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: