Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 1. An in silico bioinformatics screen identifies mitotic proteins essential for EWS growth. (A) The DepMap portal was used to access the RNA expression data across different cancer cell lines. Expression in Ewing sarcoma is highlighted in red. Capillary-based analysis of protein lysates from EWS cell lines indicating expression of (B) KIF11 and AURKA and (C) KIF15 and TPX2 protein levels. The uncropped blots are shown in Figure S3.

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: In Silico, RNA Expression, Expressing

Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 2. Synergistic inhibition of EWS growth by VIC-1911 and different KIF11 inhibitors. Dose– response curves, dose–response matrix, and heat map indicating synergy scores in TC-71 EWS cell

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: Inhibition

Journal: Cancers

Article Title: Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

doi: 10.3390/cancers15204911

Figure Lengend Snippet: Figure 6. Analysis of protein expression post-drug treatment. (A) CHLA-10 and (B) TC-71 cells treated with drugs were assessed for changes in protein expression 24 h post-treatment via capillary electrophoresis-based Wes analysis. Increased protein levels of KIF11, p-KIF11Thr926 AURKA, and p-AURKAThr288 were observed for the drug combination group, whereas KIF15 levels were noticeably lower. Similarly, enhanced cleaved-PARP expression was observed with the combination treatment. The uncropped blots are shown in Figures S8 and S9.

Article Snippet: After this denaturation step, the prepared samples; blocking reagent; 1:50 diluted primary antibodies KIF11 or Eg5 (Cell Signaling Technology, Danvers, MA, USA, Cat# 4203, RRID:AB_10545760), p-KIF11 (Thermo Fisher Scientific Cat# PA5-105186, RRID:AB_2816659), KIF15 (Proteintech, Rosemont, IL, USA, Cat# 55407-1- AP, RRID:AB_11182836), AURKA (Novus Cat# NBP1-51843SS, RRID:AB_11018019), pAURKA (Novus Cat# NBP3-05434) PARP(46D11) (Cell Signaling Technology Cat# 9532, RRID:AB_659884), and β-actin (Cell Signaling Technology Cat# 12262, RRID:AB_2566811); HRP-conjugated secondary antibodies anti-rabbit (Biotechne, Minneapolis, MN, USA, #DM-001) and anti-mouse (Biotechne, #DM-002); and chemiluminescent substrate were dispensed into designated wells in an assay plate.

Techniques: Expressing, Electrophoresis

Journal: eLife

Article Title: Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies

doi: 10.7554/eLife.79402

Figure Lengend Snippet: ( A ) Motor heads are fused to optogenetic proteins such that activation with light causes the formation of motor tetramers (dimer of dimers). Motors are shown walking toward the microtubule plus-end. K401 and Kif11 walk in this direction, however Ncd is minus-end directed. ( B ) Images of the microtubule fluorescence for asters formed with each of the motors excited with a disk either 50 or 600 μm in diameter. ( C ) Image of the microtubule fluorescence from an aster with the measured size represented with the outer black circle. The plot on the right shows the radial microtubule concentration as revealed by fluorescence intensity; the threshold concentration used to determine the aster size is shown as a black horizontal line. ( D ) Mean aster size ( n ≈ 5 asters for each condition) for the three motors and different excitation diameters; the error bars represent the standard deviation.

Article Snippet: Human kinesin-5 (Kif11/Eg5) 5–513 was PCR amplified from mCherry-Kinesin11-N-18 plasmid (gift from Michael Davidson, Addgene # 55067).

Techniques: Activation Assay, Fluorescence, Concentration Assay, Standard Deviation

Journal: eLife

Article Title: Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies

doi: 10.7554/eLife.79402

Figure Lengend Snippet: Properties of motor proteins used in this study. Speeds were measured by us by gliding assay, the processivity and directionality are literature values.

Article Snippet: Human kinesin-5 (Kif11/Eg5) 5–513 was PCR amplified from mCherry-Kinesin11-N-18 plasmid (gift from Michael Davidson, Addgene # 55067).

Techniques: Gliding Assay

Journal: eLife

Article Title: Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies

doi: 10.7554/eLife.79402

Figure Lengend Snippet: ( A ) Schematic of the radial microtubule organization in an aster. Modeling applies to locations outside the disordered core region at the aster center. Components of the schematic are not drawn to scale. ( B ) Motor states and transitions between them. ( C ) Governing equations for the bound and free motor populations, along with our solution for the total motor distribution at steady state, expressed via effective parameters K d = k off / k on and λ 0 = D / v (see SI section ‘Model formulation’ for details). ( D ) Demonstration of the model fitting procedure on an example Kif11 aster. Fits to the average motor profile as well as to 5 out of 16 wedge profiles are shown. The outlier case with a lower concentration corresponds to wedge 13 in the fluorescence images. ( E ) Mean fitting errors for all asters calculated from the fits to the wedge profiles. The error is defined as the ratio of the mean residual to the concentration value at the inner boundary. ( F ) Inferred parameters K d and λ 0 grouped by the kind of motor. Box plots indicate the quartiles of the inferred parameter sets. The fitting error and the inferred parameters for the Kif11 aster in panel ( D ) are shown as white dots in panels ( E ) and ( F ).

Article Snippet: Human kinesin-5 (Kif11/Eg5) 5–513 was PCR amplified from mCherry-Kinesin11-N-18 plasmid (gift from Michael Davidson, Addgene # 55067).

Techniques: Concentration Assay, Fluorescence

Journal: eLife

Article Title: Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies

doi: 10.7554/eLife.79402

Figure Lengend Snippet: ( a,b ) Fluorescence images of an example Kif11 aster in tubulin ( a ) and motor ( b ) channels. Sixteen different wedges are separated and numbered. ( c ) Landscape of fit residuals when varying the two effective parameters K d and λ 0 . For each pair, an optimal scaling coefficient C is inferred before calculating the residual. The dot at the brightest spot stands for the optimal pair (or, the arrow indicates the location of the optimal pair in the landscape). ( d ) Average motor profile and the model fit, along with the inferred parameters. ( e ) Collection of fits to separate wedge profiles using the optimal ( K d , λ 0 ) pair inferred from the average profile.

Article Snippet: Human kinesin-5 (Kif11/Eg5) 5–513 was PCR amplified from mCherry-Kinesin11-N-18 plasmid (gift from Michael Davidson, Addgene # 55067).

