Journal: EMBO Reports

Article Title: UBAP2L ‐dependent coupling of PLK1 localization and stability during mitosis

doi: 10.15252/embr.202256241

Figure Lengend Snippet: A, B Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (A) and quantification of the relative PLK1 intensity at the inner centromere (A.U.) (B). ROIs are shown in the corresponding numbered panels. Scale bar, 5 μm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/INCENP intensity ratio at a single centromere. The measurements of three biological replicates are combined, red bars represent the mean (Mann–Whitney test **** P < 0.0001). C, D Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (C) and quantification of the relative PLK1 intensity at the centrosomes (A.U.) (D). ROIs are shown in the corresponding numbered panels. Scale bar, 5 μm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/Pericentrin intensity ratio at a single centrosome. The measurements of three biological replicates are combined, red bars represent the mean (Mann–Whitney test **** P < 0.0001). E, F Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR (E) and quantification of the relative PLK1 intensity at the midbody (A.U.) (F). ROIs are shown in the corresponding numbered panels. Scale bar, 4 μm. At least 50 cells were quantified per condition for each experiment. Each dot represents PLK1/PRC1 intensity ratio at the midbody. The measurements of three biological replicates are combined, red bars represent the mean (Mann–Whitney test ** P < 0.01). G Representative IF pictures of WT or UBAP2L KO HeLa cells synchronized in mitosis with DTBR ( n = 3) to qualitatively assess the localization of PLK1 at the mitotic spindle with α‐tubulin staining. ROIs are shown in the corresponding numbered panels. Scale bar, 5 μm. Source data are available online for this figure.

Article Snippet: Primary antibodies used in this study are the following: rabbit anti‐UBAP2L (this study), rabbit monoclonal anti‐PLK1 (4513, Cell Signaling Technology), mouse monoclonal anti‐PLK1 (sc‐17783, Santa Cruz Biotechnology), mouse monoclonal anti‐Cyclin B1 (sc‐245, Santa Cruz Biotechnology), rabbit monoclonal anti‐Aurora A (4718, Cell Signaling Technology), mouse monoclonal anti‐Flag M2 (F1804, Sigma‐Aldrich), rabbit polyclonal anti‐GAPDH (G9545, Sigma‐Aldrich), mouse monoclonal anti‐alpha‐tubulin (T9026, Sigma‐Aldrich), rabbit polyclonal anti‐BubR1 (612502, BD Biosciences), human polyclonal anti‐centromere (15‐234‐0001, Antibodies Incorporated), mouse monoclonal anti‐ubiquitin (FK2) (ST1200, Millipore), mouse monoclonal anti‐AIM‐1 (Aurora B) (611083, BD Biosciences), rabbit polyclonal anti‐Lamin B1 (ab16048, Abcam), rabbit polyclonal anti‐phospho‐PLK1 (5472, Cell Signaling Technology), goat polyclonal anti‐PLK4 (NB100‐894, Novus Biologicals), rabbit polyclonal anti‐PLK3 (NBP2‐32530, Novus Biologicals), rabbit polyclonal anti‐PLK2 (GTX112022, GeneTex), rabbit polyclonal anti‐Cyclin A (sc‐751, Santa Cruz Biotechnology), mouse monoclonal anti‐cyclin E (sc‐247, Santa Cruz Biotechnology), rabbit polyclonal anti‐GFP (ab290, Abcam), rabbit anti‐CUL3 (From Sumara et al , ), rabbit polyclonal anti KLHL22 (16214‐1‐AP, Proteintech), mouse monoclonal anti‐ubiquitin (clone P4D1) (3936, Cell Signaling Technology), mouse monoclonal anti‐G3BP1 (611126, BD Biosciences), rabbit polyclonal anti‐G3BP2 (A302‐040A‐M, Thermo Fisher Scientific), rabbit polyclonal anti‐INCENP (2807, Cell Signaling Technology), rabbit polyclonal anti‐pericentrin (ab4448, Abcam), rabbit polyclonal anti‐gamma‐tubulin (T3559, Sigma‐Aldrich), rabbit polyclonal anti‐PRC1 (sc‐8356, Santa Cruz Biotechnology).

Techniques: MANN-WHITNEY, Staining

Journal: PLoS ONE

Article Title: DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

doi: 10.1371/journal.pone.0013064

Figure Lengend Snippet: (A) Western analysis of DAAM1 expression in different cell lines. Total protein lysate (30 µg) were loaded per lane. (B) Domain organization of DAAM1. (C) Left: confocal images of COS-7 cell taken at different planes. Right: co-localization of DAAM1 with centrosomes in prophase (after centrosome duplication) marked by anti-γ-tubulin. White arrowheads indicate centrosomes. (D) Confocal images of U2OS and DAAM1-null H460 cells, as well as COS-7 and COS-7 cells with DAAM1 knockdown by siRNA (COS-7 + siDAAM1). Endogenous DAAM1 was detected by indirect immuno-fluorescence with anti-DAAM1 under identical conditions. (E) Full-length Flag-DAAM1 or Flag-hDia1 (in green) was expressed in COS-7 cells and co-stained for actin (red). Bars = 10 µm.

Article Snippet: Monoclonal anti-α-tubulin, anti-γ-tubulin and rabbit polyclonal anti-gamma tubulin were from Sigma.

Techniques: Western Blot, Expressing, Fluorescence, Staining

Journal: PLoS ONE

Article Title: DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

doi: 10.1371/journal.pone.0013064

Figure Lengend Snippet: (A) Efficacy of two different siRNA against DAAM1 in COS-7 cells by Western analysis at 24 hours and 48 hours post-transfection. (B) COS-7 cells transfected with control (Ctr) or human DAAM1 siRNA (si2318) for 48 hours were scratched and observed using time-lapse imaging for 12 hours. Tracks of individual cells are shown as colored lines. Bar = 40 µm. (C) Wound-edge COS-7 cells subjected to GSK inhibitor (SB216763, 20 µM) and PKC inhibitor (RO-320432, 20 µM), or si2318 (40 pmol) were stained with anti-α-tubulin to visualize the microtubule network. Loss of DAAM1 was associated with random protrusions versus the more organized broad extensions in controls. Wound-edge U2OS cells subjected to siRNA treatment are shown in the bottom panel. Bar = 10 µm. (D) Forward Golgi orientation of COS-7 cells transfected with control (Ctr) or DAAM1 siRNA (si2318 or si2832, 40 pmol) at 0 and 1 hour post-wounding. Cells were scored for Golgi re-orientation using a 120° sector centered on the nucleus as shown on the right. (E) Right: Western analysis of DAAM1 knockdown in U2OS cells. Left: Golgi re-orientation in COS-7 and U2OS cells treated with various inhibitors 1 hour before wounding. PKC inhibitor (PKCi) and GSK inhibitors (GSKi) were used as in (c). In these analyses, cells were scored for Golgi re-orientation in three separate experiments. Error bars indicate standard deviation from the mean.

Article Snippet: Monoclonal anti-α-tubulin, anti-γ-tubulin and rabbit polyclonal anti-gamma tubulin were from Sigma.

Techniques: Western Blot, Transfection, Imaging, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

doi: 10.1371/journal.pone.0112428

Figure Lengend Snippet: (A) Endogenous NA14 and spastin (red) co-localize at the centrosome in HeLa cells, which were co-stained for the centrosomal markers pericentrin or γ-tubulin (green). Merged images are at the right, and boxed areas are enlarged in the insets. Images were acquired using confocal immunofluorescence microscopy, and relative fluorescence intensities for the indicated linear regions in the merged images, measured using Zeiss LSM710 software, are graphed. Note the high degree of line-scan overlap (right). AU, arbitrary units. (B) HeLa cells co-immunostained for NA14 (two different antibodies), spastin and γ-tubulin are shown. Merged images with DAPI nuclear staining are at the right. Boxed areas are enlarged in the upper right-hand corner insets. Scale bar: 10 µm.

Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-spastin (1∶1000; IgG 2a , clone Sp 6C6, Sigma-Aldrich, St. Louis, MO, USA), mouse polyclonal anti-SSNA1 (1∶500; Abnova, Taipei, Taiwan), rabbit polyclonal anti-SSNA1 (1∶1000; Proteintech Group, Chicago, IL, USA), mouse monoclonal anti-c-Myc epitope (1∶1000; IgG 1 , clone 9E10, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-c-Myc epitope (1∶500; A190-104A, Bethyl Laboratories, Montgomery, TX, USA), mouse monoclonal anti-HA probe (1∶1000; IgG 2a , clone F-7, Santa Cruz Biotechnology), rabbit polyclonal anti-HA probe (1∶1000; ab9110-100, Abcam, Cambridge, MA, USA), mouse monoclonal anti-α-actin (1∶1000; clone AC-40, Sigma-Aldrich), mouse monoclonal anti-β-tubulin (1∶2000; IgG 1 , clone D66, Sigma-Aldrich), rabbit polyclonal anti-pericentrin (1∶1000, Abcam), mouse monoclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), rabbit polyclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), mouse monoclonal anti-tau-1 (1∶1000; IgG 2a , clone PC1C6, Chemicon International, Temecula, CA, USA), mouse monoclonal anti-MAP2 (1∶1000; IgG 1 , clone HM-2, Sigma-Aldrich), rabbit polyclonal anti-PLCγ1 (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-ankyrin G (1∶1000; IgG 1 , Life Technologies Invitrogen, Grand Island, NY, USA).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence, Software

Journal: PLoS ONE

Article Title: Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

doi: 10.1371/journal.pone.0112428

Figure Lengend Snippet: (A) Merged images of endogenous NA14 (green) accumulated at the centrosome during interphase and at the midbodies during late cytokinesis, along with β-tubulin (red). (B) Endogenous spastin (green) localizes to the centrosome and the midbodies, as shown by co-staining for β-tubulin (red). Merged images are at the right. (C) HeLa cell lines stably expressing control shRNA (shCTL) or shRNAs against NA14 (sh3 shown) were immunostained for endogenous β-tubulin (green) and γ-tubulin (red), with merged images at the right. Bar: 10 µm. (D) Cell extracts from cell lines stably expressing the indicated shRNAs were immunoblotted for NA14. PLCγ1 (149 kDa) and β-tubulin levels were monitored to control for protein loading. # denotes a cross-reacting band. (E and F) Mitotic and multinucleated cells were quantified in control and NA14 shRNA stable cell lines (means ±SEM; n = 3, with 100 cells per experiment). Nuclei were identified by co-staining with DAPI. (G) Quantification of cell death in control and NA14 shRNA stable cells lines by measuring lactate dehydrogenase release from cells (means ±SEM; n = 3, with 100 cells per experiment). * p <0.05; ** p <0.01.

Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-spastin (1∶1000; IgG 2a , clone Sp 6C6, Sigma-Aldrich, St. Louis, MO, USA), mouse polyclonal anti-SSNA1 (1∶500; Abnova, Taipei, Taiwan), rabbit polyclonal anti-SSNA1 (1∶1000; Proteintech Group, Chicago, IL, USA), mouse monoclonal anti-c-Myc epitope (1∶1000; IgG 1 , clone 9E10, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-c-Myc epitope (1∶500; A190-104A, Bethyl Laboratories, Montgomery, TX, USA), mouse monoclonal anti-HA probe (1∶1000; IgG 2a , clone F-7, Santa Cruz Biotechnology), rabbit polyclonal anti-HA probe (1∶1000; ab9110-100, Abcam, Cambridge, MA, USA), mouse monoclonal anti-α-actin (1∶1000; clone AC-40, Sigma-Aldrich), mouse monoclonal anti-β-tubulin (1∶2000; IgG 1 , clone D66, Sigma-Aldrich), rabbit polyclonal anti-pericentrin (1∶1000, Abcam), mouse monoclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), rabbit polyclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), mouse monoclonal anti-tau-1 (1∶1000; IgG 2a , clone PC1C6, Chemicon International, Temecula, CA, USA), mouse monoclonal anti-MAP2 (1∶1000; IgG 1 , clone HM-2, Sigma-Aldrich), rabbit polyclonal anti-PLCγ1 (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-ankyrin G (1∶1000; IgG 1 , Life Technologies Invitrogen, Grand Island, NY, USA).

Techniques: Staining, Stable Transfection, Expressing, shRNA

Journal: PLoS ONE

Article Title: Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

doi: 10.1371/journal.pone.0112428

Figure Lengend Snippet: (A) Mixed cortical neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (green) and γ-tubulin (red); NA14 localizes to the centrosome at 6DIV. Scale bars: 40 µm. Relative fluorescence intensities for the indicated linear regions in merged images were measured using Zeiss LSM710 software and graphed (at the right). AU, arbitrary units. (B) Neurons at 1, 3 and 6DIV were immunostained for endogenous NA14 (red), neurofilaments (gray) and pericentrin (green). The merged images are indicated. Scale bars: 40 µm.

Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-spastin (1∶1000; IgG 2a , clone Sp 6C6, Sigma-Aldrich, St. Louis, MO, USA), mouse polyclonal anti-SSNA1 (1∶500; Abnova, Taipei, Taiwan), rabbit polyclonal anti-SSNA1 (1∶1000; Proteintech Group, Chicago, IL, USA), mouse monoclonal anti-c-Myc epitope (1∶1000; IgG 1 , clone 9E10, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-c-Myc epitope (1∶500; A190-104A, Bethyl Laboratories, Montgomery, TX, USA), mouse monoclonal anti-HA probe (1∶1000; IgG 2a , clone F-7, Santa Cruz Biotechnology), rabbit polyclonal anti-HA probe (1∶1000; ab9110-100, Abcam, Cambridge, MA, USA), mouse monoclonal anti-α-actin (1∶1000; clone AC-40, Sigma-Aldrich), mouse monoclonal anti-β-tubulin (1∶2000; IgG 1 , clone D66, Sigma-Aldrich), rabbit polyclonal anti-pericentrin (1∶1000, Abcam), mouse monoclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), rabbit polyclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), mouse monoclonal anti-tau-1 (1∶1000; IgG 2a , clone PC1C6, Chemicon International, Temecula, CA, USA), mouse monoclonal anti-MAP2 (1∶1000; IgG 1 , clone HM-2, Sigma-Aldrich), rabbit polyclonal anti-PLCγ1 (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-ankyrin G (1∶1000; IgG 1 , Life Technologies Invitrogen, Grand Island, NY, USA).

Techniques: Fluorescence, Software

Journal: PLoS ONE

Article Title: Spastin-Interacting Protein NA14/SSNA1 Functions in Cytokinesis and Axon Development

doi: 10.1371/journal.pone.0112428

Figure Lengend Snippet: (A) Representative neurons at developmental stages I, II, and III were co-stained for tau-1 (red) and MAP-2 (green). Merged images are at the right. Scale bar: 40 µm. (B) Pie graphs showing the percentages of cortical neurons in stages I, II, and III in each experimental group ( n >100). More neurons remained in stages I and II in untransfected and NA14 (33–119) expressing cultures than in HA-NA14 expressing neurons. (C) β-tubulin staining (black) reveals processes of transfected and control cultured neurons at 3 and 6DIV. Scale bar: 40 µm. (D and E) Quantifications of primary axon length as well as number of primary axon branches in cortical neurons in primary culture (means ±SEM; n = 3, with 30–60 neurons per trial). (F) Numbers of dendrites per cell are shown graphically (means ±SD; n = 3, with 30–60 neurons per trial). * p <0.05, *** p <0.001.

Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-spastin (1∶1000; IgG 2a , clone Sp 6C6, Sigma-Aldrich, St. Louis, MO, USA), mouse polyclonal anti-SSNA1 (1∶500; Abnova, Taipei, Taiwan), rabbit polyclonal anti-SSNA1 (1∶1000; Proteintech Group, Chicago, IL, USA), mouse monoclonal anti-c-Myc epitope (1∶1000; IgG 1 , clone 9E10, Santa Cruz Biotechnology, Dallas, TX, USA), goat polyclonal anti-c-Myc epitope (1∶500; A190-104A, Bethyl Laboratories, Montgomery, TX, USA), mouse monoclonal anti-HA probe (1∶1000; IgG 2a , clone F-7, Santa Cruz Biotechnology), rabbit polyclonal anti-HA probe (1∶1000; ab9110-100, Abcam, Cambridge, MA, USA), mouse monoclonal anti-α-actin (1∶1000; clone AC-40, Sigma-Aldrich), mouse monoclonal anti-β-tubulin (1∶2000; IgG 1 , clone D66, Sigma-Aldrich), rabbit polyclonal anti-pericentrin (1∶1000, Abcam), mouse monoclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), rabbit polyclonal anti-γ-tubulin (1∶1000; Sigma-Aldrich), mouse monoclonal anti-tau-1 (1∶1000; IgG 2a , clone PC1C6, Chemicon International, Temecula, CA, USA), mouse monoclonal anti-MAP2 (1∶1000; IgG 1 , clone HM-2, Sigma-Aldrich), rabbit polyclonal anti-PLCγ1 (Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-ankyrin G (1∶1000; IgG 1 , Life Technologies Invitrogen, Grand Island, NY, USA).

Techniques: Staining, Expressing, Transfection, Cell Culture