Journal: Scientific Reports

Article Title: CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells

doi: 10.1038/s41598-019-48438-0

Figure Lengend Snippet: MMP14 expression in degradative focal adhesions in RPE cells. ( A , B ) Representative images of ARPE19 plated on a fluorescein-conjugated gelatin coverslip for 5 hours and immunostained with anti-vinculin (in A ) or anti-MMP14 (in B ) antibodies followed by Alexa 568-secondary antibodies. Black-and-white single-channel images and the merged color images are shown. Enlarged views are from the boxed areas showing the overlapping vinculin signal and degradation foci. ( C ) Representative low-power (insets) and high-power images of ARPE19 cells plated on non-fluorescent gelatin-coated coverslips for 5 hours and labeled for MMP14 (green) and vinculin (red). ( D , E ) ARPE19 cells transfected with MMP14-mCherry for one day were plated on fluorescein-conjugated gelatin coverslips for 5 hours. Both low-power ( D ) and high-power ( E ) views are shown. Arrows in ( D ) point to the cells with massive gelatin degradation activity caused by the ectopic expression of MMP14-mCherry. Arrows in ( E ) point to the MMP14-mCherry-labeled tubulovesicles that match the gelatin degradation footprints. ( F ) Representative images of ARPE19 cells plated on non-coated coverslips and immunostained for endogenous MMP14 (green) and CD63 (red). Blue (in A , B , D – F ): DAPI nuclear stain. Scale bars (in A – F ) = 10 µm.

Article Snippet: Antibodies used in this study included actin (mouse, Santa Cruz, sc-8432, 1:250), CD63 (mouse H5C6, DSHB, 1:1000), mCherry (goat, Sicgen, AB0040-200, 1:1000), CLIC4 (rabbit, Millipore ABS2088;1:500), Flag (mouse Sigma F3165, 1:2000), MMP14 (rabbit, Abcam ab51074, 1:500 for IFA, 1:1000 for WB; epitope: amino acid ~150–180); MMP14 (mouse, Santa Cruz sc-373908, 2 μg for immunoprecipitation; epitope: amino acids 431–462), vinculin (mouse, Sigma V9131, 1:500), flotillin-1 (mouse, BD Transduction Laboratories, Clone 18, 1:1,000), GAPDH (rabbit, Cell Signaling Technology, 14C10, 1:5000), Tsg101 (goat, Santa Cruz M-19, 1:100), Bestrophin1 (rabbit; Proteintech 21910-1-AP, 1: 1,000), RPE65 (rabbit, gift of Jian-Xing Ma, 1:1000), β-tubulin (mouse, Sigma-Aldrich Cat# T5201, 1:2,000), ZO-1 antibody (rabbit Zymed 61–7300 or mouse Zymed 33–9100, 1:500), Alexa-dye conjugated secondary antibodies (made in donkey, Thermo Fisher Scientific, 1:400), Alexa-dye conjugated phalloidin (Thermo Fisher Scientific, 1:1000), and IRDye-conjugated secondary antibodies (LI-COR; 1:10,000).

Techniques: Expressing, Labeling, Transfection, Activity Assay, Staining

Journal: Scientific Reports

Article Title: CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells

doi: 10.1038/s41598-019-48438-0

Figure Lengend Snippet: CLIC4 interacts with MMP14 and is required for LE luminal expression of MMP14. ( A ) ARPE19 cell lysates and the immunoprecipitants pulled down by anti-MMP14 or control mouse antibody were analyzed by immunoblotting with the indicated rabbit antibodies. ( B ) The lysates of 293 T cells transfected with Flag-CLIC4 and MMP14-mCherry (left) and the immunoprecipitants pulled down by anti-Flag or control antibody (right) were immunoblotted with the indicated antibodies. Asterisks point to the MMP14-mCherry specifically co-immunoprecipitated with CLIC4. ( C ) Live snapshot image of co-transfected GFP-CLIC4 and MMP14-mCherry in ARPE19 cells. The low magnification photograph of a single cell is shown in the bottom right panel; the dashed lines mark the cell border. The boxed area is magnified to highlight the overlapping GFP-CLIC4 and MMP14-mCherry signals. ( D ) A still image of live ARPE19 cells transfected with mCherry-CLIC4 (red) and Lamp1-GFP (green). The low-magnification images taken from a single cell and the enlarged views of the boxed area (insets) are shown. ( E ) A snapshot of live images shown in Supplementary Movie . Fluorescein-dextran was added to the culture medium of ARPE19 cells transiently transfected with mCherry-CLIC4. After washing and a 6-hour chase, the cells were imaged in recording buffer. ( F ) Immunoblots of Dox treated (+) or untreated (−) ARPE19 cells stably expressing inducible CLIC4-sh1 plasmid (no fluorescent reporter) probed with the indicated antibodies are shown. ( G ) The ARPE19 cell line described in ( F ) was treated with (i.e., CLIC4 KD) or without (CLIC4 WT) Dox for 3 days. These cells were then transfected with MMP14-mCherry, GFP-CD63, and Flag-Rab5 Q79L for 1 day in the same Dox conditions before fixation and staining. Representative super-resolution confocal images of mCherry and GFP stained cells are shown. Arrowheads and arrows point to LE luminal and LE limiting membrane signals of MMP14, respectively. ( H ) Quantification of targeting of MMP14 in ( G ). For quantification, a threshold was adjusted to exclusively reveal the limiting membrane signal of CD63. In 120 randomly selected LEs, the ratio of the MMP14 signal within the LE lumens was deducted by subtracting its intensity on limiting membrane from total intensity on the entire LE globe. n = 120 endosomes from 24 cells. Scale bars (in C – E , G ) = 10 µm.

Article Snippet: Antibodies used in this study included actin (mouse, Santa Cruz, sc-8432, 1:250), CD63 (mouse H5C6, DSHB, 1:1000), mCherry (goat, Sicgen, AB0040-200, 1:1000), CLIC4 (rabbit, Millipore ABS2088;1:500), Flag (mouse Sigma F3165, 1:2000), MMP14 (rabbit, Abcam ab51074, 1:500 for IFA, 1:1000 for WB; epitope: amino acid ~150–180); MMP14 (mouse, Santa Cruz sc-373908, 2 μg for immunoprecipitation; epitope: amino acids 431–462), vinculin (mouse, Sigma V9131, 1:500), flotillin-1 (mouse, BD Transduction Laboratories, Clone 18, 1:1,000), GAPDH (rabbit, Cell Signaling Technology, 14C10, 1:5000), Tsg101 (goat, Santa Cruz M-19, 1:100), Bestrophin1 (rabbit; Proteintech 21910-1-AP, 1: 1,000), RPE65 (rabbit, gift of Jian-Xing Ma, 1:1000), β-tubulin (mouse, Sigma-Aldrich Cat# T5201, 1:2,000), ZO-1 antibody (rabbit Zymed 61–7300 or mouse Zymed 33–9100, 1:500), Alexa-dye conjugated secondary antibodies (made in donkey, Thermo Fisher Scientific, 1:400), Alexa-dye conjugated phalloidin (Thermo Fisher Scientific, 1:1000), and IRDye-conjugated secondary antibodies (LI-COR; 1:10,000).