Techniques: Fluorescence

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kif11 inhibitor reduces transport of EdU-labeled PSVs to mitotic chromosomes. HaCaT cells were infected with WT EdU-labeled PsVs for 24 h and then treated with 1.5 µM Kif11 inhibitor (Eg5 inh+) or left untreated (Ctrl) for 10 min prior to the cells being stained and subjected to the Click-iT reaction as previously described in Materials and Methods. ( A ) AF555 dye was used to stain EdU-labeled pseudogenomes (red), mouse anti-α-tubulin antibody was used to stain for α-tubulin (white), and rabbit anti-Kif11 antibody was used to stain for Kif11 (green). Left-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in prophase, and the right-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in metaphase. Since Eg5 inh+ treatment locks cells into a monoastral phenotype , both the left-hand and right-panels of the Eg5 inh+ row of ( A ) are of pre-monoastral cells. Two smaller close-up images, rotated at a 45° angle on the x-, y-, and z-axes, are included on the right-side of the main image(s) to better show close association, or lack thereof in Eg5 inh+, between EdU-labeled HPV genome and Kif11. ( B ) AF555 dye was again used to stain EdU-labeled pseudogenomes in cells undergoing mitosis at prophase or metaphase (red), and mitotic chromosomes were visualized using Hoechst (blue). Images represent single medial slices. ( C ) Quantification of EdU-labeled HPV16 PsV in whole cells +/−Eg5 inh+ treatment; at least 20 cells per group, control and Eg5 inh+ were counted, with 69 total cells counted (group numbers of 21, 26, and 22). Localization of EdU puncta (red) was analyzed with IMARIS using spots/surface analysis. The number of pseudogenomes in whole cells was established based on alpha tubulin and EdU-labeled HPV genome signals; determining genome and the localization on mitotic chromosomes was based on Hoechst staining. Lines in graph represent mean with SEM, and statistical significance was assessed by Student t -test, N = 3, ns: P > 0.05. ( D ) Quantification of EdU-labeled HPV16 PsV (red) co-localized with mitotic chromatin (blue) upon Kif11 inhibitor treatment using IMARIS. At least 20 cells per group, control and Eg5 inh+ were counted, with 66 total cells counted (group numbers of 21, 25, and 20). Lines in graph represent mean with SEM, and statistical significance was assessed by Mann Whitney test, N = 3, ***: P < 0.001. ( E ) Uninfected HaCaT cells were treated with 1.5 µM Eg5 inhibitor for 10 min and then stained for Kif11 and Kif18a as previously described in Materials and Methods, except cells were also subjected to the Click-iT reaction, but without the AF555 dye to stain for kinesins under denaturing conditions. Either Kif11 or Kif18A was stained using anti-rabbit Kif11 or anti-rabbit Kif18A antibodies, respectively (red). Mouse anti-α-tubulin antibody was used to stain α-tubulin (green), and cell nuclei were visualized using Hoechst (blue). Images represent single medial slices.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Labeling, Infection, Staining, Control, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kinesin motor proteins are in proximity to HPV16 L2 protein during infection. ( A ) HEK293TT cells were transfected with plasmids encoding HA-tagged HPV16 L2 and FLAG-tagged Kif11 to perform a co-immunoprecipitation (CoIP) assay using anti-HA beads; pCMV6 plasmid was used for the CoIP empty vector controls. CoIPs were performed with (right-hand panel) or without (left-hand panel) transfected cells treated with Kif11 inhibitor (1.5 µM; “Eg5i”) 1 or 2 h prior to cell harvesting for CoIP. Tagged HPV16 L2 and Kif11 proteins were probed for via Western blot using anti-HA and anti-FLAG antibodies, respectively, in both whole-cell lysates (“Lysate”) and anti-HA bead elutions (“elute”); anti α-tubulin Western