Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Schematic diagram of podosome structure. b, Dyn2 forms belt-like structures around the actin cores of synaptic podosomes. Myoblasts were seeded on laminin-coated coverslips and subjected to differentiation. Differentiated myotubes were fixed and stained to visualize endogenous Factin, Dyn2, and Tks5. Images were acquired from z-stack confocal microscopy. Boxed areas were magnified and shown in lower panels to display the Z-projection and orthogonal view of a single podosome. Arrowhead, podosome with a Dyn2 belt. Arrow, podosome without a Dyn2 belt. Scale bar, 2 μm. c, Dyn2 localizes differently from other podosome components. Differentiated myotubes were fixed and stained to visualize endogenous F-actin, Dyn2, Arp2, cortactin, vinculin, and myosin IIA. Scale bar, 2 μm. d-f, Level of Dyn2-belt formation is correlated with podosome height and width. Podosomes were grouped into three categories according to amounts of Dyn2 surrounding the actin core. Data are presented as mean ± s.d. of podosome height and width. Each dot represents one podosome. At least 40 podosomes in 10 different cells were analyzed. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test. ** P < 0.01; *** P < 0.001. The representative images are shown in the lower panels, with Dyn2 in green and F-actin in red. g-h, Snapshots of time-lapse images of a single podosome showing the temporal distribution of Cortactin and Dyn2 in myotubes. Myoblasts were transfected with Lifeact-GFP and Cortactin-mCherry (g) or Dyn2 WT -mCherry (h), respectively, and were seeded on laminin-coated glassbottom dishes, subjected to differentiation for 5-7 days, and imaged by inverted fluorescence microscopy at 37 °C with 1 min frame intervals. Scale bar, 2 μm. i, Frequency distribution of podosome lifespan in myotubes (n = 37 podosomes). j, Percentage of Dyn2 appearance during podosome lifespans in myotubes. Each dot represents one podosome.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Staining, Confocal Microscopy, Transfection, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Dyn2 bundles actin filaments. Reconstituted F-actin (5 μM) was incubated with Dyn2 at indicated concentrations for 30 min and subjected to 20 min centrifugation at 14,000 x g . S, supernatant; P, pellet. b, Dyn2 (1 μM) shows significant actin bundling activity. Mean ± s.d. of percentage of sedimented actin was quantified in ImageJ as the ratio of actin in the pellet versus total actin. c-d, F-actin sedimentation assay and quantification result of Dyn2 bundling activity. Reconstituted F-actin (5 μM) was incubated with 1 μM Dyn2 for 30 min followed by 15 min incubation with 1 mM GTP or GMPPCP. e, Real-time visualization of the actin bundling activity of Dyn2 under confocal microscopy. Scale bar, 10 μm. f, TEM images of negative-stained actin-Dyn2 bundles. Scale bar, 100 nm. g, TEM images of negative-stained branched actin-Dyn2 bundles. Scale bar, 100 nm. h-k, Branched F-actin sedimentation assay and quantification result of Dyn2 bundling activity. Concentrations of individual components were 5 μM actin, 1 μM Dyn2, 30 nM Arp2/3 complex, 80 nM VCA and 1 mM nucleotides. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons test. ns, not significant; * P < 0.05; *** P < 0.001. l, Morphology of Dyn2-bundled linear and branched actin under confocal microscopy. Scale bar, 1 μm.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Incubation, Centrifugation, Activity Assay, Sedimentation, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Dynamin-2 regulates synaptic podosome maturation to facilitate neuromuscular junction development

doi: 10.1101/2020.05.30.125062

Figure Lengend Snippet: a, Distribution of phospho-deficient Dyn2 Y597F -mCherry in myotubes. b, Percentage of synaptic podosomes with Dyn2-mCherry enrichment in myotubes (n = 19 podosomes per condition). c, Actin bundling ability of Dyn2 mutants with or without GTP. The percentage of sedimented actin was quantified and is shown in d. e, TEM images of negative-stained actin-Dyn2 bundles with or without 15 min GTP incubation. Scale bar, 100 nm. f, Dyn2 functions as a molecular girdle and checkpoint for podosome maturation and turnover, respectively. (i) Interactions between ECM and integrins initiate podosome formation. Actin polymerization proteins such as Arp2/3 complex and cortactin drive podosome initiation. (ii) Tks5 is recruited to the nascent podosome and promotes podosome maturation. (iii) Phosphorylated Dyn2 is recruited to the podosome to bundle the actin core and facilitate its growth. Dyn2 forms a belt-like structure around the actin core to enhance the function of podosome. (iv, v) After dephosphorylation, GTP hydrolysis triggers Dyn2 dissociation from the podosome and induces its turnover. g, Functional role of Dyn2 in postsynaptic NMJ morphogenesis. (i) During NMJ prepatterning, AChR clusters are induced by extracellular signals, such as laminin or Wnt. (ii) During NMJ development, perforation and remodeling of AchR clusters is facilitated by synaptic podosomes whose maturation and turnover is governed by Dyn2.

Article Snippet: To reconstitute branched actin, 160 nM WASP VCA domain protein (#VCG03, Cytoskeleton) and 60 nM Arp2/3 protein complex (#RP01P, Cytoskeleton) were also added to the actin polymerization buffer.

Techniques: Staining, Incubation, De-Phosphorylation Assay, Functional Assay

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: (A) Flow cytometry dot plot showing CD45 phosphatase activity versus CD27 expression on gated CD19 + human peripheral B cells. (B and C) CD45 phosphatase activity (B) and CD45 surface expression (C) of CD27 − (blue) and CD27 + B cells (red). Numbers in histograms represent CD45 activity (pCAP-SP1) (B) or CD45 surface expression (C) as the mean fluorescence intensity (MFI) ratio of CD27 + /CD27 − B cells. Bottom graphs: pCAP-SP1 or CD45 surface expression (MFI) in CD27 + relative to CD27 − B cells. (D) CD45 expression versus CD45 phosphatase activity in gated CD27 + MBCs; CD45 hi and CD45 lo expression gates are shown. (E) CD45 phosphatase activity and (F) CD45 expression in CD27 + MBCs expressing low (blue open histogram) or high (red open histogram) levels of surface CD45 compared to CD27 − B cells (filled blue histogram). Graphs show pCAP-SP1 or CD45 MFI relative to CD27 − B cells. n = 12. Related to . ****p < 0.0001.

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Flow Cytometry, Activity Assay, Expressing, Fluorescence

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FITC Mouse monoclonal anti human CD27 (clone MT271) , Miltenyi Biotec , Cat# 130–093-184; RRID:AB_1036205.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 1. A genetic screen in Cos-7 cells to isolate cDNAs whose overexpression interferes with CME. Cos-7 cells are transfected with a library of partial cDNAs fused to eGFP (1). One day after transfection, cells accumulating the TfR at the cell surface are selected using a monoclonal antibody against the TfR and anti-mouse IgG antibody-coated dishes or Cy5-labeled Tf and FACS (2). Selected cells are lysed and plasmids are recovered by electroporation into E. coli (3). Plasmids from individual colonies are either pooled and subjected to a new round of selection (4) or are separately purified to analyze the effect of overexpressing individual eGFP fusion proteins on the internalization of TR-Tf by fluorescence microscopy (5).

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Over Expression, Transfection, Labeling, Electroporation, Selection, Purification, Fluorescence, Microscopy

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 2. Identification of short peptides whose overexpression inhibits CME by panning with antibody-coated plates. (A) Cos-7 cells transfected with pEGFP-N1 (C), pEGFP-C2-EΔ95/295 (eps15) or the plasmids isolated from the library (numbers) were incubated in the presence of TR-Tf. eGFP-expressing cells exhibiting normal, reduced or no uptake were scored. The graph represents the percentage of eGFP-expressing cells showing no Tf uptake. Bar = 10 µm. (B) Peptide sequences in frame with the eGFP initiation codon encoded by the inserts of the isolated plasmids. Yxx∅ and LL motifs are highlighted.

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Over Expression, Transfection, Isolation, Incubation, Expressing

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 3. Peptide 38 bears a bona fide Yxx∅ endocytosis motif. (A) Cos-7 cells transfected with pEGFP-N1 (C) or the indicated plasmids were assayed for their ability to internalize TR-Tf. The fluorescent signals from the eGFP and TR-Tf were analyzed by fluorescence microscopy using the appropriate filters. The graph represents the percentage of eGFP-expressing cells showing no uptake. Bar = 10 µm. (B) GST or GST fused to peptide 38 was bound to glutathione–Sepharose beads and incubated with a rat brain extract. Proteins bound to the beads were separated by SDS–PAGE and analyzed by immunoblotting using an antibody against adaptin β. Two micrograms of total rat brain protein extract were loaded as total (TE).

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Fluorescence, Microscopy, Expressing, Incubation, SDS Page, Western Blot

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 4. Isolation of two cDNAs from human brain whose overexpression inhibits CME using FACS. (A) Cos-7 cells transfected with pEGFP-C2 (eGFP) or pEGFP-C2-EΔ95/295 (eGFP-EΔ95/295) were incubated in the presence of Cy5-Tf and analyzed by FACS. The x-axis represents the fluorescence intensity of the Cy5-Tf surface labeling per cell. The y-axis represents the number of transfected cells showing a particular fluorescence intensity. The bar indicates the intensity threshold used to screen the brain library (>2 × 101). (B) Cos-7 cells transfected with pEGFP-C2 (C) or the plasmids isolated from the human brain library (numbers) were incubated in the presence of TR-Tf. eGFP-expressing cells exhibiting normal, reduced or no uptake were scored. The graph represents the percentage of eGFP-expressing cells showing no uptake. (C) Cos-7 cells transfected with plasmids 13 and 24 were incubated with TR-Tf. The fluorescent signals from eGFP and TR-Tf were analyzed by fluorescence microscopy using the appropriate filters. Bar = 10 µm. (D) Scheme showing the structure of the eGFP fusion proteins encoded by plasmids 13 and 24.

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Isolation, Over Expression, Transfection, Incubation, Fluorescence, Labeling, Expressing, Microscopy

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: CXCR4 is internalized into endosomes by interacting with EBOV GP. (A) GFP-LC3 was co-transfected with 2 μg HA-tagged CXCR4 (or vector control) into HEK 293T cells, and the formation of autophagosomes indicated by GFP-LC3 puncta was determined by confocal microscopy. (B) HEK 293T cells was pre-transfected with NP, VP35, VP30, L and HAVCR1, and were infected with EBOV trVLPs (10 6 copies/mL) for 24 h, and dual luciferase reporter assay was carried out to evaluate the success of infection indicated by relative luciferase activity of Renilla to firefly. (C) and (D) HEK 293T cells was treated the same as (B), and levels of CXCR4 mRNA (C) and CXCR4 protein (D) 48 h and 72 h post infection were measured by RT-qPCR and western blotting (WB). The ACTB gene and TUBA/α-tubulin were used as internal controls. (E) HEK 293T cells were treated the same as (B), and the subcellular distributions of CXCR4 (red) and cell membrane (indicated by DiD staining, far-red) were determined by confocal microscopy. (F), (G) and (H) two μg flag-tagged EBOV GP (F), EBOV GP1 (G) and EBOV GP2 (H) was individually expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells. EBOV GP (F), GP1 (G) and GP2 (H) proteins were immunoprecipitated by anti-flag antibodies and their interactions with CXCR4 were analyzed by WB. (I) two μg HA-tagged CXCR4 was expressed with 2 μg either GP1 or GP1 with receptor binding domain (RBD) deletion in HEK 293T cells. GP1 and GP1ΔRBD proteins were immunoprecipitated by anti-flag antibodies and their interactions with CXCR4 were analyzed by WB. GP, GP1, GP2 and GP1ΔRBD were detected by anti-flag, CXCR4 was detected by anti-HA. TUBA/α-tubulin was used as an internal control. (J) two μg HA-tagged CXCR4 was either expressed with 2 μg flag-tagged eGFP or flag-tagged GP fused with eGFP in HEK 293T cells. Subcellular localization of eGFP (green), HA-CXCR4 (red), EBOV GP (green) were determined by confocal microscopy. (K) HEK 293T cells were treated as (J), and whole cell lysate, membrane and cytoplasmic proteins were isolated and detected by WB. (L) HEK 293T cells were transfected with either 2 μg flag-tagged eGFP or flag-tagged EBOV GP fused with eGFP plasmids. Subcellular localization of eGFP (green), EBOV GP (green) and endogenous CXCR4 (red) were determined by confocal microscopy. (M) Huh7 cells were treated as (L), and whole cell lysate, membrane and cytoplasmic proteins were isolated and detected by WB. TUBA/α-tubulin was used as an internal control. (N) flag-tagged GP fused with eGFP was expressed alone or with a growing amount of HA-tagged CXCR4 (100 ng, 400 ng and 1.6 μg) in HEK 293T cells. Subcellular localization of eGFP (green), HA-CXCR4 (red), EBOV GP (green) were determined by confocal microscopy. (O) two μg flag-tagged GP fused with eGFP was expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells. Subcellular localization of HA-CXCR4 (red), EBOV GP (green), and EEA1 (far-red) were determined by confocal microscopy. (P) two μg flag-tagged GP fused with eGFP was expressed in wild-type and CXCR4 gene knockout HEK 293T cells. Subcellular localization of CXCR4 (red), EBOV GP (green), and EEA1 (far-red) were determined by confocal microscopy. DAPI indicated the nucleus and scale bars for (A), (E), (J), (L), (N), (O) and (P): 5 μm. Data are presented as means±standard error of measurements (SEMs) of three independent experiments. Two-tailed Student’s t test was used. **** p < 0.0001, NS, non-significant.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Transfection, Plasmid Preparation, Control, Confocal Microscopy, Infection, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Western Blot, Membrane, Staining, Immunoprecipitation, Binding Assay, Isolation, Gene Knockout, Two Tailed Test

