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

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: bioRxiv

Article Title: Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

doi: 10.1101/865394

Figure Lengend Snippet: ( A ) Spindle (top block) in a HeLa cell stably expressing PRC1-GFP (magenta) immunostained for Kif4A-AF-647 (green). Arrows in the enlargements of the boxed region point to Kif4A outside chromosomes, at the positions where PRC1-GFP is found. Spindle (middle block) in a HeLa cell stably expressing Kif18A-GFP (green) and stained for SiR-tubulin (not shown) and spindle (bottom block) in a HeLa cells stably expressing MKLP1-GFP (green) and stained for SiR-tubulin (magenta). Arrows in the enlargements of the boxed regions point to the bridging fiber. ( B ) Time-lapse images of: unlabeled U2OS cell with transient expression of opto-PRC1 (magenta), iLID-CAAX and GFP-Kif4A (green), and stained with SiR-DNA (not shown) (first row); HeLa cell with stable expression of EGFP-CLASP1 (green) with transient expression of opto-PRC1 and iLID-CAAX, and stained with SiR-DNA (second row); unlabeled U2OS cell with transient expression of opto-PRC1, iLID-CAAX and EGFP-Kif18A (green) (third row); unlabeled U2OS cell with transient expression of opto-PRC1, iLID-CAAX and GFP-CENP-E (green) (fourth row); HeLa BAC cell stably expressing MKLP1-GFP (green) and with transient expression of opto-PRC1 and iLID-CAAX, and stained with SiR-DNA (fifth row). All time points are before (0 min, Dark), at the end of continuous exposure (20 min, Light) and 10 min after cessation of exposure to the blue light (30 min, Dark). ( C ) Images of mock (left column) and PRC1 siRNA treated (right column): HeLa BAC cell expressing Kif4A-GFP (green) stained with SiR-tubulin (first row); HeLa cell stably expressing EGFP-CLASP1 (green) stained with SiR-DNA (second row); HeLa BAC cell expressing Kif18A-GFP (green) (third row); HeLa BAC cell expressing CENP-E-GFP (green) stained with SiR-DNA (fourth row); HeLa BAC cell expressing MKLP1-GFP (green) stained with SiR-tubulin (fifth row). ( D ) Spindles from HeLa cells stably expressing PRC1-GFP (green), treated with mock (top), Kif4A (middle), and Kif18A (bottom) siRNA. Graph shows the lengths of PRC1 streaks per treatment. Numbers in brackets; number of measurements and cells, respectively. Images in A (bottom), B , and C are maximum intensity projections of 2-3 z-planes, smoothed with 0.5-pixel-sigma Gaussian blur. Scale bars; 2 µm.

Article Snippet: Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody (1:100; C-1; sc-376983, Santa Cruz Biotechnology), rabbit anti-α-tubulin polyclonal antibody (1:100; SAB4500087, Sigma-Aldrich Corporation, St. Louis, MO, USA), mouse monoclonal anti-Kif4A antibody (1:100; E-8; sc-365144, Santa Cruz Biotechnology), rabbit polyclonal anti-MKLP1 antibody (1:100; N-19; sc-867, Santa Cruz Biotechnology), mouse monoclonal anti-Eg5 antibody (1:100; A-1; sc-365681, Santa Cruz Biotechnology).

