were treated with TGF-β1 (1 ng/μL), exosomes (0.4 mg), or co-cultured with BMSCs (5 × 10 4 /well) in a Transwell plate for 24 h. GW4869 (GLPBIO, Montclair, CA, USA) was used as an inhibitor of exosome biogenesis and release.

Techniques: Transplantation Assay, Activation Assay, Expressing, Plasmid Preparation, Transfection, Control, Cell Culture, Luciferase, Reporter Gene Assay, Western Blot, Quantitative RT-PCR