Journal: Scientific Reports

Article Title: CRISPR-mediated gene targeting of CK1δ/ε leads to enhanced understanding of their role in endocytosis via phosphoregulation of GAPVD1

doi: 10.1038/s41598-020-63669-2

Figure Lengend Snippet: Intracellular localization of endogenous CK1δ-mNG and CK1ε-mNG. ( A–C ) Representative images of fixed HEK293 cells at indicated cell cycle stages producing CK1δ-mNG or CK1ε-mNG stained with ( A ) DAPI and anti-γ-tubulin, ( B ) DAPI and anti-γ-tubulin, or ( C ) DAPI and anti-MKLP1 antibodies. Scale bars, 10 μm. Insets correspond to centrosomes in A and B or the midbody in C. Scale bars, 0.5 μm. ( D ) Representative single z-sections of live-cell images of HEK293 CK1δ-mNG and CK1ε-mNG cells. Yellow arrows indicate examples of vesicle-like structures. Scale bars, 10 μm.

Article Snippet: Cells were incubated with antibodies against γ-tubulin (Sigma-Aldrich, GTU88; 1:500), Flag M2 (Sigma-Aldrich, 1:1500), or MKLP1 (Bethyl laboratory, 1:1000).

Techniques: Staining

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 1. Increasing the concentration of PEG6000 distorts the MKLP1-GFP signal and MBR structure (A) Cleared conditioned medium from HeLa-MKLP1-GFP cells was incubated with increasing concentrations of PEG6000 and settled onto coverslips. Increasing PEG concentration resulted in decreased transparency. The scale bar represents 5 mm. (B) Higher PEG6000 concentrations caused an increased, blurred fluorescent background. The coverslips were imaged at 203 magnification on a Revolve microscope. The scale bar represents 100 mm. (C) MBRs isolated from HeLa-MKLP1-GFP cell culture medium by increasing PEG6000 concentrations imaged at 1003 magnification by N-SIM Z projection. The scale bar represents 10 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Concentration Assay, Incubation, Microscopy, Isolation, Cell Culture

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 2. Isolated MBRs are midbodies (MB) formed during the late telophase stage of mitosis (A) Representative images of MBRs isolated from HeLa-CCL-2 cells stained with anti-MKLP1 (magenta) and anti-CD9 (cyan). Images were taken at 603 magnification on a confocal microscope. The scale bar represents 24 mm, and the inset represents an 83 enlargement of the dotted square. The same slide images of isolated MBRs from HeLa-CCL-2 cell culture labeled with anti-MKLP1 (Magenta) and anti-CD9 (Cyan) were taken at 1003 magnification on an N-SIM microscope. The scale bar represents 10 mm, and the inset represents a 43 enlargement of the dotted square. (B) Representative images of mitotic HeLa-CCL-2 cells labeled with anti-MKLP1 (green), anti-ɑ-tubulin 555 (red), and DAPI (gray). The green midbody in the first image attaches two daughter cells in the final stages of mitosis. Green MBR is taken up by a cell in interphase post-mitosis. Images were taken at 1003 magnification on an N-SIM microscope. The scale bar represents 10 mm. (C) Representative images of isolated MBRs from HeLa-CCL-2 cells using transmission electron microscopy confirm the electron-dense property of MBs at 40,0003 magnification. The scale bar represents 500 nm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Isolation, Staining, Microscopy, Cell Culture, Labeling, Transmission Assay, Electron Microscopy

Journal: STAR protocols

Article Title: A protocol for isolating and imaging large extracellular vesicles or midbody remnants from mammalian cell culture.

doi: 10.1016/j.xpro.2023.102562

Figure Lengend Snippet: Figure 3. Comparison of GOLD PEG5000 nanoparticle concentrations in the isolation of midbody remnants (MBRs) (A) HeLa CCL-2 clear conditioned medium was incubated with decreasing concentrations of PEG-coated gold nanoparticles overnight at 4C. MBRs were stained with anti-MKLP1 and imaged at 203 magnification. The scale bar represents 90 mm. (B) HeLa MKLP1-GFP clear conditioned medium was incubated with PEG5000-coated gold nanoparticles at a 1:5000 (v/v) dilution overnight at 4C. MBRs were stained with anti-CD9 and imaged at 203 magnification. The scale bar represents 90 mm. (C) HeLa MKLP1-GFP clear conditioned medium was incubated with PEGylated gold nanoparticles at a 1:5000 dilution (v/v) overnight at 4C. MBRs were stained with anti-CD9 and imaged at 1003 magnification. The scale bar represents 10 mm. Inset shows a 103 zoomed view of the MBR.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-MKLP1 Novus Biologicals Cat#NBP2-56923 Mouse anti-CD9 Santa Cruz Biotechnology Cat#SC-13118 Mouse anti-a-tubulin Alexa Fluor 555 Millipore Sigma Cat#05-829X-555 Alexa Fluor 488 goat anti-mouse IgG Jackson ImmunoResearch Cat#115-545-003 Alexa Fluor 647 goat anti-rabbit IgG Jackson ImmunoResearch Cat#111-605-003 Chemicals, peptides, and recombinant proteins DMEM/F12 medium Gibco Cat#11330-057 DMEM Gibco Cat#11965092 Fetal bovine serum (FBS) Fisher Scientific Cat#26140079 Penicillin/Streptomycin Life Technologies Cat#15140-122 Geneticin Gibco Cat#10131-035 Phosphate-buffered saline (PBS) no calcium no magnesium Gibco Cat#14190250 0.25% Trypsin-EDTA (1X) Gibco Cat#25200-056 Poly(ethylene glycol), BioUltra, 6000 (PEG6000) Millipore Sigma Cat#81253 Gold nanoparticles, 30 nm diameter, methyl terminated, PEG 5000 coated Sigma-Aldrich Cat#765732 Poly-L-lysine solution, mol. wt.

Techniques: Comparison, Isolation, Incubation, Staining

Journal: bioRxiv

Article Title: A persistent intracellular bridge and cell cycle misregulation enable polar body cell divisions and tumor formation in Mos -deficient eggs

doi: 10.1101/2025.08.26.672293

Figure Lengend Snippet: (A-D) Representative immunofluorescence images of wildtype (A) and mos −/− eggs (B-D) showing localization of MKLP1 (magenta), tubulin (green), and DNA (white). Arrowheads indicate structure features of MKLP1. Wildtype and mos −/− oocytes were fixed 9h-11h30min after prophase I release. Scale bars, 10μm. (E) Percentage of wildtype and mos −/− eggs with normal or defective MKLP1 structure (n indicates the number of eggs imaged per condition; 3 or more independent experiments conducted per condition; ****, P < 0.0001, two-tailed Fisher’s exact test).

Article Snippet: The primary antibodies used were rabbit anti-MKLP1 polyclonal antibody (Novus NBP2–56923; 1:100) and rabbit anti-PRC1 polyclonal antibody (Proteintech 15617–1-AP; 1:100).

Techniques: Immunofluorescence, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Identification of Overexpressed Genes in Malignant Pleural Mesothelioma

doi: 10.3390/ijms22052738

Figure Lengend Snippet: Multivariate Cox analysis of the three gene signatures: ( A ) unreduced; ( B ) FDR-reduced; ( C ) GO-reduced.

Article Snippet: The membranes were blocked with 5% milk TBST and probed overnight at 4 °C with the specific primary antibody: anti-CIT rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-CTHRC1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-E selectin rabbit polyclonal antibody (1:750; Proteintech, Rosemont, IL, USA); anti-Midkine (MDK) rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-SPARC rabbit polyclonal antibody (1:750; Proteintech, Rosemont, IL, USA); anti-TRAF2 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-UHRF1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-DSC3 mouse polyclonal antibody (1:500; Genetex, Irvine, CA, USA); anti-KIF23 rabbit polyclonal antibody (1:500; OriGene, Rockville, MD, USA); anti-PRSS23 rabbit polyclonal antibody (1:500; Abcam, Cambridge, MA, USA); anti-ADAMTS1 rabbit polyclonal antibody (1:500; GeneTex, Irvine, CA, USA); anti-PODXL rabbit polyclonal antibody (1:500; Proteintech, Rosemont, IL, USA); anti-BAG2 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-TNNT1 rabbit polyclonal antibody (1:1000; Proteintech, Rosemont, IL, USA); anti-MAD2L1 rabbit polyclonal antibody (1:800; Proteintech, Rosemont, IL, USA).

Techniques:

Journal: bioRxiv

Article Title: Evolutionarily conserved midbody reorganization precedes ring canal formation during gametogenesis

doi: 10.1101/2022.06.03.494691

Figure Lengend Snippet: (A) Stills from a time-lapse movie of a germarium expressing Pav::GFP and Histone::RFP. In the plane of focus are two actively dividing cells of a four-cell cyst; the planes of division are oriented perpendicular to each other as depicted in the schematic at the lower right of t=0 minutes. Arrowheads indicate the progression of cleavage furrows to ring canals. 1.2% of Pav-labeled structures are midbodies. B) Mouse testis sections were fixed and stained to detect microtubules (MTs, green), MKLP1 (magenta), and DNA (blue). (B-B’) A dividing germ cell at anaphase. (C-C’) MKLP1-labeled midbodies are present at the intercellular bridge connected segregated sister chromatids; 2.4% (n=242) of MKLP1-positive structures are midbodies. (D-D’) Nascent ring canals (arrowheads) are marked by the association of open ring canal with luminal microtubules. (E-F’) Hydra testes were fixed and stained to detect KIF23 (magenta) and DNA (blue). (E-E’) A dividing spermatocyte with enrichment of KIF23 at the intercellular bridge (arrowhead). (F-F’) Ring canals (“RC”) and a midbody (“M”) are present in the field of view; 3.2% (n=572) of KIF23-labeled structures in dividing cells appeared as midbodies

Article Snippet: The following primary antibodies were used: 1:200 rabbit anti-MKLP1 (clone 7C9, Bioss Antibodies, cat. BSM-52401R) and 1:10 mouse anti-alpha Tubulin (clone 4A1, Developmental Studies Hybridoma Bank).

Techniques: Expressing, Labeling, Staining

Journal: PLoS ONE

Article Title: Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/C Cdh1 Complex

doi: 10.1371/journal.pone.0057532

Figure Lengend Snippet: (A) HeLa cells were transfected with vectors expressing Gas2l3-EGFP, Gas2l3-DM4-EGFP, or EGFP (control). Thirty-two hrs post-transfection, cells were fixed and immunolabeled, as described in . The slides were scanned using a Zeiss AxioImager upright microscope and 100X oil lens for imaging. Transfected (green) cells were counted in order to detect 500 positive daughter cell pairs connected by a midbody (positives). The proportion of attached daughter cells equals 500/ N , where N = the number of transfected cells ( N - 500 = the number of negatives). (B) HeLa cells were transfected with vectors expressing EGFP-tagged Gas2l3 (wt) or Gas2l3-DM4. Thirty-two hours post-transfection, cells were fixed, immunolabeled with anti-MKLP1 (stembody marker) and anti-Tubulin, and stained with DAPI. We used a Zeiss AxioImager Z1 upright microscope and 100X oil lens for imaging.

Article Snippet: The following primary antibodies were used for IF: rabbit polyclonal anti-hGas2l3 (serum, custom-made by Covance), mouse monoclonal and rabbit polyclonal anti-Aurora B (Abcam, ab3609, ab2254), rabbit polyclonal MKLP1 (Gene Tex, GTX30315), mouse monoclonal anti-αTubulin (Abcam, ab7291), mouse monoclonal anti-FLAG® M2 (Sigma-Aldrich, F3165), and mouse monoclonal anti-Myc (DSHB, 9E10).

Techniques: Transfection, Expressing, Immunolabeling, Microscopy, Imaging, Marker, Staining