Journal: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Robust expression exhibiting a comparative CD63-tdTomato-labeled vesicle load in resting microglia across species. ( A – C ) Confocal microscopy images of immortalized human microglia in resting state showing the presence of CD63-tdTomato-labeled vesicles (red). Cells were counterstained with Alexa Fluor™ 488 Phalloidin (green) to visualize microglial cell bodies and Hoechst (blue) to label nuclei. ( A , B ) Low-magnification view showing widespread intracellular distribution of CD63-tdTomato-labeled with majority exhibiting a perinuclear accumulation. Higher-magnification (40×) image of HuMG parent cells ( C ) demonstrating punctate CD63-positive vesicles localized along actin filaments and concentrated in the perinuclear region. ( D , E ) Resting mouse microglia exhibiting robust expression of CD63-tdTomato-labeled intracellular vesicles. ( D ) Phalloidin-488 staining defines cell boundaries and morphology, revealing widespread CD63-positive puncta throughout the cytoplasm. ( E ) Hoechst counterstaining confirms intracellular vesicle localization surrounding the nucleus. ( F ) Quantification of intracellular vesicle load in resting microglia of both mouse and human origin displayed significantly higher cellular load of CD63-expressing intracellular vesicles in microglia of mouse origin compared to that of human origin (** p < 0.01). Bars represent mean ± SEM; acquired from images obtained from independent biological replicates from each species. Scale bar: 15 µm ( A , B , D , E ) and 5 µm ( C ).

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Expressing, Labeling, Confocal Microscopy, Staining

Journal: Cells

Article Title: Comparative Profiling of Mouse and Human Microglial Small Extracellular Vesicles Reveals Conserved Core Functions with Distinct miRNA Signatures

doi: 10.3390/cells15020184

Figure Lengend Snippet: Time-dependent uptake of CD63-tdTomato-labeled MGEVs by HuSCs. ( A – F ) Confocal images of HuSC exposed to purified CD63-tdTomato-labeled MsMGEVs (red) for 24 h ( A , B ), 48 h ( C , D ), or 72 h ( E , F ). Cells were stained with Alexa Fluor™ 488 Phalloidin (green) to mark the cell body and Hoechst to label nuclei (blue). Internalized CD63-tdTomato-positive sEVs appear as red puncta distributed throughout the cytoplasm and concentrated in perinuclear regions. The size of fluorescent puncta reflects intracellular endosomal accumulation of multiple internalized sEVs rather than aggregation of individual vesicles. Uptake of MsMGEVs showed a trend in an increase in sEVs overtime. Confocal images of HuSC treated with purified CD63-tdTomato-labeled HuMGEVs, red) for 24 h ( G , H ), 48 h ( I , J ), or 72 h ( K , L ). Like MsMGEVs, HuMGEV uptake was evident as intracellular red puncta; however, the overall accumulation pattern remained relatively stable across time points. ( M , N ) shows untreated HuSC microglia (control with no MGEV exposure) showing phalloidin-488 and Hoechst staining but no detectable CD63-tdTomato signal, confirming that red puncta in treated groups represent internalized CD63-tdTomato-labeled MGEVs. ( O ) Quantification of CD63-tdTomato fluorescence intensity in HuSC cells following 24 h, 48 h, or 72 h exposure to MsMGEVs or HuMGEVs. Both vesicle types were taken up by HuSC microglia, with MsMGEVs showing a modest trend toward increased accumulation over time. Bars represent mean ± SEM; individual points represent biological replicates. Scale Bar: 15 µm.

Article Snippet: Parent human (HMC3) and mouse (BV2) microglial cells stably expressing an sEV reporter were generated by transduction with a lentiviral vector encoding a CD63-tdTomato fusion (LentifectTM Purified Lentiviral Particles, Cat. # LP772-025, Genecopoeia, Rockville, MD, USA) and cultured to ~90% confluence.

Techniques: Labeling, Purification, Staining, Control, Fluorescence

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Two Hybrid Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Western Blot, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid analysis was carried out to examine interactions between IFITM1 and ACBD3, PI4KB, OSBP, or CERT, and the results are shown as described in . Data are the mean ± SD of at least three independent experiments. (B) HA-tagged IFITM1 and FLAG-tagged ACBD3, PI4KB, OSBP, or CERT were cotransfected into 293T cells, and the cell lysates were subjected to immunoprecipitation with anti-HA antibody or control IgG. The resulting immunocomplexes and whole-cell lysates were detected by anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h, the cells were labeled with anti-FLAG and anti-ACBD3 (top), anti-PI4KB (middle), or anti-OSBP (bottom) antibodies. (D) Vero IFITM1 cells were incubated with Tet (−) or Tet (+) for 72 h, and the cells were fixed and double stained with the indicated antibodies. (E) Vero cells were transfected with HA-IFITM1. At 24 h, the cells were labeled using anti-HA, anti-OSBP and anti-EEA1, or anti-CD63 antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation (C-E).

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Immunoprecipitation, Control, Transfection, Labeling, Incubation, Staining, Standard Deviation

Journal: Burns & Trauma

Article Title: Hypoxia macrophage-derived exosomal miR-26b-5p targeting PTEN promotes the development of keloids

doi: 10.1093/burnst/tkad036

Figure Lengend Snippet: HMDE promoting HKFs’ migration and invasion depends on PTEN. HKFs were treated with PBS, exosomes derived from normoxic or hypoxic macrophages. ( a ) Western blot analysis measures the p-AKT, AKT and PTEN expression in HKFs. ( b ) HKF1 were transfected with si-PTEN and the negative control or infected with adenoviral vector PTEN-GFP and adenoviral vector GFP. Western blot analysis measures the p-AKT, AKT and PTEN expression in HKFs. ( c, d ) CCK-8 assay represents the cell proliferation ability when PTEN is downregulated or overexpressed. Knockdown of PTEN in HKF1 by siRNA treatment. ( e ) Wound-healing assay. Scale bar, 100 μm. ( f ) Cell migration and invasion assays utilizing Transwell or Matrigel-coated Transwell. Scale bar, 100 μm. Overexpression of PTEN in HKF1 by adenoviral vector PTEN-GFP. ( g ) Wound-healing assay. Scale bar:100 μm. ( h ) Cell migration and invasion assays utilizing Transwell or Matrigel-coated Transwell. Scale bar: 100 μm. ( i ) Western blot analysis represents E-cadherin, MMP9 and α-SMA expression. ns not significant, * p < 0.05, * * p < 0.01, * * * p < 0.001. si-PTEN small interfering RNA-PTEN, LV PTEN lentivirus-PTEN, LV NC lentivirus-negative control, Norm M-exo normaxic macrophage derived exosomes, Hypo M-exo hypoxic macrophage derived exosomes, HKF human keloid fibroblast, α-SMA α-smooth muscle actin, MMP9 matrix metalloprotein 9, E-cadherin epithelial cadherin

Article Snippet: After being blocked in 5% milk for 1 h, the membranes were then probed at 4°C overnight with antibodies against PTEN (1:500; Santa Cruz Biotechnology, USA, sc-7974), AKT (1:1000; Proteintech Group, USA, 10176-2-AP), p-AKT (1:1000; Proteintech Group, USA, 66444-1-lg), HIF-1α (1:1000; Proteintech Group, USA, 20960-1-AP), MMP9 (1:1000; Proteintech Group, USA, 10375-2-AP), Collagen Type I (1:1000; Proteintech Group, USA, 14695-1-AP), E-cadherin (1:1000; Proteintech Group, USA, 20874-1-AP), Smooth Muscle Actin (1:1000; Proteintech Group, USA, 14395-1-AP), CD63 (1:1000; Proteintech Group, USA,25682-1-AP), CD81 (1:1000; Proteintech Group, USA, 66866-1-lg), HSP70 (1:1000; Abcam , USA, ab5439), β-Tubulin (1:5000; Cell Signaling Technology, USA, 2146) and GAPDH (1:5000; Abcam , USA, ab8245).

Techniques: Migration, Derivative Assay, Western Blot, Expressing, Transfection, Negative Control, Infection, Plasmid Preparation, CCK-8 Assay, Knockdown, Wound Healing Assay, Over Expression, Small Interfering RNA

Journal: Nature Communications

Article Title: NDP52 mediates an antiviral response to hepatitis B virus infection through Rab9-dependent lysosomal degradation pathway

doi: 10.1038/s41467-023-44201-2

Figure Lengend Snippet: a Schematic presentation of HBV three envelope proteins. The preS1, preS2 and S regions are indicated. Glycosylation within S and preS2 is indicated. b GST pull-down assays using cell extract of HEK293T cells transfected with plasmids encoding Flag-tagged S, M or L and GST or GST fusion of NDP52. Coprecipitated proteins were detected with indicated antibodies. c Coimmunoprecipitation assays with anti-NDP52 antibody in either Huh7 cells transfected with Flag-tagged vector (V), S, M or L (upper panel), or HepAD38 cells without doxycycline (Dox) (lower panel). Immunoprecipitates were detected by indicated antibodies. d Immunofluorescence with anti-NDP52 and anti-preS2 antibodies shows colocalization of NDP52 with M and L in HepAD38 cells without Dox and with Dox. The scale bar is 10 µm for full cell images. e Representative fluorescence micrographs of Huh7 cells transfected with plasmids coding for GFP fusion of L, M or S proteins and immunostained for NDP52. The scale bar is 10 µm for full cell images. f Pearson’s correlation coefficients for colocalizations in e ( n = 7 biological replicates). ns non significance. g Representative fluorescence micrographs of HepAD38 cells replicating HBV stained with anti-NDP52 and anti-HBs antibodies and reagents labeling ER (ER-Tracker), Golgi (anti-TGN46 antibody) and multivesicular body (anti-CD63). The scale bar is 10 µm for full cell images, 2.5 µm for zoomed images. Data are means ± SD. Statistical significance in f is determined by a two-sided unpaired t-test. Source data for b , c and f are provided as a Source Data file.

Article Snippet: The reagents used for colocalization of HBs and NDP52 in ER, Golgi or multivesicular body (MVB) are: HBsAg antibody (HB3) conjugated with Alexa FluorTM 594 (Novus Biologicals NB500-474AF594), ER Staining Kit - Green Fluorescence – Cytopainter (Abcam, ab139481), TGN46 antibody (Abcam, ab50595), CD63 antibody (E-12) (Santa Cruz, sc-365604).

Techniques: Glycoproteomics, Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining, Labeling

Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: Heparanase stimulates the production of syntenin-1-containing exosomes. (A) Exosome production was evaluated after overnight conditioning of MCF-7 cells with increasing concentrations of proheparanase (0.04-25 nM) and compared to that of cells not receiving proheparanase (0 nM). Exosomes were collected from equivalent amounts of culture medium, conditioned by equal numbers of cells, for equal lengths of time. For each condition both the lysate and exosomal fractions were analyzed by western blot, using cognate antibodies against heparanase, monitoring the conversion of proheparanase (Prohep) into mature heparanase (Hep) and against different exosomal markers: syntenin-1 (Synt1), syndecan-1 (SDC1), syndecan-4 (SDC4), CD63, flotillin-1 (Flo1), CD9 and CD81. Syndecan-1, which is a hybrid heparan sulfate (HS)/chondroitin sulfate proteoglycan, was analyzed using two different approaches. In one approach, the samples were digested with both heparitinase and chondroitinase ABC, removing all glycosaminoglycan chains and enabling visualization of the full-length syndecan core proteins (SDC1 FL) as sharp bands. In the other approach, the samples were digested with chondroitinase ABC only, leaving the HS on the syndecans (SDC1 with HS); comparison of 'SDC1 with HS' and 'SDC1 FL' yields information on the mass of HS on syndecans. Because of the heterogeneity in HS chain length, syndecan-1 with HS is smeared over a wide mass range in the absence of heparanase activity (and is therefore hardly visible in western blot, as illustrated by lane 1 of the lysates). With increasing heparanase activity, the HS chains on syndecan-1 are trimmed to shorter chains of more or less the same length, syndecan-1 with HS migrating as one or a few bands that are readily visualized in western blot (as illustrated by lane 6 of the lysates). Note that cell lysates contain mainly full-length syndecan core proteins; the opposite is true for exosomes, where hardly any full-length syndecan is detected and C-terminal fragments (CTFs) represent the dominant form. β-actin was used as a loading control for the lysates. Western blots are representative of five independent experiments. (B) Histogram representing the quantification of the exosomal levels of syntenin-1 (Synt1), syndecan-1 CTF (SDC1 CTF), CD63, syndecan-4 CTF (SDC4 CTF) and flotillin-1 (Flo1) in response to the addition of increasing concentrations (0 nM till 25 nM) of proheparanase. Values are relative to the exosomal levels measured in absence of exogenously added proheparanase. Bar heights represent mean values, calculated from five independent experiments. Individual data points are shown as white dots on top of the corresponding bars. * P < 0.1, ** P < 0.05, *** P < 0.01 (Student's t -test, assuming normal distribution of the data points). (C) Knockdown of endogenous heparanase reduces the production of syntenin-1-containing exosomes, which can be rescued by the addition of exogenous proheparanase. Duplicate lanes show the results of two independent experiments, run side by side. B16-F10 cells are sham-transfected (−) or stably transfected with a shRNA targeting murine heparanase (+). To rescue the effects of endogenous heparanase knockdown, 10 nM human proheparanase was added to the cells. Heparanase, syntenin-1, syndecan-1 full-length (SDC1 FL), syndecan 1 CTF (SDC1 CTF) and CD63 were analyzed by western blot. Positions of molecular weight markers (in kDa) are indicated on the right of each blot. Note that (because of differences in glycosylation) the Mr of human heparanase is slightly larger than that of mouse heparanase.

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Western Blot, Activity Assay, Transfection, Stable Transfection, shRNA, Molecular Weight

Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: The effect of heparanase on exosome production depends on the modification of the heparan sulfate (HS) on syndecans. (A) The role of heparanase enzymatic activity on exosome production was investigated by comparing MCF-7 cells stably expressing wild-type heparanase (WT), catalytically dead heparanase (Cat) or empty vector (Φ) in western blot, in the absence (−) or presence (+) of 10 nM exogenously added proheparanase. (B) The importance of HS was analyzed by treating MCF-7 cells with RNAi targeting EXT1 and EXT2 (KD). Non-targeting RNAi (NT) was used as a control. Cells were challenged with 10 nM proheparanase (+) or left untreated (−). Heparanase activity, reducing the HS on syndecan, was apparent from the migration of chondroitinase ABC-treated syndecan-1 present in cell lysates (SDC1 with HS). EXT1 and EXT2 knockdown leads to the appearance of syndecan-1 that is not substituted with HS (a band running slightly > 70 kDa, after chondroitinase ABC digestion only), not detectable in cells treated with non-targeting RNAi, where all syndecan is substituted with HS (and is larger than SDC FL). A small amount of the syndecan-1 still carried HS and was affected by heparanase addition (yielding a band slightly > 100 kDa, after chondroitinase ABC digestion only), indicating an incomplete knockdown of EXT1 and EXT2. Western blots are representative of three independent experiments. (C) Quantification of the effect of EXT1 and EXT2 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF, CD63 and flotillin-1 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi and heparanase, dark gray bars; EXT1 and EXT2 RNAi, light gray bars; EXT1 and EXT2 RNAi and heparanase, white bars). Values are relative to the exosomal levels measured in cells treated with non-targeting RNAi and in the absence of exogenously added proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05, *** P < 0.01, n.s., not significant (Student's t -test, assuming normal distribution of the data points). (D) Likewise, the role of syndecans was investigated by treating MCF-7 cells with RNAi targeting syndecan-1 and -4 (KD). Non-targeting RNAi (NT) was used as a control. Cells were treated with increasing concentrations of proheparanase (0 to 25 nM) and both the lysates and exosomal fractions were analyzed. Western blots are representative of three independent experiments. Note that lysate and exosome samples derived from NT-and KD-treated cells, separated by a blank space in , were run in the same gel, but not side by side, and that the band intensities in each row are directly comparable. (E) The ability of glypican-1 to rescue the effect of heparanase on exosome production in absence of syndecans was investigated by knocking down both syndecan-1 and -4 (KD) and overexpressing glypican-1 (GPC1) in the same cells. Glypican-1, when deglycanated by heparitinase and chondroitinase ABC treatment (GPC1) or when unmodified (because of overexpression) has a mass of ∼ 60 kDa. Treatment with chondroitinase ABC only reveals 'GPC1 with HS' of a mass of ∼ 90 kDa in heparanase-exposed cells, indicating the HS on glypican-1 is trimmed the same way as the HS on syndecans. Yet, extra glypican fails to rescue the exosomal accumulations of syntenin and CD63. Rescue by expression of mouse syndecan-1 (SDC1) was used as a positive control. Cells treated with non-targeting RNAi (NT) served as reference. Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of two independent experiments. Note that MCF-7 cells use mainly Man-6-P receptors for heparanase internalization, and that neither the knock down of EXT1 and EXT2 nor the knock down of syndecans has an effect on the uptake of proheparanase and its conversion into mature active form.

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Modification, Activity Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Migration, Derivative Assay, Over Expression, Positive Control, Molecular Weight

Journal: Cell Research

Article Title: Heparanase activates the syndecan-syntenin-ALIX exosome pathway

doi: 10.1038/cr.2015.29

Figure Lengend Snippet: Heparanase influences the biogenesis of vesicles of endosomal origin, enhancing intraluminal budding. (A) To investigate whether the extracellular vesicles affected by heparanase were of endosomal origin, RAB7 was knocked down ( RAB7 RNAi) in MCF-7 cells. Non-targeting RNAi (−) served as a control. Cells were left untreated (−) or treated with proheparanase (10 nM). In both experiments, heparanase activity was evaluated using the migration pattern of chondroitinase ABC-, but not heparitinase-treated syndecan-1 present in cell lysates (SDC1 with HS). Molecular weight markers (in kDa) are indicated on the right of each blot. Western blots are representative of three independent experiments. (B) Quantification of the effect of RAB7 knockdown. Histograms representing the exosomal levels of syntenin-1, syndecan-1 CTF and CD63 in the different conditions tested (non-targeting RNAi, black bars; non-targeting RNAi with heparanase, dark gray bars; RAB7 knockdown, light gray bars; RAB7 knockdown with heparanase, white bars). Values are relative to the levels (intensities of the signals) measured in exosomes derived from cells treated with non-targeting RNAi in the absence of proheparanase. Bar heights represent mean values, calculated from three independent experiments. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05, n.s., not significant (Student's t -test, assuming normal distribution of the data points). (C) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge) and Cerulean-RAB5 Q79L (green in merge). Note the presence of mCherry-syntenin-1, a cytosolic protein, inside the Cerulean-RAB5 Q79L endosomes upon heparanase treatment (50 nM). (D) Confocal micrographs of MCF-7 cells co-transfected with mCherry-syntenin-1 (red in merge), Cerulean-RAB5 Q79L (blue in merge) and syndecan-1 (green in merge), scoring the accumulations (budding) of mCherry-syntenin-1 and of syndecan-1 cytoplasmic domain inside vacuoles outlined by Cerulean-RAB5 Q79L . (E) Quantification of intraluminal budding of mCherry-syntenin-1, as in , by measuring the fluorescence of mCherry-syntenin-1 in the lumen of RAB5 Q79L -positive endosomes, corrected for the size of the RAB5 Q79L -positive endosomes (mean gray value per pixel). Bar heights represent mean values calculated from six independent experiments, scoring at least 30 cells per experiment. Individual data points (mean luminal fluorescence intensity per individual experiment) are shown as white dots on top of the corresponding bars. *** P < 0.01 (Student's t -test, assuming normal distribution of the data points). (F) Quantification of intraluminal budding of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain, as in , by measuring the fluorescence intensity of mCherry-syntenin-1 and syndecan-1 cytoplasmic domain in the lumen of RAB5 Q79L -positive endosomes. Bar heights represent mean values calculated from three independent experiments, scoring at least 30 cells per experiment. Individual data points are shown as white dots on top of the corresponding bars. ** P < 0.05 (Student's t -test, assuming normal distribution of the data points).

Article Snippet: Other antibodies were from commercial sources and were used as recommended by the manufacturer: antibodies against RAB7 (H-50) and β-actin (AC-15) were from Santa Cruz; antibody against the intracellular domain of syndecan 4 was from Abnova; antibodies against human CD63 (MEM-259) was from Abcam; antibody against HA (16B12) was from Covance; anti-flotillin-1 was from BD Biosciences.

Techniques: Activity Assay, Migration, Molecular Weight, Western Blot, Derivative Assay, Transfection, Fluorescence

Journal: Scientific Reports

Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

doi: 10.1038/srep31172

Figure Lengend Snippet: ( a ) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. ( b ) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured rESCs expressed GFP. ( c ) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. ( d ) Adult female chimaeric rat from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a Wistar wild type (Wt) male (arrows). ( e ) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size marker.

Article Snippet: Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson).

Techniques: Construct, Control, Transfection, Cell Culture, Microinjection, Derivative Assay, Injection, Plasmid Preparation, Marker

Journal: Scientific Reports

Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

doi: 10.1038/srep31172

Figure Lengend Snippet: ( a ) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). ( b ) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus.

Article Snippet: Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson).

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

doi: 10.1038/srep31172

Figure Lengend Snippet: ( a ) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. ( b ) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. ( c , d ) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours ( c ). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs ( d ; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 ( d ; right). β-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression .

Article Snippet: Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson).

Techniques: Cell Culture, Immunostaining, Isolation, Western Blot, Expressing, Control, Over Expression

Journal: Scientific Reports

Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

doi: 10.1038/srep31172

Figure Lengend Snippet: ( a , b ) Western blotting analysis of the EVs isolated from serum ( a ), breast milk and AF ( b ) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. ( c ) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm.

Article Snippet: Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson).

Techniques: Western Blot, Isolation, Immuno-Electron Microscopy, Derivative Assay

Journal: Scientific Reports

Article Title: Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

doi: 10.1038/srep31172

Figure Lengend Snippet: ( a ) EVs of Wt rats, Tg rats and vehicle PBS (−) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67-labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars = 20 μm. ( b ) Images of co-localization of EVs with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the insets. Scale bars = 25 μm. Nuclei were counterstained with Hoechst 33342 (blue).

Article Snippet: Primary antibodies specific to the following proteins were used: rat CD63 (1:200; AbD Serotec) and human CD63 (1:200; Becton Dickinson).

Techniques: Control, Incubation

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Schematic depicting the workflow for the screening method. b Left, immunofluorescence of EGFR-HA (green) and CD63 (red) in the indicated stable HeLa cells transiently expressing EGFR-HA under normal condition. Middle, the ratio of co-localization of EGFR-HA with CD63-positive late endosome and MVE (LE/MVE) in Vector ( n = 12 fields) and RAB31 Q65L ( n = 13 fields). Right, diameter of CD63-positive LE/MVE in Vector ( n = 120) and RAB31 Q65L ( n = 150). c Left, immunofluorescence of EGFR-HA (green) and CD63 (red) in the indicated stable HeLa cells transiently expressing EGFR-HA under serum starvation (SS). Middle, the ratio of co-localization of EGFR-HA with CD63-positive LE/MVE in Vector ( n = 16 fields) and RAB31 Q65L ( n = 16 fields). Right, diameter of CD63-positive LE/MVE in Vector ( n = 150) and RAB31 Q65L ( n = 180). d Western blotting analyses of whole-cell lysates (WCL) and immunoprecipitates (IP) from the indicated stable HeLa cells under SS upon EGF treatment for the indicated time points. e Left, immunofluorescence of endogenous EGFR (green) and CD63 (red) in the indicated stable HeLa cells under SS. Middle, the ratio of co-localization of EGFR with CD63-positive LE/MVE in Vector ( n = 7 fields) and RAB31 Q65L ( n = 8 fields). Right, diameter of CD63-positive LE/MVE in Vector ( n = 142) and RAB31 Q65L ( n = 147). f Left, immunofluorescence of endogenous EGFR (green) and CD63 (red) in the indicated stable A431 cells under SS. Middle, the ratio of co-localization of EGFR with CD63-positive LE/MVE in Vector ( n = 7 fields) and RAB31 Q65L ( n = 11 fields). Right, diameter of CD63-positive LE/MVE in Vector ( n = 150) and RAB31 Q65L ( n = 154). g Up panels, immunofluorescence of endogenous EGFR (green) and LAMP1 (red) in the indicated stable A431 cells under SS. Low panel, the ratio of co-localization of EGFR with LAMP1-positive lysosome in Vector ( n = 6 fields) and RAB31 Q65L ( n = 9 fields). h Left, immunofluorescence of Flag-RAB31 (green) with CD63 (red) in the indicated stable HeLa cells under SS. Middle, the ratio of co-localization of Flag-RAB31 with CD63-positive LE/MVE in RAB31 WT ( n = 7 fields) and RAB31 Q65L ( n = 13 fields). Right, diameter of CD63-positive LE/MVE in Vector ( n = 150), RAB31 WT ( n = 165) and RAB31 Q65L ( n = 180). i Left, immunofluorescence of Flag-RAB31 (red) with EGFR-HA (green) in the indicated stable HeLa cells transiently expressing EGFR-HA under SS. Middle, the ratio of co-localization of Flag-RAB31 with EGFR-HA-positive vesicle in RAB31 WT ( n = 7 fields) and RAB31 Q65L ( n = 10 fields). Right, diameter of EGFR-HA-positive vesicle in Vector ( n = 129), RAB31 WT ( n = 139) and RAB31 Q65L ( n = 179). j Immunofluorescence of Flag-RAB5A Q79L and Flag-RAB22A Q64L (green) with CD63 (red) in the indicated stable HeLa cells under SS. k Immunofluorescence of Flag-RAB5A Q79L and Flag-RAB22A Q64L (red) with EGFR-HA (green) in the indicated stable HeLa cells transiently expressing EGFR-HA under SS. All data are means ± SD. Unpaired t -test was used to analyze the difference between the two groups. **** P < 0.0001, NS, no statistical significance. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Immunofluorescence, Expressing, Plasmid Preparation, Western Blot

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Up panels, immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). Low panel left, the ratio of co-localization of EGFR-HA with CD63-GFP-positive LE/MVE in Vector ( n = 6 fields), RAB31 WT ( n = 8 fields) and RAB31 Q65L ( n = 11 fields). Low panel middle, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in RAB31 WT ( n = 8 fields) and RAB31 Q65L ( n = 11 fields). Low panel right, the ratio of entry of Flag-RAB31 into CD63-GFP-positive LE/MVE in RAB31 WT ( n = 8 fields) and RAB31 Q65L ( n = 11 fields). b – d Immunofluorescence of the localization of EGFR-HA (red) with CD63-GFP (green) ( b ), Flag-RAB31 Q65L (red) with CD63-GFP (green) ( c ), and Flag-RAB31 Q65L (green) with EGFR-HA (red) ( d ) in Flag-RAB31 Q65L stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under SS using three-dimensional structured illumination microscopy (3D-SIM). e Immunoelectron microscopy of the localization of EGFR-HA in Vector stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. f Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31 WT in Flag-RAB31 WT stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. g Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31 Q65L in Flag-RAB31 Q65L stable HeLa cells transiently expressing EGFR-HA under SS. h Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells under SS. i NanoSight nanoparticle tracking analysis of the concentrated conditional media from the indicated stable HeLa cells under SS. j Transmission electron microscopy analysis of the concentrated conditional media from Flag-RAB31 Q65L stable HeLa cells under SS. All data are means ± SD. Unpaired t -test was used to analyze the difference between the two groups. **** P < 0.0001. Scale bars, 10 μm ( a – d ), 200 nm ( e – g ), 100 nm ( j ).

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Immunofluorescence, Expressing, Plasmid Preparation, Microscopy, Immuno-Electron Microscopy, Western Blot, Transmission Assay, Electron Microscopy

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Left, immunofluorescence of EGFR-HA (red) and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in Flag-RAB31 Q65L stable HeLa cells stably expressing shNC (negative control), shFLOT1, shFLOT2 or shFLOT1 and shFLOT2 and transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). Right up panel, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in shNC ( n = 9 fields), shFLOT1 and shFLOT2 ( n = 9 fields), shFLOT1 ( n = 12 fields), shFLOT2 ( n = 12 fields). Right low panel, the ratio of entry of Flag-RAB31 Q65L into CD63-GFP-positive LE/MVE in shNC ( n = 9 fields), shFLOT1 and shFLOT2 ( n = 9 fields), shFLOT1 ( n = 12 fields), shFLOT2 ( n = 12 fields). b Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells used in a . c Up panels, immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with FLOT1-GFP (green) in the indicated stable HeLa cells transiently expressing EGFR-HA and FLOT1-GFP under SS. Low panel left, the ratio of co-localization of EGFR-HA with FLOT1-GFP-positive vesicle in Vector ( n = 7 fields), RAB31 WT ( n = 8 fields) and RAB31 Q65L ( n = 9 fields). Low panel right, the ratio of co-localization of Flag-RAB31 with FLOT1-GFP-positive vesicle in RAB31 WT ( n = 8 fields) and RAB31 Q65L ( n = 9 fields). d Up panels, immunofluorescence of FLOT1-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing FLOT1-HA and CD63-GFP under SS. Low panel left, the ratio of co-localization of FLOT1-HA with CD63-GFP-positive LE/MVE in Vector ( n = 7 fields), RAB31 WT ( n = 7 fields) and RAB31 Q65L ( n = 8 fields). Low panel right, the ratio of entry of FLOT1-HA into CD63-GFP-positive LE/MVE in Vector ( n = 7 fields), RAB31 WT ( n = 7 fields) and RAB31 Q65L ( n = 8 fields). e Left, immunofluorescence of EGFR-HA (red) and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in Flag-RAB31 Q65L stable HeLa cells transiently expressing EGFR-HA and CD63-GFP and treated with DMSO, 5 μM GW4869, 5 μM simvastatin or 10 μM lovastatin under SS. Right up panel, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in DMSO ( n = 8 fields), GW4869 ( n = 11 fields), simvastatin ( n = 13 fields) and lovastatin ( n = 12 fields). Right low panel, the ratio of entry of Flag-RAB31 Q65L into CD63-GFP-positive LE/MVE in DMSO ( n = 8 fields), GW4869 ( n = 11 fields), simvastatin ( n = 13 fields) and lovastatin ( n = 12 fields). f Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells used in e . All data are means ± SD. Unpaired t -test was used to analyze the difference between the two groups. **** P < 0.0001, *** P < 0.001, NS, no statistical significance. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Negative Control, Western Blot, Plasmid Preparation

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a , b Western blotting analyses of whole-cell lysates (WCLs) and immunoprecipitates (IP) at their endogenous levels from NCI-H1975 cells using anti-FLOT1 antibody ( a ) or anti-FLOT2 antibody ( b ). c , d Western blotting analyses of WCL and IP from HEK-293T cells co-expressing the indicated plasmids. e Immunofluorescence of erlin1-HA, erlin2-HA, prohibitin1-HA or prohibitin2-HA (green) with Flag-RAB31 Q65L (red) in HeLa cells stably expression Flag-RAB31 Q65L and transiently expressing the indicated plasmids under serum starvation (SS). f Immunofluorescence of stomatin-HA or STOML3-HA (red) and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in HeLa cells stably expression Flag-RAB31 Q65L and transiently expressing the indicated plasmids under SS. g Immunofluorescence of FLOT1-HA, FLOT2-HA or stomatin-HA and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing the indicated plasmids under SS. h Immunofluorescence of FLOT1-SPFH-HA, FLOT2-SPFH-HA, FLOT1-flotillin-HA, or FLOT2-flotillin-HA (red) and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing the indicated plasmids under SS. i Immunofluorescence of Sto-flotillin1-HA (red) chimeras and Flag-RAB31 Q65L (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing the indicated plasmids under SS. j Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells stably re-introduced with the indicated plasmids. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Western Blot, Expressing, Immunofluorescence, Stable Transfection

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Immunofluorescence of EGFR-HA (red) and Flag-RAB31 WT (magenta) with CD63-GFP (green) in Flag-RAB31 WT stable HeLa cells transiently expressing EGFR-HA and CD63-GFP stimulated with EGF for 30 min as indicated. b Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells treated with 100 ng/mL of EGF at the indicated times. c Western blotting analyses of whole-cell lysates (WCL) and immunoprecipitates (IP) from HEK-293T cells co-expressing the indicated plasmids treated with 100 ng/mL of EGF at the indicated times. p-Tyr, anti-phosphotyrosine antibody. d Immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated Flag-RAB31 stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). e – g Immunofluorescence of EGFR M2 -HA (red) with CD63-GFP (green) ( e ), Flag-RAB31 WT (red) with CD63-GFP (green) ( f ) and Flag-RAB31 WT (green) with EGFR M2 -HA (red) ( g ) in Flag-RAB31 WT stable HeLa cells transiently expressing EGFR M2 -HA and CD63-GFP under SS using 3D-SIM. h Western blotting analyses of WCL and IP from HEK-293T cells co-expressing EGFR M2 -HA and Flag-RAB31 WT treated with the indicated EGFR-tyrosine kinase inhibitors under SS. i Immunofluorescence of EGFR M2 -HA (red) and Flag-RAB31 WT (magenta) with CD63-GFP (green) in Flag-RAB31 WT stable HeLa cells transiently expressing EGFR M2 -HA and CD63-GFP treated with the indicated inhibitors under SS. j , k Western blotting analyses of WCL and IP from HEK-293T cells co-expressing the indicated plasmids under SS. l Western blotting analyses of WCL and IP from HEK-293T cells co-expressing the indicated plasmids treated with EGF for the indicated times. m Western blotting (WB) and Coomassie brilliant blue (CBB) analyses of the purified different EGFR and RAB31 forms as indicated after in vitro kinase assay, as described in Materials and Methods section. n Mass spectrometry analysis of the phosphorylated tyrosine sites in RAB31 purified from in vitro kinase assay. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Immunofluorescence, Expressing, Western Blot, Purification, In Vitro, Kinase Assay, Mass Spectrometry

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Western blotting analyses of whole-cell lysates (WCL) from the indicated NCI-H1975 cells stably expressing shNC, shRAB31 or shFLOT1 and shFLOT2. b Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells used in a under serum starvation (SS). c Immunofluorescence of endogenous EGFR (green) and CD63 (red) in the indicated stable NCI-H1975 cells used in a under SS. d Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells under serum starvation (SS). e Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells under serum starvation (SS). f Western blotting analyses of WCL and immunoprecipitates (IP) from NCI-H1975 cells treated with afatinib or erlotinib under SS. g Western blotting analyses of the concentrated conditional media from the indicated stable NCI-H1975 cells under SS. h Representative clone images of PC9-GFP cells treated with the concentrated conditional media derived from the indicated stable NCI-H1975 cells without or with erlotinib. i Quantification of the numbers of each clone for h . Data are means ± SD of cell numbers in each clone with PBS ( n = 75), Vector ( n = 82), RAB31 WT ( n = 62), RAB31 Q65L ( n = 62), RAB31 R77Q ( n = 65) or RAB31 3YF ( n = 84). j Representative clone images of PC9-GFP cells treated with the pure small EV (sEV) derived from the indicated stable NCI-H1975 cells without or with erlotinib. k Quantification of the numbers of each clone for j . Data are means ± SD of cell numbers in each clone with Vector ( n = 71), RAB31 WT ( n = 80) or RAB31 3YF ( n = 80). Unpaired t -test was used to analyze the difference between the two groups. **** P < 0.0001, NS, no statistical significance. Scale bars, 10 μm ( c ) and 100 μm ( h and j ).

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Western Blot, Stable Transfection, Expressing, Immunofluorescence, Derivative Assay, Plasmid Preparation

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Western blotting analyses of whole-cell lysates (WCL) from the indicated stable HeLa cells treated with 100 ng/mL of EGF at the indicated time points. b Western blotting analyses of WCL from the indicated stable HeLa cells treated with 100 ng/mL of EGF or pre-treated with Bafilomycin A1 (Baf A1) for 6 h and then treated with 100 ng/mL of EGF at the indicated time points. c The ratio of co-localization of EGFR-HA with CD63-positive LE/MVE in Vector and Flag-RAB31 WT stable HeLa cells treated with 100 ng/mL of EGF at the indicated times for Supplementary information, Fig. . d The ratio of co-localization of EGFR-HA with LAMP1-positive lysosome in Vector and Flag-RAB31 WT stable HeLa cells treated with 100 ng/mL of EGF at the indicated times for Supplementary information, Fig. . e Immunofluorescence of EGFR-HA (green) with Flag-RAB31 WT (red) in Flag-RAB31 WT stable HeLa cells transiently expressing EGFR-HA treated with 100 ng/mL of EGF at the indicated times. Western blotting analyses of WCL and immunoprecipitation (IP) at their endogenous levels from NCI-H1975 cells using anti-EGFR ( f ) or anti-RAB31 ( g ) antibodies. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Western Blot, Plasmid Preparation, Immunofluorescence, Expressing, Immunoprecipitation

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: a Immunofluorescence of endogenous RAB7 (red) and endogenous CD63 (magenta) with GFP-RAB31 WT (green) in the indicated stable NCI-H1975 cells. b Western blotting analyses of whole-cell lysates (WCL) and streptavidin pull-down (PD) from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. c Immunofluorescence of endogenous RAB7 (red) and HA-RILP (magenta) with GFP-RAB31 (green) in the indicated stable HeLa cells transiently expressing HA-RILP. d Western blotting analyses of WCL and streptavidin PD from HEK-293T cells co-expressing the indicated plasmids with SBP-RILP. Coomassie brilliant blue (CBB) analyses of the PD of SBP-RILP. e Immunofluorescence of endogenous RAB7 (red) and endogenous TBC1D2B (magenta) with GFP-RAB31 (green) in the indicated stable NCI-H1975 cells. f Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB31 (green) in NCI-H1975 cells. g Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. h Immunofluorescence of endogenous TBC1D2B (red) with endogenous RAB7 (green) in NCI-H1975 cells stably expressing shNC or shRAB31. i Western blotting analyses of WCL and IP using the indicated antibody at their endogenous levels from NCI-H1975 cells. j Western blotting analyses of WCL and GTP agarose PD at their endogenous levels from NCI-H1975 and MDA-MB231 cells stably expressing shNC or shRAB31. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Immunofluorescence, Western Blot, Expressing, Stable Transfection

Journal: Cell Research

Article Title: RAB31 marks and controls an ESCRT-independent exosome pathway

doi: 10.1038/s41422-020-00409-1

Figure Lengend Snippet: EGFR are endocytosed into cells to form signaling endosomes (SE) and early endosomes (EE) regulated by RAB5, and then are transported from early to late endosomes (LE) regulated by transition from RAB5 to RAB7. a At this time, ESCRT machinery sorts the ubiquitylated EGFR into intraluminal vesicles (ILVs) that are destined to lysosomes for degradation by the fusion of multivesicular endosomes (MVEs) with lysosomes regulated by RAB7. b However, high RAB31, guarding on the late endosomes, encounters active EGFR and can be activated via tyrosine phosphorylation by EGFR, and then active RAB31 engages FLOTs in lipid rafts to drive EGFR entry into MVEs to form ILVs. Meanwhile, RAB31 recruits TBC1D2B to inactivate RAB7 preventing the fusion of MVEs with lysosomes, thereby enabling that the sequestered EGFR ILVs are secreted as exosomes. c Representative image of MVE membrane budding to form ILVs driven by the active RAB31 in NCI-H1975 cells. The white triangles indicate the budding moments of MVE membrane. Immunofluorescence of endogenous RAB31 (green) and CD63 (red) in NCI-H1975 cells. Scale bar, 5 μm.

Article Snippet: The following primary antibodies were used for immunofluorescence: Flag rabbit antibody (1:500; Cell Signaling; 14793), Flag mouse antibody (1:500; Cell Signaling; 8146), HA rabbit antibody (1:500; Cell Signaling; 3724), HA mouse antibody (1:500; Cell Signaling; 2367), V5 rabbit antibody (1:500; Cell Signaling; 13202), EGFR rabbit antibody (1:100; Cell Signaling; 4267), RAB31 rabbit antibody (1:50; GeneTex; GTX55929), TBC1D2B mouse antibody (1:50; Santa Cruz; sc-398906), RAB7 rabbit antibody (1:200; Abcam; ab137029), CD63 mouse antibody (1:500; Santa Cruz; sc-5275), EEA1 rabbit antibody (1:300; Cell Signaling; 3288), LAMP1 rabbit antibody (1:300; Cell Signaling; 9091), LAMP1 mouse antibody (1:50; Santa Cruz; sc-20011).

Techniques: Phospho-proteomics, Membrane, Immunofluorescence