blots were used as a loading control for the lysates. The enclosed immunoblots are representative of at least two independent experiments ( N = 2). ( B ) Mock-infected (“Mock”) and PsV16-infected (“Infected”) HaCaT cells were processed using the proximity ligation assay (PLA) protocol 21 hpi. PLAs were performed using an antibody cocktail to target the HPV16 L2 protein and an individually selected kinesin, either Kif11, Kif18a, or Kif25. The HPV16L2 antibodies used are the same throughout all PLAs, while the specific kinesin antibody used for the indicated PLA is identified in the left-hand margin of the images. PLA puncta were visualized in the representative images as red dots. Confocal images were acquired as z-stacks and processed as described in Materials and Methods. The white arrows highlight mitotic cells in their representative images for the PLAs. ( C ) The average PLA puncta numbers per interphase cells were counted for at least 200 cells per group in one experiment and are presented as median with 95% CI in the corresponding graphs for the PLAs in the middle panels adjacent to their representative images. The average PLA puncta numbers per mock-infected versus PSV-infected interphase cell were (median) 0.43 vs 6.8 (Kif11), 0.83 vs 5.8 (Kif18A), and 1.8 vs 4.1 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, ( N = 3); **** P < 0.0001. ( D ) The average PLA puncta numbers were counted per at least 20 mitotic cells for each condition and are presented as median with 95% CI. Mock-infected vs PSV-infected PLA puncta numbers per mitotic cell were (median) 0.0 vs 6.0 (Kif11), 0.0 vs 5.0 (Kif18A), and 1.0 vs 5.5 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, **** P < 0.0001.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Infection, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Cell Harvesting, Western Blot, Control, Proximity Ligation Assay, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Knocksideways experimental layout. 1) HeLa(Mito)–Kif11–FKBP–3x Flag cells were first treated with doxycycline (doxy) for 24 h to induce expression of the Kif11–FKBP–3x Flag transgene. 2) An endogenous Kif11-targeting siRNA pool was then reverse-transfected into doxy-treated cells to decrease endogenous Kif11 population over 24 h. 3) The doxy and Kif11 siRNA-treated cells, now containing reduced levels of endogenous Ki11 and increased amounts of exogenous Kif11–FKBP–3x Flag protein, were then infected with EdU-labeled PsV16s for either 16 h or infected with PsV16s containing a luciferase reporter genome for 24 h. Infected cells were then treated 16 hpi with 200 nM rapamycin for 1 h or added along with PsV16s for the longer 24 h infection. 4) Rapamycin-induced relocalization of Kif11–FKBP–3x Flag proteins to MitoTrap via dimerization of their respective FKBP and FRB domains. Not all Kif11–FKBP–3x will be re-localized to MitoTrap, some transgenic Kif11 protein will remain on microtubules to transport HPV16. 5) Optimally, the initial goal of the knocksideways experiment was to detect relocalization of EdU-labeled HPV16 genome to mitochondria in infected HeLa(Mito)–Kif11–FKBP–3x Flag cells treated with doxy and rapamycin. 6) Without rapamycin, Kif11–FKBP–3x Flag should perform the same functions as endogenous Kif11, especially during mitosis. 7) With rapamycin addition, a significant portion of the Kif11–FKBP–3x Flag cellular pool will be relocalized to MitoTrap. As a result of cells containing less endogenous Kif11, due to Kif11 siRNA, rapamycin treatment would increase levels of monoastral cells due to less endogenous and transgenic Kif11 available to assist in spindle microtubule sliding. Diagram was generated using BioRender and its available templates.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Expressing, Transfection, Infection, Labeling, Luciferase, Transgenic Assay, Generated

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Relocalization of Kif11 via knocksideways significantly decreases transport of EdU-labeled PsVs to mitotic chromosomes. HeLa(Mito) cells containing the tet-inducible Kif11–FKBP–3x Flag construct, were treated with doxycycline (doxy, 5 µg/mL) for 24 h, reverse-transfected with 2.5 pmol of Kif11-targeting siRNA for 24 h, and then infected with PsV16 for 16 h. Cells were then treated with either DMSO or rapamycin (200 nM) for 1 h prior to processing for confocal microscopy or Western blotting (WB) as described in Materials and Methods. ( A ) Doxy-treated (24 h) HeLa(Mito) cells were harvested approximately 42 h post-siRNA-transfection (1–10 pmol control scrambled (SCR) or Kif11-targeting [Kif11] siRNAs) and probed via WB for presence of endogenous Kif11 (WB:Kif11) or exogenous Kif11–FKBP–3x Flag (WB: Flag). Blot is representative of two separate experiments. ( B ) For panels left to right, for HeLa(Mito): Kif11–FKBP–3x flag cells treated with doxy, than 2.5 pmol Kif11 siRNAs, and finally treated with either DMSO or rapamycin; mouse anti-HA antibody was used to stain for MitoTrap protein (green), rabbit anti-Kif11 was used to stain for Kif11 (red), and nuclei were stained with Hoechst (blue). Images with combined staining are on the right-hand side, with a close-up of a selected cell. Colocalization of Kif11 and MitoTrap are visualized as yellow puncta in the combined image, with representative signal(s) shown in the z-stacks provided with the close-up images. White arrows in the DMSO panels show metaphase cells, and blue arrows in the rapamycin panels indicate monoastral cells. ( C,E ) The percentages of monoastral cells for each condition (DMSO or rapamycin treatment) were determined by Hoechst33342 staining. Confocal images were acquired as z-stacks with Olympus CSU W1 Spinning Disk Confocal System using a 20× objective. Monoastral quantifications in ( C ) and ( E ) are based on Hoechst staining and represent individual experiments. Differences in monoastral cell percentages in each condition (DMSO or rapamycin treatment) were analyzed using the Mann–Whitney test; *, P < 0.05. ( D, F, G ) Quantification of EdU-labeled PsV16(s) on mitotic chromatin of either monoastral or metaphase cells, with or without rapamycin treatment. Approximately 23 cells per condition per experiment were counted, with ( D ) 11, 6.3, 7.4 , and 2.2 (median) EdU puncta counted in monoastral, −/+ rapamycin and metaphase, −/+ rapamycin-treated cells, respectively, in one independent experiment and ( F ) 22, 16, 17, and 8.0 (median) EdU puncta counted in monoastral, −/+ rapamycin and metaphase, −/+ rapamycin-treated cells, respectively, in another independent experiment. ( G ) Confocal images were acquired and processed as described in Materials and Methods. AF555 dye coupled with Click-iT® chemistry was used to stain EdU-labeled pseudogenomes (red), and genome localization was determined by proximity to mitotic chromatin stained with Hoechst (blue). Differences of EdU puncta amounts on mitotic chromosomes in each condition were analyzed using the Mann–Whitney test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Labeling, Construct, Transfection, Infection, Confocal Microscopy, Western Blot, Control, Staining, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Knocksideways relocalization of Kif11 over 24 h also significantly decreases PsV infectivity of cells. HeLa(Mito): Kif11–FKBP–3x Flag cells were subjected to the same general knocksideways protocol as in , with the main differences being the infection time, and rapamycin time treatments were extended, 24 h each; the resultant infection readout is based on luciferase activity instead of EdU signal on mitotic chromosomes. ( A ) HeLa(Mito) cells containing the tet-inducible Kif11–FKBP–3x Flag construct were treated with or without doxycycline (doxy, 5 µg/mL) and then transfected with control (SCR) or Kif11-targeting siRNAs as before. Cells were then infected with luciferase-containing PsV16s at a VGE of 500 with or without rapamycin (200 nM). At 24 hpi, cells were harvested and lysed using OneGlo substrate, with subsequent luciferase activity ascertained with a Tecan Spark plate reader. “100%” infectivity was set for the luciferase activity of infected cells transfected with control (SCR) siRNAs, with or without doxycycline; the luciferase activity (infectivity) of infected cells for subsequent conditions, with or without doxycycline, was charted relative to the respective “100%” infectivity set points of SCR siRNAs. The % infectivity and standard deviation values for SCR siRNA transfection, −/+ rapamycin, and Kif11siRNA transfection, −/+ rapamycin, under “no doxycycline” conditions were 100.0% ± 14.8, 82.9% ± 14.5, 52.7% ± 46.2, and 31.5% ± 31.3, respectively. The % infectivity and standard deviation values for SCR siRNA transfection, −/+ rapamycin, and Kif11siRNA transfection, −/+ rapamycin, under “with doxycycline” conditions were 100.7% ± 9.3, 94.8% ± 15.5, 166.2% ± 37.9, and 64.1% ± 70.5, respectively. Differences of “% Infectivity” amounts in each condition were analyzed using Student’s t -test ( N = 4, with technical triplicates per condition); **, P < 0.01; ****, P < 0.0001. (B and C) Representative images of cells (10× magnification) in the stated conditions (−/+ rapamycin, with SCR or Kif11-siRNA transfected cells) in the ( B ) “no doxycycline” or ( C ) “with doxycycline” conditions were taken immediately before cell harvesting and luciferase activity determination. The white arrows show representative cells and clusters of cells that are in the monoastral phenotype. Brightfield images were acquired with a Leica DMI6000 B microsope using a 10x objective.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Infection, Luciferase, Activity Assay, Construct, Transfection, Control, Standard Deviation, Cell Harvesting