Techniques: Expressing, Control, Western Blot, Transfection, Immunoprecipitation, Stable Transfection, Plasmid Preparation, Staining, Membrane

Journal: Scientific Reports

Article Title: CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells

doi: 10.1038/s41598-019-48438-0

Figure Lengend Snippet: Late domains of CLIC4 modulate the LE lumen sorting step of MMP14 trafficking. ( A ) Amino acid alignments of CLIC4 from different species. The two overlapping late domains (PPXY and YXXL) are highly conserved in vertebrates, but less conserved in invertebrates. ( B ) The putative dual late domain sequence in human CLIC4. The CLIC4 protein structure was imaged (Protein Data Bank ID: 2AHE. Doi: 10.2100/pdb2ahe/pdb) using online JSmol software. ( C ) (Left panels) The representative immunoblots of total lysates of 293 T cells transfected with mCherry-Tsg101 together with Flag-CLIC4-WT or Flag-CLIC4-Y104A and probed by the indicated antibodies. (Middle panel) The immunoblots of the immunoprecipitants pulled down by the control or anti-Flag antibody from the indicated transfected cell lysates. (Right panel) Immunoblots of Flag immunoprecipitants isolated from the indicated transfected cell lysates. Compared to its WT counterpart, the Flag-CLIC4-Y104A pulled down less mCherry-Tsg101. ( D ) The total lysates (left panels) as well as the immunoprecipitants pulled down by anti-Flag or control antibody (right panels) were immunoblotted by the indicated antibodies. Asterisks point to the MMP14-mCherry species that was specifically co-immunoprecipitated with Flag-CLIC4. ( E ) ARPE19 cell line expressing inducible CLIC4-sh was treated with Dox for 3 days and then transfected with MMP14-mCherry, GFP-CD63, together with CLIC4-WT-IRES-FLAG-Rab5 Q79L, or CLIC4-Y104A-IRES-FLAG-Rab5 for 1 day in the same Dox conditions. Representative confocal images of mCherry and GFP stained cells are shown. Arrowheads and arrows point to LE luminal and LE limiting membrane signals of MMP14, respectively. ( F ) MMP14 intensity ratio in LE lumen for ( E ). n = 120 endosomes from 24 cells. Bars show means ± S.D. from three independent experiments. p value, t-test. Scale bar = 10 µm. ( G ) Immunoblots demonstrate the same amount of ectopically expressed proteins in the rescue experiments (in E , F ).

Article Snippet: Antibodies used in this study included actin (mouse, Santa Cruz, sc-8432, 1:250), CD63 (mouse H5C6, DSHB, 1:1000), mCherry (goat, Sicgen, AB0040-200, 1:1000), CLIC4 (rabbit, Millipore ABS2088;1:500), Flag (mouse Sigma F3165, 1:2000), MMP14 (rabbit, Abcam ab51074, 1:500 for IFA, 1:1000 for WB; epitope: amino acid ~150–180); MMP14 (mouse, Santa Cruz sc-373908, 2 μg for immunoprecipitation; epitope: amino acids 431–462), vinculin (mouse, Sigma V9131, 1:500), flotillin-1 (mouse, BD Transduction Laboratories, Clone 18, 1:1,000), GAPDH (rabbit, Cell Signaling Technology, 14C10, 1:5000), Tsg101 (goat, Santa Cruz M-19, 1:100), Bestrophin1 (rabbit; Proteintech 21910-1-AP, 1: 1,000), RPE65 (rabbit, gift of Jian-Xing Ma, 1:1000), β-tubulin (mouse, Sigma-Aldrich Cat# T5201, 1:2,000), ZO-1 antibody (rabbit Zymed 61–7300 or mouse Zymed 33–9100, 1:500), Alexa-dye conjugated secondary antibodies (made in donkey, Thermo Fisher Scientific, 1:400), Alexa-dye conjugated phalloidin (Thermo Fisher Scientific, 1:1000), and IRDye-conjugated secondary antibodies (LI-COR; 1:10,000).

Techniques: Sequencing, Software, Western Blot, Transfection, Control, Isolation, Immunoprecipitation, Expressing, Staining, Membrane

Journal: eLife

Article Title: Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

doi: 10.7554/eLife.71982

Figure Lengend Snippet: ( A ) Representative microscope images from U2OS cells expressing mChery-RAB5 Q79L . Confocal micrographs of cells expressing mCherry-RAB5 Q79L , alone (upper row) or with EGFP (lower row). Cells are stained with anti-CD63 (upper row) or with anti-GFP (lower row). ( B ) Confocal micrographs of U2OS cells expressing mChery-RAB5 Q79L and YFP-YBX1. ( C ) Over-expression of YBX1 in U2OS cells increased the secretion of YBX1 in EVs. Immunoblots for the indicated protein markers in cells and high-speed pellet fractions. The numbers under the YBX1 blot represent quantification analysis of endogenous YBX1, YFP-YBX1, and YFP-YBX1-F85A in cells and sedimentable particles by Fiji software. ‘*’ is a non-specific band; Blue arrow represents endogenous YBX1; Red arrow represents fusion YBX1 or YBX1-F85A. ( D ) IDR-driven YBX1 phase separation is required for YBX1 secretion in EVs. Immunoblots for the indicated protein markers in U2OS cells and high-speed pellet fractions. The numbers under the YFP blot represent quantification analysis of endogenous YBX1 and variants in cells and sedimentable particles by Fiji software. ( E ) Proteinase K protection assay on high-speed pellet fractions from U2OS cells. Triton X-100 (0.5%) was used to disrupt the membranes. Immunoblots for YBX1, ALIX, Flotillin-2, and CD9 are shown. ( F ) Proteinase K protection assay on high-speed pellet fractions from U2OS cells expressing YFP-YBX1. Triton X-100 (0.5%) was used to disrupt the membranes. Immunoblots for YBX1, ALIX, Flotillin-2, and CD9 are shown. ( G ) Schematic showing exosome purification with buoyant density flotation in a sucrose step gradient. ( H ) Nanoparticle tracking analysis (NTA) quantification of exosomes from cultured U2OS cells. ( I ) YFP-YBX1 detected in sucrose post-flotation fraction from U2OS cells. Immunoblots for YBX1, ALIX, and CD63 from buoyant exosomes are shown. ( J ) Schematic showing exosome purification with buoyant density flotation in a linear iodixanol gradient. ( K ) Immunoblots across the iodixanol gradient from U2OS cells for classical exosome markers CD9, CD63 and ALIX (the left panel). Collection of fractions F15-F17 corresponding to high density vesicles and immunoblots for YBX1 and CD9. The numbers under YBX1 blot and CD9 blot represent quantification analysis of YFP-YBX1-WT or YFP-YBX1-F85A and CD9 in HD vesicles, respectively, by Fiji software. Scale bars, 3 µm. Figure 4—source data 1. Uncropped Western blot images corresponding to . Figure 4—source data 2. Uncropped Western blot images corresponding to . Figure 4—source data 3. Uncropped Western blot images corresponding to . Figure 4—source data 4. Uncropped Western blot images corresponding to . Figure 4—source data 5. Uncropped Western blot images corresponding to . Figure 4—source data 6. Uncropped Western blot images corresponding to .

Article Snippet: Primary antibodies used in this study were as follows: anti-YBX1 (Cell Signaling Technology, Danvers, MA, #4202); anti-YBX1 (Abcam, Cambridge, MA, ab12148); anti-CD9 (Cell Signaling Technology, Danvers, MA, #13,174 S); anti-ALIX (Santa Cruz Biotechnology, CA, Sc-53540); anti-flotillin-2 (BD Biosciences, San Jose, CA, #610383); anti-CD63 (Abcam, Cambridge, MA, ab134045); anti-CD63 (Fisher Scientific, BDB556019, #H5C6); anti-Actin (Abcam, Cambridge, MA, ab8224); anti-DDX6 (Bethyl Laboratories, Inc, A300-461A); anti-EDC4 (Santa Cruz Biotechnology, CA, Sc-376382); anti-G3BP1 (Santa Cruz Biotechnology, CA, Sc-81940); anti-GFP (Torrey Pines Biolabs, Inc, Houston, TX, TP401); anti-GFP (Santa Cruz Biotechnology, CA, Sc-9996); anti-GM130 (BD Biosciences, 610823); anti-Flag (Sigma, St. Louis, MO, F9291).

Techniques: Microscopy, Expressing, Staining, Over Expression, Western Blot, Software, Purification, Cell Culture

Journal: eLife

Article Title: Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates

doi: 10.7554/eLife.71982

Figure Lengend Snippet:

Article Snippet: Primary antibodies used in this study were as follows: anti-YBX1 (Cell Signaling Technology, Danvers, MA, #4202); anti-YBX1 (Abcam, Cambridge, MA, ab12148); anti-CD9 (Cell Signaling Technology, Danvers, MA, #13,174 S); anti-ALIX (Santa Cruz Biotechnology, CA, Sc-53540); anti-flotillin-2 (BD Biosciences, San Jose, CA, #610383); anti-CD63 (Abcam, Cambridge, MA, ab134045); anti-CD63 (Fisher Scientific, BDB556019, #H5C6); anti-Actin (Abcam, Cambridge, MA, ab8224); anti-DDX6 (Bethyl Laboratories, Inc, A300-461A); anti-EDC4 (Santa Cruz Biotechnology, CA, Sc-376382); anti-G3BP1 (Santa Cruz Biotechnology, CA, Sc-81940); anti-GFP (Torrey Pines Biolabs, Inc, Houston, TX, TP401); anti-GFP (Santa Cruz Biotechnology, CA, Sc-9996); anti-GM130 (BD Biosciences, 610823); anti-Flag (Sigma, St. Louis, MO, F9291).

Techniques: Cell Culture, CRISPR, Recombinant, Plasmid Preparation, shRNA, Software

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Preparation of sEVs derived from PC3 cells and determination of their size and concentration. (A) sEVs from PC3 cells were isolated from the cell culture supernatant by ultrafiltration and ultracentrifugation. Tetraspanins tagged with Halo7 in sEVs were fluorescently labeled with SaraFluor650T (SF650T). (B) Negative-staining TEM images of sEVs revealed that the mean size of the sEVs was 83 ± 19 nm (mean ± SD). (C) The mean size of the sEVs determined by qNano was 69 ± 17 nm, as indicated by the arrowhead. (D) Single-particle fluorescence images of sEVs by TIRFM. Only when sEVs expressed CD63-Halo7, single particles labeled with SF650T (sEVs–CD63Halo7-SF650T) could be observed. (E, G, and I) We directly measured the number of sEV-tetraspanin-Halo7-SF650T particles bound to the antibody-coated glass. After incubating the sEV solution (2 × 10 10 particles/ml) on antibody-coated glass at a dilution factor (df) of 1, 3, or 9, followed by three washes with HBSS, we obtained TIRFM images of single sEV–CD63-Halo7-SF650T particles. Single-particle fluorescence images of three concentrations of sEV–CD63Halo7-SF650T particles (E), sEV–CD81Halo7-SF650T particles (G), and sEV–CD9Halo7-SF650T particles (I), which attached to glass coated with anti-CD63 antibody, anti-CD81 antibody, and anti-CD9 antibody, respectively. df indicates the dilution factor. (F, H, and J) The number of sEVs bound to the glass decreased in accordance with the dilution factor (df), which allowed us to obtain a calibration curve. The numbers of sEV–CD63Halo7-SF650T particles (F), sEV–CD81Halo7-SF650T particles (H), and sEV–CD9Halo7-SF650T particles (J) at three df values attached to the CD63 antibody, anti-CD81 antibody, and anti-CD9 antibody-coated glass, respectively ( n = 16 images). Data are presented as the mean ± SE. The sEV concentration was adjusted according to the calibration line.

Article Snippet: WT PC3 cells and integrin KO PC3 cells were transfected with 1,500 ng of cDNA-encoding CD63-Halo7, CD81-Halo7, or CD9-Halo7 using a 4D-Nucleofector (LONZA) according to the manufacturer’s recommendations. cDNA plasmids for human CD63 (NCBI accession no. NM_001780 ), human CD81 ( NM_004356 and pFN21AE5228), and human CD9 ( NM_001769 and pFN21AE1546) were purchased from Kazusa DNA Res.

Techniques: Derivative Assay, Concentration Assay, Isolation, Cell Culture, Labeling, Negative Staining, Single Particle, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Integrin α2β1 in sEVs derived from PC3 cells is responsible for the binding of CD63-containing sEVs to collagen type I, and integrins α6β1 and α6β4 are responsible for the binding to laminin. (A, C, E, and G) Single-particle fluorescence images of sEV–CD63Halo7-SF650T on glass coated with fibronectin, collagen type I, and laminin before and after the KO of integrin β1 (A), integrin α2 (C), integrin α6 (E), and integrin β4 (G). (B, D, F, and H) The numbers of sEVs attached to glass coated with these ECM molecules before and after the KO of integrin β1 (B), integrin α2 (D), integrin α6 (F), and integrin β4 (H) ( n = 16 images). Data are presented as the mean ± SE. n.s., nonsignificant difference; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: WT PC3 cells and integrin KO PC3 cells were transfected with 1,500 ng of cDNA-encoding CD63-Halo7, CD81-Halo7, or CD9-Halo7 using a 4D-Nucleofector (LONZA) according to the manufacturer’s recommendations. cDNA plasmids for human CD63 (NCBI accession no. NM_001780 ), human CD81 ( NM_004356 and pFN21AE5228), and human CD9 ( NM_001769 and pFN21AE1546) were purchased from Kazusa DNA Res.

Techniques: Derivative Assay, Binding Assay, Single Particle, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Neither laminin nor fibronectin is present on PC3-derived sEV surfaces. (A) Schematic diagram of the isolation of specific sEVs. sEVs were isolated by ultracentrifugation at 200,000 × g for 4 h (UC sample). Then, special sEVs were isolated from sEVs by a bead-conjugating antibody (IP: immunoprecipitation sample). (B) Western blot analysis of tetraspanin (CD63, CD81, and CD9), fibronectin (FN), and laminin (LN) in UC and IP samples. sEVs were isolated by IP using an anti-CD63 antibody. (C) Western blot analysis of sEVs isolated by IP using anti-fibronectin antibody. Source data are available for this figure: .

Article Snippet: WT PC3 cells and integrin KO PC3 cells were transfected with 1,500 ng of cDNA-encoding CD63-Halo7, CD81-Halo7, or CD9-Halo7 using a 4D-Nucleofector (LONZA) according to the manufacturer’s recommendations. cDNA plasmids for human CD63 (NCBI accession no. NM_001780 ), human CD81 ( NM_004356 and pFN21AE5228), and human CD9 ( NM_001769 and pFN21AE1546) were purchased from Kazusa DNA Res.

Techniques: Derivative Assay, Isolation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Talin-1 in sEVs does not regulate the binding affinity of integrins for laminin. (A) Western blot analysis of PC3 and BxPC3 cells after talin-1 KD by siRNA. (B and C) Cell spreading assay of WT and talin-1 (Tln1) KD PC3 cells (B) and BxPC3 cells (C) on glass coated with ECM components: fibronectin (FN), laminin (LN), or collagen typeⅠ (COL1). Cells were observed after 2 h of incubation, and cell areas were quantified ( n = 35 cells). (D) Western blot analysis of PC3 cell–derived sEVs after talin-1 KD by siRNA or overexpression of talin-1. (E–G) The fluorescence images (E) and the numbers of PC3-sEVs attached to glass coated with laminin before and after (F) talin-1 KD or (G) overexpression of talin-1 ( n = 14 images). Halo7-integrin β1 in sEVs was labeled with SF650T. (H–J) The fluorescence images (H) and the numbers of CD63-labeled sEVs attached to glass coated with laminin before and after (I) talin-1 KD and (J) overexpression of talin-1 ( n = 16 images). (K and L) The numbers of EV–CD63Halo7-SF650T particles attached to glass coated with collagen type I before and after (K) talin-1 KD or (L) overexpression of talin-1 ( n = 21 images). (M) Western blot analysis of BxPC3 cell–derived sEVs after talin-1 KD by siRNA. (N–P) Fluorescence images (N) and the numbers of sEVs-BxPC3 bound to laminin (O) or collagen type I (P) on glass before and after talin-1 KD ( n = 7 images). The membranes of sEVs were stained with Exosparkler DeepRed. (Q) Western blot analysis of the phosphorylation of Ser425 on talin-1 in PC3 cells and sEVs. Roscovitine: an inhibitor of CDK5 that phosphorylates Ser425 of talin-1. (R) Western blot analysis of kindlin-2 in PC3 cells and PC3-derived sEVs before and after kindlin-2 KD and talin-1 KD. Data are presented as the mean ± SE. n.s., nonsignificant difference; *P < 0.05; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided). Source data are available for this figure: .

Article Snippet: WT PC3 cells and integrin KO PC3 cells were transfected with 1,500 ng of cDNA-encoding CD63-Halo7, CD81-Halo7, or CD9-Halo7 using a 4D-Nucleofector (LONZA) according to the manufacturer’s recommendations. cDNA plasmids for human CD63 (NCBI accession no. NM_001780 ), human CD81 ( NM_004356 and pFN21AE5228), and human CD9 ( NM_001769 and pFN21AE1546) were purchased from Kazusa DNA Res.

Techniques: Binding Assay, Western Blot, Incubation, Derivative Assay, Over Expression, Fluorescence, Labeling, Staining, Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Extracellular vesicles adhere to cells primarily by interactions of integrins and GM1 with laminin

doi: 10.1083/jcb.202404064

Figure Lengend Snippet: Cholesterol impairs the binding of sEVs to laminin and the MRC-5 cell PM. (A) Fluorescence images of sEV–CD63Halo7-SF650T particles bound to laminin (LN) on glass before and after cholesterol depletion by MβCD and the numbers of attached sEVs per image (82 × 82 μm). The cholesterol content in PC3 cell–derived sEVs was reduced to 16% after treatment with MβCD. (B and C) The numbers of sEV–CD63-Halo7-SF650T particles attached to glass coated with laminin before and after treatment with saponin (B) and the addition of cholesterol by MβCD–cholesterol complex (C).The cholesterol content was increased to 186% after treatment with the MβCD–cholesterol complex. (D) Fluorescence images of an MRC-5–GFP cell and sEV–CD63Halo7-SF650T particles on the MRC-5 cell after 30 min of incubation. (E and F) Time course of the number of sEV–CD63-Halo7-SF650T particles per 1,000 μm 2 attached to the MRC-5 cell membrane before and after treatment with MβCD ( n = 16 cells) (E) or the MβCD–cholesterol complex ( n = 8 cells) (F). Data are presented as the mean ± SE. n.s., nonsignificant difference; **P < 0.01; ***P < 0.001 according to Welch’s t test (two-sided).

Article Snippet: WT PC3 cells and integrin KO PC3 cells were transfected with 1,500 ng of cDNA-encoding CD63-Halo7, CD81-Halo7, or CD9-Halo7 using a 4D-Nucleofector (LONZA) according to the manufacturer’s recommendations. cDNA plasmids for human CD63 (NCBI accession no. NM_001780 ), human CD81 ( NM_004356 and pFN21AE5228), and human CD9 ( NM_001769 and pFN21AE1546) were purchased from Kazusa DNA Res.

Techniques: Binding Assay, Fluorescence, Derivative Assay, Incubation, Membrane

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