Techniques: Blocking Assay, Stable Transfection, Expressing, Staining

Journal: bioRxiv

Article Title: Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

doi: 10.1101/865394

Figure Lengend Snippet: ( A ) Time-lapse of unlabeled U2OS cell with transient expression of opto-PRC1 (magenta), iLID-CAAX and EGFP-Kif18A (green) as in . Note the co-localization of opto-PRC1 and EGFP-Kif18A in the bridging fibers. ( B ) Enlargements of control (left) and PRC1 siRNA treated (right) HeLa BAC cells expressing Kif18A-GFP (green). Note the presence of Kif18A-GFP in the bridging fibers of control cells, and its absence in PRC1 siRNA treated cells (white arrowheads). ( C ) Spindles in fixed HeLa cells stably expressing MKLP1-GFP (yellow) and immunostained for PRC1 (AF-640, magenta), tubulin (AF-560, green) and stained with DAPI (blue) in untreated (top) and PRC1 siRNA treated (bottom) cells (left). All images are sum intensity projections of five z-planes. Graph (right) shows PRC1 intensity in untreated (magenta) and PRC1 siRNA treated (green) cells. Numbers inside bars; number of cells. ( D ) Spindle in a fixed unlabeled untreated HeLa cell (top left) and HeLa BAC cell expressing MKLP1-GFP (bottom left), immunostained for MKLP1 (AF-594, green), PRC1 (AF-647, magenta) and stained with DAPI (not shown). Images are sum-intensity projections of 5 z-planes smoothed with 0.5-pixel-Gaussian blur. Graph (right) shows MKLP1 intensity in untreated unlabeled (magenta) and BAC cells (green). The signal intensity of MKLP1 in these cells was 6% higher compared to endogenous MKLP1. ( E ) Time-lapse of HeLa BAC cell expressing MKLP1-GFP, depleted for endogenous PRC1, with transient expression of opto-PRC1 and iLID-CAAX (only MKLP1-GFP is shown) in time-points as in (top). Graphs (middle) correspond to membrane-to-membrane intensity profiles of MKLP1-GFP (green) and opto-PRC1 (magenta) retrieved from the same position perpendicular to the midzone in time-points as in , acquired by 1-µm wide line on maximum projection of three z-planes. Schematic representation of performed measurement is given on the right. Graphs (bottom) show individual MKLP1-GFP (left) and opto-PRC1 intensities. ( F ) Time-lapse of a cell as in E , which progresses to cytokinesis during time-points as in E . ( G ) Time-lapse images of U2OS cells with transient expression of opto-PRC1 (magenta) and GFP-Kif4A (green). Top (merge tgRFPt and SiR-DNA), bottom (GFP). Anaphase onset is set as time 0 min. ( H ) Spindle in a fixed untreated unlabeled U2OS cell (top) and unlabeled U2OS cell, depleted for endogenous PRC1 and transiently expressing Kif4A-GFP (not shown) and opto-PRC1 (not shown) (middle), immunostained for Kif4A (AF-647, green) and stained with DAPI (blue). Images are sum-intensity projections of 5 z-planes smoothed with 0.5-pixel- Gaussian blur. Graph (bottom) shows Kif4A intensity in untreated (magenta) and cells with transient expression of Kif4A-GFP (green). The signal intensity of Kif4A in these cells was 30% higher compared to endogenous Kif4A. ( I ) Time-lapse of U2OS cell as in (only GFP-Kif4A channel) (top). Graphs (middle) correspond to membrane-to-membrane intensity profiles of GFP-Kif4A (green) and opto-PRC1 (magenta) retrieved from the same position perpendicular to the midzone and in time-points as in , acquired by 1-µm wide line on a single z-plane. Schematic representation of performed measurement is given on the right. Graphs (bottom) show individual GFP-Kif4A (left) and opto-PRC1 (right) intensities. ( J ) GFP-Kif4A intensity on chromosomes in opto (black) and control (gray) cells 4 min after anaphase onset. Anaphase onset is defined as a timeframe of sister chromatid separation. Numbers in brackets; number of cells. Scale bars; 2 µm.

Article Snippet: Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody (1:100; C-1; sc-376983, Santa Cruz Biotechnology), rabbit anti-α-tubulin polyclonal antibody (1:100; SAB4500087, Sigma-Aldrich Corporation, St. Louis, MO, USA), mouse monoclonal anti-Kif4A antibody (1:100; E-8; sc-365144, Santa Cruz Biotechnology), rabbit polyclonal anti-MKLP1 antibody (1:100; N-19; sc-867, Santa Cruz Biotechnology), mouse monoclonal anti-Eg5 antibody (1:100; A-1; sc-365681, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Stable Transfection, Staining, Membrane

Journal: bioRxiv

Article Title: Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

doi: 10.1101/865394

Figure Lengend Snippet: ( A ) We propose that the interactions between k-fibers and bridging fibers regulate the movement of bi-oriented chromosomes by forces that depend on the length of the antiparallel overlaps (purple). Shorter overlaps lead to more precise alignment of kinetochores (cyan) than longer ones. ( B ) If a kinetochore pair is displaced away from the equatorial plane towards one pole, the overlap between the k-fiber and the bridging fiber (purple) is shorter on this side and longer on the opposite side. More motors and/or crosslinkers accumulate in the longer overlap, pulling the kinetochore back to the center ( F , pulling force). The efficiency of centering depends on the relative asymmetry in the overlap length on either side. This asymmetry is larger if the overlap is short, which explains why short overlaps lead to better alignment than long ones (A). ( C ) The overlap length is regulated by Kif4A and Kif18A at the plus ends of bridging microtubules. Kif4A and Eg5 within the bridging fiber possibly slide the microtubules apart, whereas PRC1 stabilizes the overlaps probably together with MKLP1, Eg5, and other crosslinkers.

Article Snippet: Following primary antibodies were used: mouse anti-PRC1 monoclonal antibody (1:100; C-1; sc-376983, Santa Cruz Biotechnology), rabbit anti-α-tubulin polyclonal antibody (1:100; SAB4500087, Sigma-Aldrich Corporation, St. Louis, MO, USA), mouse monoclonal anti-Kif4A antibody (1:100; E-8; sc-365144, Santa Cruz Biotechnology), rabbit polyclonal anti-MKLP1 antibody (1:100; N-19; sc-867, Santa Cruz Biotechnology), mouse monoclonal anti-Eg5 antibody (1:100; A-1; sc-365681, Santa Cruz Biotechnology).

Techniques:

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: