Journal: bioRxiv

Article Title: Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

doi: 10.1101/2020.06.16.155259

Figure Lengend Snippet: A . Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α- tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. Scale bars, 10 μm. Time, hour:min. B. Representative immunoblot of cell lysates obtained before and after doxycycline induction validating the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants stained using anti-KIF4A antibody, with anti-vinculin used as a loading control. C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K64A (30, 4), shKIF4A + KIF4A K64A (32, 4), uninduced KIF4A ΔZip1 (32, 3), shKIF4A + ΔZip1 (30, 4). P-values were calculated using Student’s t -test. n.s. - not significant, **** P ≤ 0.0001. D. Model illustrating chromosome arms-localized KIF4A driving MT-flux.

Article Snippet: U2OS photoactivatable GFP (PA-GFP)-α-tubulin ( ) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5’-GCAAGATCCTGAAAGAGAT-3’ was generated using a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids ( , ).

Techniques: Live Cell Imaging, Expressing, shRNA, Staining, Western Blot, Construct, Control

Journal: bioRxiv

Article Title: Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

doi: 10.1101/2020.06.16.155259

Figure Lengend Snippet: A . Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α- tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 μm. Time, min:sec. B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3). P-values were calculated using one-way ANOVA. n.s., not significant, ** P ≤ 0.01, **** P ≤ 0.0001. C . Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4). P-values were calculated using one-way ANOVA. n.s., not significant, **** P ≤ 0.0001. D. Representative kymographs of the photoactivated spindles from (B) displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. E. Percentage of cells with asynchronous flux movements calculated from (B). F. Model illustrating the role of MT-crosslinking activities of NuMA and HSET in uniform distribution of poleward forces across the mitotic spindle.

Article Snippet: U2OS photoactivatable GFP (PA-GFP)-α-tubulin ( ) cell line expressing stable inducible short hairpin RNA (shKIF4A) targeting KIF4A sequence 5’-GCAAGATCCTGAAAGAGAT-3’ was generated using a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR) using pENTR-THT-III (Addgene plasmid #55791) and pGLTR-X-Puro (Addgene plasmid #58246) plasmids ( , ).

Techniques: Stable Transfection, Expressing, Transfection, Activity Assay, Control, Construct