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: CXCR4 acts as an entry factor of EBOV. (A) schematic representation on HEK 293T and HeLa cells infected with EBOV trVLPs for attachment and entry detection by quantifying GP and/or VP40 RNA levels. (B) and (C) HEK 293T cells stably overexpressing CXCR4 and wild-type HEK 293T cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 4°C. After washed with prechilled PBS, total RNAs were isolated and reverse transcribed. Relative levels of GP RNA (B) and VP40 RNA (C) were detected by RT-qPCR. (D) and (E) HEK 293T cells stably overexpressing CXCR4 and wild-type HEK 293T cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 37°C and cells were collected by trypsin digestion and centrifugation. After washed with PBS, total RNAs were isolated and reverse transcribed. Relative levels of GP RNA (D) and VP40 RNA (E) were detected by RT-qPCR. (F) CXCR4 KO HEK 293T cells and control cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 4°C for attachment detection. (G) CXCR4 KO HEK 293T cells and control cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 37°C for entry detection. (H) CXCR4 KO HeLa cells and control cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 4°C for attachment detection. (I) CXCR4 KO HEK 293T cells and control cells were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 37°C for entry detection. (J) and (K) HEK 293T cells (J) and HeLa cells (K) were pretreated with 20 μmol/L motixafortide and were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 4°C for attachment detection. (L) and (M) HEK 293T cells (L) and HeLa cells (M) were pretreated with 20 μmol/L motixafortide and were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 37°C for entry detection. (N) and (O) HEK 293T cells (J) and HeLa cells (K) were pretreated with 15 μg/mL 12G5 and were infected with EBOV trVLPs (10 6 copies/mL) for 2 h at 4°C for attachment detection. The ACTB gene was used as an internal control. Data are presented as means±standard error of measurements (SEMs) of three independent experiments. Two tailed Student’s t test for (B) to (E), one-way ANOVA and post hoc Tukey test for (F) to (O). ** p < 0.01, *** p < 0.001, ** **p < 0.0001.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Infection, Stable Transfection, Isolation, Reverse Transcription, Quantitative RT-PCR, Centrifugation, Control, Two Tailed Test

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: CXCR4 promotes EBOV replication without influencing viral factory assembly. (A) HEK 293T cells stably expressing CXCR4-HA or control cells were pre-transfected with NP, VP35, VP30, L and HAVCR1, and were infected with EBOV trVLPs (10 6 copies/mL) for 48 h. Levels of VP40, GP and HA-CXCR4 were analyzed by western blotting (WB). (B) Wild-type and CXCR4 gene knockout HEK 293T cells were treated as in (A), and levels of VP40, GP and CXCR4 were analyzed by WB. (C) two μg flagged-tagged GP was transfected into CXCR4 gene knockout HEK 293T cells or control cells and both groups of cells were infected with EBOV trVLPs (10 6 copies/mL) for 48 h. Levels of VP40, GP, CXCR4 and HA-CXCR4 were analyzed by WB. (D) two μg flag-tagged GP fused with eGFP was expressed alone or with a growing amount of HA-tagged CXCR4 (100 ng, 400 ng and 1.6 μg) in HEK 293T cells. The fluorescence intensity of GP was analyzed using image pro plus 6.0 software. (E) two μg HA-tagged CXCR4 was expressed with 2 μg flag-tagged GP (or vector control) in HEK 293T cells. Levels of CXCR4 and GP mRNA were measured by RT-qPCR. The ACTB gene was used as an internal control. (F) one μg flag-tagged NP and 1 μg MYC-tagged VP35 were expressed with 1 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells. NP was immunoprecipitated with anti-flag. Interactions between NP and VP35 were determined by WB. (G) one μg flag-tagged NP and 1 μg MYC-tagged VP24 were expressed with 1 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells. Interactions between NP and VP24 were determined by WB. CXCR4 was detected by anti-HA or anti-CXCR4, VP40 was detected by anti-VP40, GP was detected by anti-GP, NP was detected by anti-flag, and VP35 and VP24 were detected by anti-MYC. TUBA/α-tubulin was used as an internal control. Data are presented as means±standard error of measurements (SEMs) of three independent experiments. One-way ANOVA and post hoc Tukey test for (D) and two tailed Student’s t test for (E). **** p < 0.0001, NS, non-significant.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Stable Transfection, Expressing, Control, Transfection, Infection, Western Blot, Gene Knockout, Fluorescence, Software, Plasmid Preparation, Quantitative RT-PCR, Immunoprecipitation, Two Tailed Test

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: CXCR4 induces EBOV GP degradation by reducing its protein stability. (A) two μg flag-tagged EBOV GP was expressed with 2 μg HA-tagged CXCR4 (or vector control) in HEK 293T and HeLa cells. Protein levels of GP and CXCR4 were analyzed by western blotting (WB). (B) two μg flag-tagged EBOV GP was expressed with a growing amount of HA-tagged CXCR4 in HEK 293T and HeLa cells. (C) two μg flag-tagged GP was expressed in CXCR4 gene knockout (KO) HEK 293T and HeLa cells. Protein levels of GP and CXCR4 were analyzed by WB. GP was detected by anti-flag, CXCR4 was detected by anti-HA in (A) and (B), and by anti-CXCR4 in (C). TUBA/α-tubulin was used as an internal control. (D) two μg flag-tagged GP was expressed with 2 μg CXCR4 (or vector control) in HEK 293T cells, and cell viability was measured by OD value at 450 nm of different groups treated with CCK-8 for 1 h, 24 h, 48 h and 72 h. (E) two μg flag-tagged GP, GP1 and GP2 were expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells. (F) two μg flag-tagged GP and its truncates were with 2 μg HA-tagged CXCR4 in HEK 293T cells. (G) two μg flag-tagged GP from various ebolaviruses was expressed with HA-tagged CXCR4 in HEK 293T cells. (H) gray value analysis of indicated bands of GP in (G) was analyzed using imageJ software (version 1.53a). (I) two μg flag-tagged GP and its mutants were expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells. (J) gray value analysis of indicated bands of GP in (I) was analyzed using imageJ software (version 1.53a). (K) and (L) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells (K) or HeLa cells (L) treated with 100 μg/mL cycloheximide (CHX) for 0 to 8 h. Gray value analysis of indicated bands of GP was analyzed using imageJ software (version 1.53a). GP and its truncates or mutants were detected by anti-flag, CXCR4 was detected by anti-HA, and TUBA/α-tubulin was used as an internal control in (E), (F), (G), (I), (K) and (L). Data are presented as means±standard error of measurements (SEMs) of three independent experiments. One-way ANOVA and post hoc Tukey test for (D), (H) and (J). * p < 0.05, ** p < 0.01, ** *p < 0.001.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Plasmid Preparation, Control, Western Blot, Gene Knockout, CCK-8 Assay, Software

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: The CXCR4 sorting and degradation pathway is partially involved in lysosomal degradation of EBOV GP. (A) and (B) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells and HeLa cells treated with 20 μmol/L MG132 (A) or 100 nmol/L bafilomycin A 1 (baf A1) (B). Protein levels of GP and CXCR4 were analyzed by western blotting (WB). GP was detected by anti-flag, CXCR4 was detected by anti-HA. TUBA/α-tubulin was used as an internal control. (C) two μg flag-tagged GP fused with eGFP was expressed with 2 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells. Subcellular localization of HA-CXCR4 (red), EBOV GP (green), and LAMP1 (lysosomal associated membrane protein 1; far-red) were determined by confocal microscopy. Scale bars: 5 μm. (D) liquid chromatography mass spectrometry (LC-MS/MS) was used to identify proteins that interacted with EBOV GP in HEK 293T cells. Numbers of proteins interacted with flag-tagged GP (or control IgG) were indicated by Venn diagram. (E), (F) and (G) gene ontology enrichment analysis of proteins potentially interacting with flag-tagged GP according to their biological process (E), molecular function (F) and cellular component (G). (H) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in HGS knockout (KO) HEK 293T cells or control cells. (I) one μg flag-tagged GP was expressed with 1 μg HA-tagged CXCR4 and/or 1 μg MYC-tagged HGS in HEK 293T cells. (J) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 in ITCH/AIP4 KO HEK 293T cells or control cells. (K) one μg flag-tagged GP was expressed with 1 μg HA-tagged CXCR4 and/or 1 μg MYC-tagged ITCH/AIP4 in HEK 293T cells. (L) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in ITCH/AIP4 KO HEK 293T cells or control cells. (M) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 in TRIM21 KO HEK 293T cells or control cells. (N) one μg flag-tagged GP was expressed with 1 μg HA-tagged CXCR4 and/or 1 μg MYC-tagged TRIM21 in HEK 293T cells. (O) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in TRIM21 KO HEK 293T cells or control cells. Protein expression was analyzed by WB. GP was detected by anti-flag, CXCR4 was detected by anti-HA. HGS, ITCH/AIP4 and TRIM21 were detected by anti-MYC or specific antibodies. TUBA/α-tubulin was used as an internal control.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Western Blot, Control, Plasmid Preparation, Membrane, Confocal Microscopy, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Knock-Out, Expressing

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: CXCR4 targets EBOV GP for reticulophagic degradation. (A) one μg GFP-LC3 was co-transfected with 1 μg flag-tagged GP and 1 μg HA-tagged CXCR4 (or vector control) into HEK 293T cells, and the formation of autophagosomes indicated by GFP-LC3 puncta was determined by confocal microscopy. (B) two μg flag-tagged GP fused with eGFP was expressed with 2 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells. Subcellular localization of HA-CXCR4 (red), EBOV GP (green), and LC3 (far-red) were determined by confocal microscopy. DAPI indicated the nucleus and scale bars: 5 μm. (C) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in ATG3 knockout (KO) HEK 293T cells or control cells. (D) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in ATG5 KO HEK 293T cells or control cells. (E) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in SQSTM1/p62 KO HEK 293T cells or control cells. (F) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in RETREG1 KO HEK 293T cells or control cells. (G) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in LAMP2 KO HEK 293T cells or control cells. (H) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells and HeLa cells treated with 10 nmol/L rapamycin. Protein expression was analyzed by western blotting. GP was detected by anti-flag, CXCR4 was detected by anti-HA. ATG3, ATG5, SQSTM1/p62, RETREG1 and LAMP2 were detected by their specific antibodies. TUBA/α-tubulin was used as an internal control. Data are presented as means±standard error of measurements (SEMs) of three independent experiments. Two tailed Student’s t test was used. **** p < 0.0001.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Transfection, Plasmid Preparation, Control, Confocal Microscopy, Knock-Out, Expressing, Western Blot, Two Tailed Test

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: RNF185 participates in the CXCR4-mediated EBOV GP polyubiquitination and degradation. (A) one μg flag-tagged GP was expressed with 1 μg his-tagged ubiquitin (ub) in the presence or absence of 1 μg HA-tagged CXCR4 in HEK 293T cells. (B) one μg flag-tagged GP was expressed with 1 μg ub, its mutants with indicated lysine residue (K6, K11, K27, K29, K33, K48 and K63) reserved, or all seven lysine residues mutated to arginine (7K/7 R) in the presence or absence of 1 μg HA-tagged CXCR4 in HEK 293T cells. GP was pulled down by anti-flag, and protein expression in whole cell lysate (input) and immunoprecipitation (IP) samples were analyzed by western blotting (WB) in (A) and (B). GP was detected with anti-flag, CXCR4 was detected with anti-HA, and ubiquitin was detected with its specific antibody. (C) one μg flag-tagged GP was expressed with 1 μg HA-tagged CXCR4 and/or 1 μg MYC-tagged RNF185 in HEK 293T cells. (D) two μg flag-tagged GP was expressed alone or with 2 μg HA-tagged CXCR4 in RNF185 knockout (KO) HEK 293T cells or control cells. (E) two μg flag-tagged GP was expressed with 2 μg HA-tagged CXCR4 (or vector control) in HEK 293T cells. GP was pulled down by anti-flag and protein expression in whole cell lysate (input) and IP samples were analyzed by WB. (F) one μg flag-tagged GP was expressed with 1 μg MYC-tagged RNF185 in the presence or absence of 1 μg HA-tagged CXCR4 in HEK 293T cells. RNF185 was pulled down by anti-MYC and protein expression in whole cell lysate (input) and IP samples were analyzed by WB. (G) one μg flag-tagged GP was expressed with 1 μg MYC-tagged HGS in the presence or absence of 1 μg HA-tagged CXCR4 in HEK 293T cells. HGS was pulled down by anti-MYC and protein expression in whole cell lysate (input) and IP samples were analyzed by WB. (H) two μg MYC-tagged RNF185 and 2 μg flag-tagged GP were expressed in CXCR4 KO cells or control cells. RNF185 was pulled down by anti-MYC and protein expression in whole cell lysate (input) and IP samples were analyzed by WB. (I) two μg flag-tagged GP was expressed in CXCR4 KO cells or control cells. GP was pulled down by anti-flag and protein expression in whole cell lysate (input) and IP samples were analyzed by WB. (J) two μg flag-tagged wild-type or K673A mutated GP was expressed with 2 μg HA-tagged CXCR4 in HEK 293T cells. GP was detected by anti-flag, CXCR4 was detected by anti-HA. RNF185 and HGS were detected by anti-MYC or their specific antibodies. SQSTM1/p62 and RETREG1 were detected by their specific antibodies. TUBA/α-tubulin was used as an internal control.

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Ubiquitin Proteomics, Residue, Expressing, Immunoprecipitation, Western Blot, Knock-Out, Control, Plasmid Preparation

Journal: Autophagy

Article Title: Dual roles of CXCR4 (C-X-C motif chemokine receptor 4) in promoting entry of ebolavirus and targeting excessive glycoprotein for reticulophagic degradation to facilitate viral fitness

doi: 10.1080/15548627.2025.2492877

Figure Lengend Snippet: The graphical abstract on the dual roles of CXCR4 in promoting EBOV entry and mediating excessively expressed mature GP degradation via reticulophagy to facilitate viral fitness. CXCR4 interacts with EBOV GP, and such interaction could mediate internalization of protein complex into early endosomes, where GP further interacts with NPC1 to release its genome to cytosol. Mature GP is transported to the cell membrane for release of the mature viral particles, whereas mature GP anchored on the cell membrane could hijack CXCR4 sorting and transporting pathway by their interactions with HGS, and then GP could be carried back to endoplasmic reticulum by CXCR4, where the E3 ubiquitin ligase RNF185 was recruited to polyubiquitinate GP in a K27- and K63-linked manner. Finally, polyubiquitinated GP was degraded in lysosomes via reticulophagy by interacting with the reticulophagic receptor RETREG1, in an ATG3- and ATG5-dependent manner. Thus, our study uncovered dual roles of CXCR4 in EBOV life cycle and provided new evidence that EBOV hijacked the host machinery through efficient virus-host interactions to promote viral fitness. (this figure was created by BioRender.com.) [the nucleus is double membrane.].

Article Snippet: Antibodies used in this study are as follows: anti-Flag M2 mouse antibody (Merck, F1804), anti-HA-tag mAb (MBL Beijing Biotechnology, M180–3), anti-TUBA/α-tubulin mAb (MBL Beijing Biotechnology, M175–3), CXCR4 monoclonal antibody (Proteintech 60,042–1-Ig), ATG3 Rabbit polyclonal antibody (Proteintech 11,262–2-AP), ATG5 Rabbit polyclonal antibody (Proteintech 10,181–2-AP), RETREG1/FAM134B Rabbit polyclonal antibody (Proteintech 21,537–1-AP), ubiquitin recombinant antibody (Proteintech 80,992–11-RR), HGS Rabbit polyclonal antibody (Proteintech 10,390–1-AP), TRIM21 Rabbit polyclonal antibody (Proteintech 12,108–1-AP), LAMP2 Mouse polyclonal antibody (Proteintech 66,301–1-Ig), anti-ITCH/AIP4 Rabbit antibody (abcam, ab108515), anti-MYC antibody (MBL, 562), RNF185 polyclonal antibody (Immunoway, YN2489), CDH2/N-cadherin (Proteintech 66,219–1-Ig), IRDye 680LT donkey anti-rabbit IgG (LICOR, 926–68023), IRDye 800CW donkey anti-mouse IgG (LICOR, 926–32212), Goat anti-mouse IgG H&L (HRP; abcam, ab6789), Goat anti-rabbit IgG H&L (HRP; abcam, ab97051).

Techniques: Membrane, Ubiquitin Proteomics, Virus

Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: (A) Increased expression of CD44 in OVCAR3 sphere forming cells. The expression of ovarian cancer stem cell marker CD44 was increased in OVCAR3 sphere forming cells as observed under fluorescence microscopy. Nuclei were stained with Hoechst (×100). (B) Analysis of surface marker expressions by flow cytometry. CD44 and CD117 were increased approximately two folds in OVCAR3 sphere cells. FITC, fluorescein isothiocyanate; APC, allophysocyanin.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Expressing, Marker, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: The expressions of stemness genes in OVCAR3 CD44 + CD117 + cells by Western blot (A) and by semiquantitative reverse transcription polymerase chain reaction (B). The amount of cDNA input was adjusted to equalize the expression level of GAPDH. Octamer-binding trascription factor 3/4 (OCT3/4), nanog homeobox (NANOG) and sex determining region Y-box 2 (SOX2) are known to be as stemness genes. The expressions of stemness genes were increased in OVCAR3 CD44 + CD117 + cells than those in OVCAR3. Each band was quantified by densitometric analysis and presented in a bar graph. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: Effect of salinomycin in growth inhibition of ovarian cancer stem-like cells. The cells were exposed to various concentrations of (A) paclitaxel (1, 10, 100 and 200 nM) and (B) salinomycin (0.1, 0.5, 1 and 5 µM) in OVCAR3 and OVCAR3 CD44 + CD117 + cells for 48 hours to evaluate effect in growth inhibition of ovarian cancer stem-like cells. (C) OVCAR3 CD44 + CD117 + cells were treated with paclitaxel (10 nM) and/or salinomycin (0.1 µM) for 48 hours. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Values are mean±standard deviation of three measurements. CTL, control; PTX, paclitaxel; Sal, salinomycin. * P <0.05.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Inhibition, MTT Assay, Standard Deviation, Control

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Activation Assay, Staining, Western Blot, Inhibition

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Membrane, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Disruption, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence