Journal: eLife

Article Title: A functional topography within the cholinergic basal forebrain for encoding sensory cues and behavioral reinforcement outcomes

doi: 10.7554/eLife.69514

Figure Lengend Snippet:

Article Snippet: Other , DAPI stain , Vectorlabs , Cat #: H-1500–10RRID: AB_2336788 , .

Techniques: Recombinant, Software, Staining

Journal: eLife

Article Title: Corticohippocampal circuit dysfunction in a mouse model of Dravet syndrome

doi: 10.7554/eLife.69293

Figure Lengend Snippet:

Article Snippet: Other , DAPI stain , Thermo Fisher Scientific Cat# D1306 , RRID: AB_2629482 , (1:50,000).

Techniques: Recombinant, Plasmid Preparation, In Vivo, Software, Imaging, Staining

Journal: eLife

Article Title: PLK4 drives centriole amplification and apical surface area expansion in multiciliated cells

doi: 10.7554/eLife.80643

Figure Lengend Snippet:

Article Snippet: Other , DAPI Stain , MilliporeSigma , Cat. no. 10236276001 , (IF) use 1:1000.

Techniques: Staining

Journal: eLife

Article Title: Dorsal raphe nucleus to anterior cingulate cortex 5-HTergic neural circuit modulates consolation and sociability

doi: 10.7554/eLife.67638

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Article Snippet: Other , DAPI stain , Boster , AR1177 , 1 μg/ml.

Techniques: Injection, Concentration Assay, Expressing, Transfection, Construct, Software, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: Loss of IFT20 in mouse breast cancer cells 4T1 induces lamellipodia formation and epithelial mesenchymal transitions (EMTs). (A) Representative immunostaining of 4T1 and mouse embryo fibroblast (MEF) cells with Ac-α-tubulin antibodies showed that no cilia were formed in 4T1 cells, unlike cilia-positive MEF cells, when treated with serum starvation for 36 h. (B) Western blots of cell lysates from 4T1 and IFT20-knockout mutants (B13 and A24) probed with IFT20 antibodies. (C) Phase-contrast photographs of 4T1 and IFT20-KO cells showed that loss of IFT20 resulted in cell morphology changes from a cobblestone appearance into spindle-like shapes. The lower panels showed the higher magnification photographs of the insets in the top panels, respectively. (D) Representative fluorescent images of F-actin stained with rhodamine B-conjugated phalloidin showed that the loss of IFT20 induced the formation of more lamellipodia. The white arrows indicate the lamellipodia. The nucleus is stained by DAPI. (E) Quantification of the cells with lamellipodia in 30 randomly selected cells in each group of (D) . Results are representative of three independent experiments. (F) Representative immunostaining images of 4T1 and IFT20-KO cells stained with the epithelial marker (E-cadherin) and mesenchymal marker (vimentin) showed that the loss of IFT20 induced EMTs. (G) Western blots of cell lysates from 4T1 and IFT20-KO cells probed with epithelial and mesenchymal markers showed that the loss of IFT20 resulted in the down-regulation of E-cadherin but the up-regulation of vimentin. β-tubulin was used as the loading control. All immunostaining experiments were performed three times. The nucleus is stained by DAPI (blue). Scale bar, 5 μm (A) ; 50 μm ( C top panel); 10 μm ( C lower panel, D,F ). Error bars represent standard deviations. The p -values indicated were calculated by Student's t -tests (unpaired). n.s. (not significant) p > 0.05; * p ≤ 0.05; ** p ≤ 0.01.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Immunostaining, Western Blot, Knock-Out, Staining, Marker, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: IFT20 localizes at the trans-Golgi, TGN, and in the post-Golgi vesicles. (A) Representative fluorescent images of 4T1 cells expressing GT-mCherry and stained with IFT20 antibodies showed that IFT20 localized at the Golgi. (B) Representative fluorescent images of 4T1 cells expressing GT-EGFP and GT-mCherry showed that GT-EGFP labels the middle-Golgi and GT-mCherry labels the middle-Golgi, trans-Golgi, and some dot signals; arrow indicates the dot signal of GT-mCherry in cell protrusion. (C) Schematic illustrating the markers used to distinguish the middle-Golgi and trans-Golgi based on their sensitivity to pH. EGFP and mCherry fluorophores were directed to the lumen of the middle-Golgi and trans-Golgi by fusing with the Golgi targeting domain of β-1,4-galactosyltransferase (GT). The fluorescence of EGFP disappeared as a result of the lower pH at the lumen of the trans-Golgi compared with middle-Golgi, whereas mCherry fluorescence was stable. In 4T1 cells, GT-mCherry was also found at the TGN and in the post-Golgi vesicles. (D) Representative fluorescent images of 4T1 cells expressing GT-EGFP and IFT20-mCherry showed that IFT20 did not localize at the middle-Golgi; arrow indicates the dot signal of IFT20-mCherry in cell protrusion. (E) Representative fluorescent images of 4T1 cells expressing IFT20-EGFP and GT-mCherry showed that IFT20 colocalized with GT-mCherry not only at the trans-Golgi/TGN, but also in the post-Golgi vesicles. (F,G) Magnified views of the indicated insets in (E) showing complete colocalization (F) or no-colocalization (G) between IFT20-EGFP and GT-mCherry post-Golgi vesicles. The white dotted lines mark the entire cell profile. (H) Representative time-lapse images showing dynamic transport of IFT20-EGFP in cell protrusion. S, second. (I) The kymograph from a representative dot signal of IFT20-EGFP indicated by the arrows in (H) . Yellow dashed lines indicate the track of IFT20-EGFP. Horizontal scale bar is 5 μm; vertical bar is 10 s. All fluorescent experiments were performed two (A,H) or three times (B,D,E) . The nucleus was stained by DAPI (blue). Scale bar, 5 μm.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Expressing, Staining, Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: IFT20 mediates the vesicle transport from the TGN to the plasma membrane. (A) Fluorescent images of 4T1 cells co-expressing the Golgi-to-plasma membrane Rab markers Rab8a-mCherry (a) or EGFP-Rab10 (b) with IFT20-EGFP/mCherry. (B) The localization pattern of the early endosome Rab marker mCherry-Rab5a with IFT20-EGFP in 4T1 cells. (C) Representative fluorescent images of 4T1 cells co-expressing the recycle endosome Rab markers EGFP-Rab11a (a) and EGFP-Rab11b (b) with IFT20-mCherry. (D) The localization of the late endosome-associated Rab markers EGFP-Rab7 (a) or EGFP-Rab9 (b) with IFT20-mCherry in 4T1 cells. (E) Fluorescent images of 4T1 cells co-expressing the Golgi-to-early endosome Rab marker EGFP-Rab31 with IFT20-mCherry. (F) The localization quantification (Pearson's R value) of IFT20 and Rab proteins in (A–E) . Data are recorded in and represented as the mean ± SD with three different optical sections on 21 cells co-expressing fluorescent proteins in three independent experiments. (G–I) Strep-pulldown assay of IFT20-Strep and Rab8a-mCherry (G) , EGFP-Rab7 (H) , or EGFP-Rab9 (I) . HEK293T cells were transfected with plasmids that express Rab8a-mCherry (G) , EGFP-Rab7 (H) , or EGFP-Rab9 (I) combined with or without IFT20-Strep expression plasmid. After 24 h, the cells were lysed and centrifuged. The supernatants (input) were incubated with Strep-Tactin beads, and the proteins bound to the beads (pulldown) were analyzed by Western blot using anti-Strep tag or anti-mCherry/EGFP antibodies. The white dotted lines mark the entire cell profile. The nucleus is stained by DAPI (blue). Scale bar, 5 μm.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Clinical Proteomics, Membrane, Expressing, Marker, Transfection, Plasmid Preparation, Incubation, Western Blot, Strep-tag, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: Identification of IFT20 interactors using the BioID method. (A) Western blots from lysates of 4T1 cells expressing IFT20-BirA*-Myc or BirA*-Myc probed with IFT20 and Myc antibodies. The arrow points to the anticipated band of IFT20-BirA*-Myc. The arrowhead points to the band of slight truncation of IFT20-BirA*-Myc. (B) Western blots from lysates of 4T1 cells expressing IFT20-BirA*-Myc or BirA*-Myc probed with HRP-streptavidin with/without biotin addition; the results showed that the amount of biotinylated proteins increased when biotin was added. The asterisks indicate the specified bands with IFT20-BirA*-Myc in the presence of biotin. The numbers below represent different columns. (C) Representative immunofluorescence of 4T1 cells expressing BirA*-Myc in the presence of biotin probed with Myc antibodies to show the cytosol localization of BirA*-Myc and probed with streptavidin 568 to show the biotinylated proteins. (D) Representative immunofluorescence images of 4T1 expressing IFT20-BirA*-Myc in the presence of biotin probed with Myc antibodies to show the localization of fusion proteins and probed with streptavidin 568 to show the localization of biotinylated proteins. The top panels show the Golgi localization, while the lower two panels (representing the single Z-stack and max projection with five Z-stacks) show the vesicle-like localization of IFT20-BirA*-Myc and biotinylated proteins. The white dotted lines mark the entire cell profile. The white arrows indicate the vesicle-like distribution near the plasma membrane of IFT20-BirA*-Myc. The z-axis series of optical sections were performed at 0.8 μm-thick sections. The β-tubulin was used as the loading control in the Western blots. All immunostaining experiments were performed two times. The nucleus is stained by DAPI (blue); scale bar, 5 μm.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Western Blot, Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Control, Immunostaining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: IFT20 is involved in transport Numb and Ctnnal1 from the TGN to the plasma membrane. (A) Representative fluorescent images of 4T1 cells co-expressing IFT20-EGFP/mCherry and Numb-mCherry or Wwox-EGFP showing that IFT20 colocalized with Numb and scarcely colocalized with Wwox at the perinuclear region. (B) The respective distribution of GT-EGFP/GT-mCherry and Numb-mCherry or Wwox-EGFP showed that the perinuclear localization of Numb was trans-Golgi/TGN and Wwox localized at the cis-Golgi. (C) Representative fluorescent images of 4T1 cells co-expressing IFT20-EGFP and Ctnnal1-mCherry showed that IFT20 colocalized with Ctnnal1 not only at the Golgi, but also in the intracellular vesicles. The white dotted line marks the entire cell profile. The white dashed rectangular box marks the zoomed images on the bottom. (D–F) Strep-pulldown assay of IFT20-Strep and Numb-mCherry (D) , EGFP-Wwox (E) , or Ctnnal1-mCherry (F) . HEK293T cells were transfected with plasmids that express Numb-mCherry (D) , EGFP-Wwox (E) , or Ctnnal1-mCherry (F) combined with or without IFT20-Strep expression plasmid. After 24 h, the cells were lysed and centrifuged. The supernatants (input) were incubated with Strep-Tactin beads, and the proteins bound to the beads (pulldown) were analyzed by Western blot using anti-Strep tag or anti-mCherry/EGFP antibodies. (G,H) Quantification of the migration efficiency in the wound healing assay (G) and transwell assay (H) ; results showed that knockdown of Numb and Ctnnal1 enhanced breast cancer cell migration. All quantifications were carried out in three independent experiments, and data are expressed as the mean ± S.D.; n.s. (not significant) p > 0.05; * p ≤ 0.05; ** p ≤ 0.01. All fluorescent experiments were performed three times. The nucleus is stained by DAPI (blue); scale bar, 5 μm.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Clinical Proteomics, Membrane, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Strep-tag, Migration, Wound Healing Assay, Transwell Assay, Knockdown, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: IFT20 Mediates the Transport of Cell Migration Regulators From the Trans-Golgi Network to the Plasma Membrane in Breast Cancer Cells

doi: 10.3389/fcell.2021.632198

Figure Lengend Snippet: Interactions of IFT20 with the F-actin associated protein Tagln2 regulates the migration of breast cancer cells. (A) Representative fluorescent images of 4T1 cells co-expressing IFT20-EGFP and Tagln2-mCherry showed that IFT20 co-localizes with Tagln2. Arrow indicates the signal of Tagln2-mCherry at the cell projection. (B) Representative fluorescent images of 4T1 cells expressing Tagln2-mCherry and Lifeact-EGFP showing the F-actin localization of Tagln2. (C) Representative fluorescent images of 4T1 cells co-expressing GT-EGFP and Tagln2-mCherry showing the adjacent localization of Tagln2-mCherry to GT-EGFP. (D) Strep-pulldown assay of IFT20-Strep and Tagln2-mCherry. HEK293T cells were transfected with plasmids that express Tagln2-mCherry combined with or without IFT20-Strep expression plasmid. After 24 h, the cells were lysed and centrifuged. The supernatants (input) were incubated with Strep-Tactin beads, and the proteins bound to the beads (pulldown) were analyzed by Western blot using anti-Strep tag or anti-mCherry antibodies. (E) Western blots of cell lysates from 4T1 and IFT20-knockout mutants (B13 and A24) probed with Tagln2 antibodies showing the downregulation of Tagln2 in IFT20-KO cells. β-tubulin was used as the loading control. (F,G) Quantification of the migration efficiency in the wound healing (F) and transwell (G) assays showed that the downregulation of Tagln2 significantly enhanced cell migration. (H) A working model of IFT20-associated vesicles transporting Numb and Ctnnal1 to the plasma membrane in breast cancer cells. (a) Trans-Golgi/TGN localized IFT20 is involved in the vesicle transport of Numb and Ctnnal1 from the Golgi to the plasma membrane, which is overlapped with the Rab8a-positive trafficking pathway. (b) The amount of Numb and Ctnnal1 decreased at the plasma membrane because of the loss of IFT20, which enhances the migration of 4T1 cells. (c) IFT20 interacts with F-actin-associated protein Tagln2; loss of IFT20 causes the downregulation of Tagln2, which enhances the migration of 4T1 cells. (d) A key showing the shapes used in the illustration and their corresponding cellular components. All quantitative graphs were constructed with data from three independent experiments and date are expressed as the mean ± S.D.; n.s. (not significant) p > 0.05; * p ≤ 0.05; ** p ≤ 0.01. All fluorescent experiments were performed three times. The nucleus was stained by DAPI (blue); scale bar, 5 μm.

Article Snippet: Other staining reagents included DAPI (4′,6-diamidino-2-phenylindole) (Beyotime), Golgi Tracker (Beyotime), and rhodamine phalloidin (Sigma).

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Strep-tag, Knock-Out, Control, Clinical Proteomics, Membrane, Construct, Staining

Journal: eLife

Article Title: Domain fusion TLR2-4 enhances the autophagy-dependent clearance of Staphylococcus aureus in the genetic engineering goat

doi: 10.7554/eLife.78044

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Article Snippet: Other , DAPI stain , Solarbio , Cat#: C0065 , 10 μg/ml.

Techniques: Bacteria, Transfection, Construct, Sequencing, Control, Extraction, Software, Staining

Journal: eLife

Article Title: Diverse inhibitory projections from the cerebellar interposed nucleus

doi: 10.7554/eLife.66231

Figure Lengend Snippet:

Article Snippet: Other , DAPI stain , Advanced Cell Diagnostics , Cat#: 323,108 , .

Techniques: Mutagenesis, Knock-In, Recombinant, Virus, RNAscope, Multiplex Assay, Sequencing, Staining