Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: A) Confocal immunofluorescence images of Hela cells depicting the localization of LIC1 (red) and LIC2 (green), shown by arrows. B) Fluorescence images of Hela cells showing the presence of LIC2 (green, using the ThermoScientific antibody) at kinetochores (CREST, red) in prometaphase. C) Fluorescence imaging after treatment of cells with anti-LIC2 siRNA, showing loss of LIC2 signal from spindle poles and kinetochores (arrow in inset). DAPI was used to visualize chromosomes. Scalebar is 5 μm in all images that are not zoomed.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Immunofluorescence, Fluorescence, Imaging

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: A) Metaphase index (% of total cells present in metaphase +/- SD) in Hela cells treated with respective siRNAs as indicated. (3 experiments, n = approximately 500 cells per experiment). B) & C) Western blots showing siRNA mediated LIC1 and LIC2 specific knockdown. LIC2a and LIC2b represent two different siRNA sequences against human LIC2 . The band marked by the arrow represents human LIC2 that gets reduced upon siRNA treatment, the upper bands are non-specifically recognized by the LIC2 antibody (ThermoScientific). Actin = loading control. D) Rescue of the LIC2-depletion induced metaphase arrest by transgenic expression of rat LIC2 (3 experiments, n = at least 500 cells per experiment). E) Western blots showing specific depletion of LIC2 upon treatment with LIC2-specific siRNAs, using a different LIC2 antibody (Abcam). LIC2a and LIC2b represent two different siRNA sequences against human LIC2 . The band marked by the arrow represents human LIC2 that appears at the same molecular weight as in C.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Western Blot, Knockdown, Control, Transgenic Assay, Expressing, Molecular Weight

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: Representative images showing accumulation of SAC proteins A) Mad1, C) Zw10 and F) BubR1 at metaphase kinetochores upon prolonged metaphase arrest following indicated siRNA treatments. B, D, E, G) Left panels: integrated fluorescence intensities of SAC proteins normalized to the respective kinetochore (CREST) intensities for B) Mad1, D) Zw10, E) Mad2 and G) BubR1 at metaphase kinetochores. Y-axis = mean fluorescence intensity (+/- SEM) from 3 independent experiments, n = at least 18 metaphase cells per experiment for LIC1 and LIC2 depletion. Right panels: fold increase in normalized fluorescence intensity over control (GFP siRNA) for the various SAC proteins. Scalebar is 5 μm in all images.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Fluorescence, Control

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: Novel functions of mitotic LIC2 uncovered from this study in silencing the spindle assembly checkpoint (SAC) are shown. LIC2-dynein strips attachment sensing SAC proteins (Mad1, Mad2, Zw10) from metaphase kinetochores, like LIC1-dynein. LIC2-dynein has the additional capability of causing removal of tension-sensing SAC protein BubR1, which is lacking in LIC1-dynein. The model offers a possible mechanistic explanation for the differential effects of the two LICs in mediating metaphase to anaphase progression.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques:

Journal: bioRxiv

Article Title: Microtubule association of TRIM3 revealed by differential extraction proteomics

doi: 10.1101/2023.07.27.549915

Figure Lengend Snippet: A: U2OS cells were transfected with 1 µ g DNA of GFP-LGALSL for 21 h. Cells were then treated with 6 μM Nocodazole (Noco, 1 h) or 6 µ M Taxol (30 min) alongside a DMSO-only treated control sample. Lysis and microtubule stabilisation buffer (LMS) was then added for 5 min at 4°C. Cells were fixed with ice-cold methanol before and after LMS addition and stained for α-Tubulin. Visualisation was performed with a 3i spinning disk confocal microscope, Plan-Apochromat 63x/1.4NA Oil Objective M27, scale bar = 10 μm. B: U2OS cells were treated with either 6 μM Nocodazole for 1 h or Taxol for 30 min alongside a DMSO-only treated control sample. Lysis and microtubule stabilisation buffer (LMS) was then added (5 min, 4 °C) to remove cytosolic proteins (Cytosol). Microtubules were then collected using 8 M urea (MT). Western blot analysis of α-Tubulin, GAPDH and DYNC1LI1. Sample proportions are as indicated. C: U2OS cells were treated with either 6 μM Nocodazole for 1 h or Taxol for 30 min alongside a DMSO control. LMS buffer was either added (5 min, 4 °C) before cells were fixed in ice-cold methanol before and after LMS addition and stained for dynein (DYNC1LI1) and Tubulin, scale bar = 10 μm.

Article Snippet: The following primary antibodies were used for western blotting at the respective concentrations: α-Tubulin (Sigma, T5168, 1:10,000), acetylated α-Tubulin (Sigma, T6793, 1:1000), ATAT1 (Proteintech, 28828-1-AP, 1:500), Detyrosinated α-Tubulin (Abcam, ab32386, 1:1000), DYNC1LI1 (Atlas, HPA035013, 1:1000), EML4 (Cell Signaling Technology, 12156S, 1:1000), GAPDH (Cell Signalling Technology, 2118S, 1:1000).

Techniques: Transfection, Control, Lysis, Staining, Microscopy, Western Blot

Journal: Oncotarget

Article Title: The mitotic tensegrity guardian tau protects mammary epithelia from katanin-like1-induced aneuploidy

doi: 10.18632/oncotarget.10728

Figure Lengend Snippet: A. RFL-6 cells were transfected with wild-type KL1 (KL1wt), KL1 (L123V), oncogenic Ras (Rasmt), oncogenic ErbB2 (ErbB2mt), or KL1(L123V)(L288V). The transformation efficiency was measured by the number of FFU ( n = 4). B. Focus assay of stable tau or TC expressing cells. ( n = 3) C. Morphological and growth characteristics of stable KL1 expressing cells growing in monolayers. Empty vector control (Ctrl), KL1wt, KL1(L123V), and Rasmt stable lines were established. The Rasmt line showed spontaneous formation of larger foci which consisted of refractile and fusiform cells in criss-crossed arrays (Rasmt: lower part). KL1(L123V) expressers showed spontaneous foci formation with massive piling up into virtually opaque multilayers. KL1wt cells showed heterogeneity; altered slender and long cells appeared. Bar, 200 μm. D. KL1wt, KL1(L123V), KL1(L123V)(L288V), KL1(L123V) and Tau, Tau, and Rasmt stable lines were plated in soft agar and macroscopic colonies were counted ( n = 3). E. KL1wt cells showed small-sized colony formation. KL1(L123V) induced colonies comparable to those by Rasmt in size. Bar, 100 μm. Arrows indicate the small-sized colonies. F. KL1 protein expression in KL1(L123V)-induced colony-derived cells (Colony d1 and d2) was analyzed by western blot. β-actin was used as a loading control. The asterisks and double asterisks indicate significant differences (Student's t -test, P < 0.01 and P < 0.05, respectively).

Article Snippet: Rabbit polyclonal antibodies against KATNAL1 (KL1) (HPA046205, Sigma, St. Louis, MO, USA), dynein (DYNC1LI1) (GTX120114, GeneTex, Irvine, CA), Eg5 (KIF11) (GTX109054, GeneTex), Neuron specific β-Tubulin III (GenScript, Piscataway, NJ), H-Ras (GeneTex), ErbB2 (GeneTex), GFP (Sigma), Flag (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Transfection, Transformation Assay, Expressing, Plasmid Preparation, Derivative Assay, Western Blot