Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) Amino acid sequences from position 174 to 186 of K2-WT, K2-S181D, K2-S181A, K2-5SA and K2-SWAP. Amino acid substitutions are shown in red. (b) WB with anti-GFP antibody of untreated EGFP-K2-WT or K2-S181D expressing HeLa cells treated with MG132 (5 µM for 6 hr) or Bafilomycin A1 (1 µM for 6 hr). (c) MTT assay showing proliferation of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD, n=3 independent experiments. (d) Representative confocal images of immunostained paxillin (green), phalloidin (red) and DAPI (blue) in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Scale bar, 10 μm. (e) Quantification of paxillin-positive FA numbers of FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD; n = 11 cells for WT and S181D, 10 cells for 5SA and SWAP pooled from 3 independent experiments. (f) Quantification of paxillin-positive FA sizes in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. n (FA number): 294 for K2-WT, 108 for K2-S181D, 426 for K2-5SA and 431 for K2-SWAP pooled from 3 independent experiments. (g) WB of GFP in untreated and STLC-arrested HeLa cell expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K1-5SA. (h) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K2-5SA quantified after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. (i) WB with anti-myc antibody of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-tagged Rap1-G12V (myc-Rap1-CA). (j) Montage of phase contrast recordings from FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA. Time (min) starts at RO3306 release. Scale bar, 20 µm. (k) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. (l) WB with anti-Rap1 antibody recognizing both Rap1A and Rap1B isoforms of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with non-targeting control (siNTC) siRNA or siRNAs targeting Rap1A and Rap1B mRNAs (siRap1). Cells were harvested 48 hr after transfection. (m) Montage of phase contrast recordings of FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1. 32 hr after siRap1 transfection, cells were synchronized with RO3306 for 16 hr and then released for imaging. Time (min) starts at RO3306 release. Scale bar, 20 µm. (n) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1, and quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. P values in (e, f, k, n) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). All WB experiments have been repeated at least three times with similar results. pH3 served as an indicator for mitosis and GAPDH as loading control.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, MTT Assay, Transduction, Transfection, Imaging

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) WB of indicated cyclins in HeLa cells released from D-THY treatment and harvested at indicated times. (b) Relative changes in GAPDH-normalized FA protein abundance in HeLa cells released from D-THY, harvested at indicated times and determined by WB. Mean ± SD; n = 3 independent experiments. Abundance of FA proteins was referenced to 0 hr after D-THY release. (c) K2 mRNA levels in D-THY-synchronized G2 phase (7 hr after release) and M-phase (9 hr after release combined with shake-off) HeLa cells quantified by RT-PCR. Mean ± SD; n = 3 independent experiments. ns: not significant by unpaired Student’s t-test (two-tailed). (d) Surface levels of integrin subunits at G2 (black, 7 hr after D-THY release) and M-phase (red, collected by mitotic shake-off 9 hr after D-THY release) in HeLa cells determined by flow cytometry. The experiment has been repeated three times with similar results. (e-i) WB of EGFP-K2 and/or endogenous K2 with anti-K2 antibody in untreated and STLC-arrested mitotic HAP1, HEK293 and HeLa (e) , U2OS and RPE1 (f) , mouse fibroblast (MF; g ) and HeLa cells seeded on (h) FN, VN, 10% FCS and (i) embedded in a 3D Matrigel or Matrigel/collagen I mixture (1:1). (j) WB of K1 and K2 in unsynchronized HeLa and U2OS cells harvested with and without mitotic shake-off. (k) WB of K3 in untreated and STLC-arrested mitotic K562 cells. (l) Montages of time-lapse live cell recordings generated by wide-field fluorescent microscopy showing the relative fluorescence of EGFP-K2 and lifeact-mRuby in HeLa cell at indicated times after RO3306 release. Scale bar, 10 μm. All WB experiments have been repeated three times with similar results. pH3 served as indicator for mitosis and GAPDH as loading control.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Flow Cytometry, Generated, Microscopy, Fluorescence

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , b , Stills of live-cell recordings generated by TIRF microscopy showing a representative interphase and mitotic (30 min after RO3306 release) HeLa cell stably expressing EGFP–KIND2 ( a ) or YPet–talin-1 ( b ), and Lifeact–mRuby and H2B–CFP. c , d , SIM images ( c ) of EGFP–KIND2 (green), the β 1 integrin activation-associated 12G10 epitope (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Co-localization of EGFP–KIND2 with the 12G10 epitope is shown in FAs (arrowheads) of interphase cells and in puncta in retraction fibres and round cell bodies of mitotic cells. To visualize EGFP–KIND2 signals in retraction fibres, the fluorescence was enhanced by increasing the exposure time. Boxes indicate areas of retraction fibres shown at high magnifications. The arrow indicates the direction of line profile analysis ( d ) of EGFP–KIND2 and 12G10 epitope (red). Each line-profile assessment was done three times. AU, arbitrary unit. e , SIM images of immunostained endogenous KIND2 (green), phalloidin (red) and pH3 (blue) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate KIND2 in FAs. KIND2 in mitotic retraction fibres was visualized by increasing the exposure time. Boxes indicate retraction fibre areas shown at high magnifications. f , Schematic of the procedure, depicting the Z positions of round mitotic (30 min after RO3306 release) HeLa cell bodies: region 1, bottom stack; region 3, stack capturing the cortical end of retraction fibres; region 2, intermediate stack between region 1 and 3. Region 1 was recorded by TIRF microscopy and regions 2 and 3 by confocal microscopy. g , Representative image sections at different regions as depicted in f of round mitotic (30 min after RO3306 release) HeLa cell bodies immunostained for the 12G10 epitope or stably expressing EGFP–KIND2, YPet–talin-1 or EGFP. h , Quantification of adhesion assays of untreated and STLC-arrested HeLa cells on FN, VN, fetal bovine serum or BSA coatings. Mean ± s.d.; n = 9 independent experiments. P values calculated by unpaired Student’s t -test (two-tailed). Scale bars, 1 μm (magnifications in c and e ) or 10 μm ( a – c , e , g ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Generated, Microscopy, Stable Transfection, Expressing, Activation Assay, Fluorescence, Confocal Microscopy, Two Tailed Test

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , Volcano plot of phospho-peptides of untreated versus STLC-arrested mitotic HeLa cells. KIND1-pS179 (K1 S179) and KIND2-pS181 (K2 S181) are highlighted in red, paxillin-pS106 and paxillin-pS109 (PXN S106/109) in blue. The black line indicates the significance cut-off (false discovery rate of 0.05, S0:1) estimated using Perseus software. b , Alignment of the mitotic phospho-motifs of KIND1 and KIND2 in different vertebrates. Asterisks indicate serine residues that were identified as phosphorylated by MS analysis. The phospho-motif is absent in KIND3. c , GFP immunoprecipitation (IP) from untreated and STLC-arrested HeLa cells expressing EGFP–KIND2 or EGFP only and analysed by WB for CDK1, cyclin B1 and cyclin A2. d , WB of KIND2-pS181 in untreated and STLC-arrested HeLa cells expressing endogenous KIND2 and overexpressing EGFP–KIND2-WT or EGFP–KIND2-S181A in the presence of 5 μg ml –1 non-phosphorylated KIND2 peptide (GSGSIYSSPGLYSKT). e , WB of KIND2 and KIND2-pS181 in untreated and STLC-arrested HeLa cell lysates treated with or without alkaline and lambda phosphatases (PPs). f , Representative confocal images of KIND2-pS181 (green), phalloidin (red) and DAPI (blue) in HeLa cells at indicated cell cycle phase. The area with nucleus/chromosomes is magnified in the bottom panel. g , SIM images of KIND2-pS181 (green) and phalloidin (red) in round mitotic HeLa cell bodies and retraction fibres (boxes show high magnifications of retraction fibres; 30 min after RO3306 release). h , Quantification of the KIND2-pS181 signal at indicated cell cycle phases of HeLa cells. n = 20 cells for each cell cycle phase pooled from 3 independent experiments. All WB experiments were repeated at least three times, with similar results obtained. pH3 served as an indicator for mitosis and GAPDH as the loading control. Scale bars, 2.5 μm (magnifications in g ) or 10 μm ( f , g ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Software, Immunoprecipitation, Expressing

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) WB of K1 and K2 in untreated and STLC-arrested mitotic HeLa cells with and without alkaline- and lambda-phosphatases (PPs) treatment. pH3 served as indicator of mitosis and successful phosphatase treatment, and GAPDH as loading control. The experiment has been repeated three times with similar results. (b) MS2 spectrum acquired under HCD fragmentation conditions for the phosphorylated peptide (KKLDDQSEDEALELEGPLIMPGSGSIYSSPGLYSK) from CDK1-cyclin B1 phosphorylated recombinant K2. Full sequence coverage for the peptide achieved by combining b- and y-ion series (blue = b-ion, red = y-ion, green = b- or y-ion with neutral loss of H2O or NH3, yellow = b- or y-ion with neutral loss of H3O4P+H2O). The ions series y7 to y21, y23, y25 and the ion b31 and b32 include mass of phosphorylation. The y7-ion identifies S181 as the phosphorylated residue. ( c,d ) ELISA of immobilized K2 (black) and in vitro CDK1 cyclin-B1 phosphorylated K2 (red) titrated with ( c ) commercial anti-K2 (clone 3A3) and d ) home-made anti-K2-pS181 antibody. n = 3 independent experiments. (e) Representative confocal images of EGFP-K2 (green), K2-pS181 (red) and DAPI (blue) in interphase and mitotic (30 min after RO3306 release) mouse fibroblasts expressing EGFP-tagged K2-WT or -S181A. Scale bar = 20 µm.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Recombinant, Sequencing, Enzyme-linked Immunosorbent Assay, In Vitro, Expressing

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) Amino acid sequences from position 174 to 186 of K2-WT, K2-S181D, K2-S181A, K2-5SA and K2-SWAP. Amino acid substitutions are shown in red. (b) WB with anti-GFP antibody of untreated EGFP-K2-WT or K2-S181D expressing HeLa cells treated with MG132 (5 µM for 6 hr) or Bafilomycin A1 (1 µM for 6 hr). (c) MTT assay showing proliferation of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD, n=3 independent experiments. (d) Representative confocal images of immunostained paxillin (green), phalloidin (red) and DAPI (blue) in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Scale bar, 10 μm. (e) Quantification of paxillin-positive FA numbers of FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD; n = 11 cells for WT and S181D, 10 cells for 5SA and SWAP pooled from 3 independent experiments. (f) Quantification of paxillin-positive FA sizes in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. n (FA number): 294 for K2-WT, 108 for K2-S181D, 426 for K2-5SA and 431 for K2-SWAP pooled from 3 independent experiments. (g) WB of GFP in untreated and STLC-arrested HeLa cell expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K1-5SA. (h) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K2-5SA quantified after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. (i) WB with anti-myc antibody of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-tagged Rap1-G12V (myc-Rap1-CA). (j) Montage of phase contrast recordings from FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA. Time (min) starts at RO3306 release. Scale bar, 20 µm. (k) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. (l) WB with anti-Rap1 antibody recognizing both Rap1A and Rap1B isoforms of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with non-targeting control (siNTC) siRNA or siRNAs targeting Rap1A and Rap1B mRNAs (siRap1). Cells were harvested 48 hr after transfection. (m) Montage of phase contrast recordings of FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1. 32 hr after siRap1 transfection, cells were synchronized with RO3306 for 16 hr and then released for imaging. Time (min) starts at RO3306 release. Scale bar, 20 µm. (n) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1, and quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. P values in (e, f, k, n) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). All WB experiments have been repeated at least three times with similar results. pH3 served as an indicator for mitosis and GAPDH as loading control.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, MTT Assay, Transduction, Transfection, Imaging

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , b , WB of GFP ( a ) and quantification ( b ) of EGFP–KIND2 stability in HeLa cells expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP treated with 10 μg ml –1 cycloheximide (CHX). Mean ± s.d.; n = 3 independent experiments. c , d , WB of GFP ( c ) and ratio ( d ) of EGFP–KIND2 levels in untreated and STLC-arrested HeLa cells expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-S181A, KIND2-5SA or KIND2-SWAP. Mean ± s.d.; n = 3 independent experiments. e , DNA contents of propidium-iodide-loaded untreated fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP. Percentage of aneuploid cells is indicated. f – h , Montage of phase-contrast recordings ( f ), percentage of rounded fibroblasts ( g ) and M-phase duration ( h ) of fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP after RO3306 release. Dashed outlines in f indicate cell margins. For g , data are the mean ± s.d.; 200 cells counted for each cell line, n = 3 independent experiments. For h , the time spans from cell margin retraction to completion of cytokinesis. n (cell number) = 61 for WT, 51 for S181D, 53 for 5SA and 54 for SWAP pooled from 3 independent experiments. i , Percentage of apoptotic fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP quantified 3 h after RO3306 release by staining for caspase 3 and caspase 7. Mean ± s.d.; 100 cells counted for each cell line, n = 3 independent experiments. j , k , Representative confocal images of immunostained paxillin (green), phalloidin (red) and DAPI (blue) ( j ) and quantification ( k ) of paxillin-positive FAs in fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP at the indicated times after RO3306 release. Mean ± s.d.; n = 10 cells of each cell line quantified at each time point pooled from 2 independent experiments ( k ). l , Circularity of unsynchronized fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP. Mean ± s.d.; interphase, n = 10 cells for each cell line; mitosis, n = 20 for WT and S181D, 10 for 5SA and 12 for SWAP pooled from 2 independent experiments. P values in d , h and i were calculated by one-way analysis of variance (ANOVA) Dunnett’s multiple comparison test (95% confidence interval (CI)). P values in l were calculated by unpaired Student’s t -test (two-tailed). All WB experiments were repeated three times, with similar results obtained. GAPDH served as loading control and pH3 as an indicator for mitosis. Scale bars, 10 μm ( j ) or 30 μm ( f ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, Staining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) Scatter plot based on fold change of EGFP fluorescent intensity in individual transfections of the siRNA screening. EGFP intensity of EGFP-K2-S181D expressing HeLa cells transfected with siNTC was set as 1 and used as reference for quantifications. 800–1200 cells were quantified in each well. CUL9 and FBXL10 genes are indicated. (b) WB with anti-GFP antibody of untreated and STLC-arrested mitotic HeLa cells expressing EGFP-tagged K2 and transfected with indicated siRNAs. pH3 served as an indicator for mitosis and GAPDH as loading control. The experiment has been repeated three times with similar results. (c) Montage of phase contrast recordings of FN-seeded mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Cells were synchronized 32 hr after transfection with RO3306 for 16 hr and then released for imaging. Time (in min) starts at RO3306 release. Dashed outlines indicate cell margins. Scale bar, 20 µm. (d) Representative confocal images of α-tubulin (red), γ-tubulin (green) and DAPI (blue) in mitotic (30 min after RO3306 release) mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10, or siC+F. Scale bar, 5 µm. (e) Percentages of mitotic (30 min after RO3306 release) mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F with normal chromosome alignments. Mean ± SD; 50 cells were measured in each transfection, n = 3 independent experiments. (f) Representative confocal images of γH2AX (red), phallodin (green) and DAPI (blue) in interphase mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Scale bar, 10 µm. (g) Percentages of γH2AX-positive cells with more than 5 foci in mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Mean ± SD; 200 cells were quantified in each transfection, n = 3 independent experiments. (h) Quantification of number of γH2AX-foci in foci-positive mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. n = 40 cells for each transfection pooled from 3 independent experiments. ( i,j ) SIM images of EGFP-K2 (green), phalloidin (magenta) and (i) CUL9 (red) or (j) FBXL10 (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Localization of K2 in mitotic retraction fibers was visualized by increasing the exposure time. Boxes indicate areas of retraction fibers shown at high magnifications. Scale bar, 10 μm; retraction fibers, 1 μm. P values in (e, g, h) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Transfection, Expressing, Imaging

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , Representative confocal microscopy images of HeLa cells expressing EGFP-tagged KIND2-S181D (green) transfected with non-targeting control siRNA (siNTC), siCUL9, siFBXL10 or siC+F and stained for paxillin (red) and DAPI (blue). Scale bar, 10 μm. b , Quantification of EGFP intensity in HeLa cells expressing EGFP-tagged KIND2-S181D transfected with siNTC, siCUL9, siFBXL10 or siC+F. n = 50 cells analysed for each transfection, pooled from 3 independent experiments. c , WB of endogenous KIND2 in D-THY-synchronized G1/S phase (0 h after release) and mitotic HeLa cells (9 h after release combined with mitotic shake-off) transfected with siNTC, siCUL9, siFBXL10 or siC+F. d , WB quantifications of endogenous KIND2 levels in D-THY synchronized G1/S phase (0 hr after release) and mitotic HeLa cells (9 hr after release combined with mitotic shake-off) transfected with siNTC, siCUL9, siFBXL10 or siC+F. Mean ± s.d.; n = 3 independent experiments. e , WB of EGFP, CUL9 and FBXL10 from mouse fibroblasts expressing EGFP-tagged KIND2-S181D and transfected with siNTC, siCUL9, siFBXL10 or siC+F. f , Percentage of rounded EGFP-tagged KIND2-S181D-expressing mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F quantified after RO3306 release. Mean ± s.d.; n = 200 cells counted for each transfection, n = 3 independent experiments. g , IP of CUL9 from untreated and STLC-arrested mitotic HeLa cells followed by WB for CUL9, FBXL10 and KIND2. h , IP of CUL9 in STLC-arrested mitotic HeLa cells expressing EGFP only, EGFP-tagged KIND2-WT or KIND2-5SA followed by WB for CUL9, FBXL10 and GFP. P values in b and d were calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). All WB experiments were repeated three times, with similar results obtained. GAPDH served as the loading control and pH3 as an indicator for mitosis.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Confocal Microscopy, Expressing, Transfection, Staining

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , GFP IP from STLC-arrested mitotic HeLa cells expressing EGFP-tagged KIND2-WT and HA-tagged ubiquitin (Ub) transfected with siNTC, siCUL9, siFBXL10 or siC+F followed by WB with anti-HA antibody. Whole-cell lysates (5%) were analysed by WB for KIND2, CUL9 and FBXL10. The arrowhead indicates non-ubiquitinated KIND2. GAPDH served as the loading control. b , In vitro ubiquitination of non-phosphorylated or CDK1–cyclin B1-phosphorylated recombinant KIND2 in the presence of WT-Ub or K48R-Ub by control rabbit IgG or anti-CUL9 precipitates and detected by anti-KIND2 WB experiments. The arrowhead indicates non-ubiquitinated KIND2 and the asterisk phosphorylated KIND2. IPs and recombinant KIND2 were analysed by WB for KIND2, CUL9 and FBXL10. c , Quantification of polyubiquitination measured by WB of in vitro ubiquitinated KIND2. Mean ± s.d.; n = 3 independent experiments. d , Percentage of rounded mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F quantified 5 min after RO3306 release. Mean ± s.d.; n = 100 cells counted for each transfection, n = 3 independent experiments. e , M-phase duration of FN-seeded mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. The time spans from cell margin retraction to completion of cytokinesis. n = 50 cells for each transfection, pooled from 3 independent experiments. P values in d and e were calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). All WB experiments were repeated three times, with similar results obtained. f , In early prophase, the CDK1–cyclin B1 complex phosphorylates KIND1 and KIND2 at the F1 loop and creates a phospho-degron. This recruits the CUL9–FBXL10 complex and results in the ubiquitination and proteasomal degradation of KIND1 and KIND2. g , KIND1 and KIND2 degradation induces FA disassembly, which is followed by the formation of retraction fibres and mitotic rounding. Cells expressing phospho-mimetic KIND2 (KIND2-S181D) display impaired retraction fibre formation, which leads to misorientation of mitotic spindles, prolonged SAC activation and either cell death or defects in chromosome segregation and aneuploidy. In cells expressing phospho-inhibitory KIND2 (KIND2-5SA or KIND2-SWAP), FA disassembly and mitotic cell rounding fail, which leads to defective spindle anchorage, aberrant chromosome capture and alignment, prolonged SAC activity and either cell death or defects in chromosome segregation and aneuploidy.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, Transfection, In Vitro, Recombinant, Activation Assay, Activity Assay

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , b , Stills of live-cell recordings generated by TIRF microscopy showing a representative interphase and mitotic (30 min after RO3306 release) HeLa cell stably expressing EGFP–KIND2 ( a ) or YPet–talin-1 ( b ), and Lifeact–mRuby and H2B–CFP. c , d , SIM images ( c ) of EGFP–KIND2 (green), the β 1 integrin activation-associated 12G10 epitope (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Co-localization of EGFP–KIND2 with the 12G10 epitope is shown in FAs (arrowheads) of interphase cells and in puncta in retraction fibres and round cell bodies of mitotic cells. To visualize EGFP–KIND2 signals in retraction fibres, the fluorescence was enhanced by increasing the exposure time. Boxes indicate areas of retraction fibres shown at high magnifications. The arrow indicates the direction of line profile analysis ( d ) of EGFP–KIND2 and 12G10 epitope (red). Each line-profile assessment was done three times. AU, arbitrary unit. e , SIM images of immunostained endogenous KIND2 (green), phalloidin (red) and pH3 (blue) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate KIND2 in FAs. KIND2 in mitotic retraction fibres was visualized by increasing the exposure time. Boxes indicate retraction fibre areas shown at high magnifications. f , Schematic of the procedure, depicting the Z positions of round mitotic (30 min after RO3306 release) HeLa cell bodies: region 1, bottom stack; region 3, stack capturing the cortical end of retraction fibres; region 2, intermediate stack between region 1 and 3. Region 1 was recorded by TIRF microscopy and regions 2 and 3 by confocal microscopy. g , Representative image sections at different regions as depicted in f of round mitotic (30 min after RO3306 release) HeLa cell bodies immunostained for the 12G10 epitope or stably expressing EGFP–KIND2, YPet–talin-1 or EGFP. h , Quantification of adhesion assays of untreated and STLC-arrested HeLa cells on FN, VN, fetal bovine serum or BSA coatings. Mean ± s.d.; n = 9 independent experiments. P values calculated by unpaired Student’s t -test (two-tailed). Scale bars, 1 μm (magnifications in c and e ) or 10 μm ( a – c , e , g ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Generated, Microscopy, Stable Transfection, Expressing, Activation Assay, Fluorescence, Confocal Microscopy, Two Tailed Test

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: ( a – f ) SIM images of EGFP-K2 (green) and phalloidin (magenta) in combination with (a) TS2/16-stained β1 integrin (red), (c) paxillin (red) or (e) vinculin (red) in interphase and mitotic HeLa cells (30 min after RO3306 release). Arrowheads indicate FAs and * plasma membrane. EGFP-K2 signals in retraction fibers (a) were enhanced by increasing exposure time. Boxes ( a , c , e ) indicate areas of retraction fibers shown at high magnifications. The arrows indicate direction of line profile analysis of (b) EGFP-K2 (green) and TS2/16 (red), (d) EGFP-K2 (green) and paxillin (red) and (f) EGFP-K2 (green) and vinculin (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. ( g – j ) SIM images of YPet-talin1 (green) and phalloidin (magenta) in combination with (g) 12G10 (red) or (i) TS2/16 β1 integrin (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads ( g , i ) indicate FAs and * (i) plasma membrane. Boxes indicate areas of retraction fibers shown at high magnifications. The arrows indicate direction of line profile analysis of YPet-talin1 (green) together with (h) 12G10 (red) or (j) TS2/16 (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. ( k , l ) SIM images of EGFP-K2 (green), zyxin (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate FAs. Boxes indicate areas of retraction fibers shown at high magnifications. The arrow indicates direction of line profile of (l) EGFP-K2 (green) and zyxin (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. (m) SIM images of pMLC (green) and phalloidin (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Boxes indicate areas of stress fibers of interphase cells and retraction fibers of mitotic cells shown at high magnifications. Scale bar, 10 µm. Scale bar of magnifications showing stress fibers and retraction fibers, 2 µm. Each line profile assessment has been done three times.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Staining

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , Volcano plot of phospho-peptides of untreated versus STLC-arrested mitotic HeLa cells. KIND1-pS179 (K1 S179) and KIND2-pS181 (K2 S181) are highlighted in red, paxillin-pS106 and paxillin-pS109 (PXN S106/109) in blue. The black line indicates the significance cut-off (false discovery rate of 0.05, S0:1) estimated using Perseus software. b , Alignment of the mitotic phospho-motifs of KIND1 and KIND2 in different vertebrates. Asterisks indicate serine residues that were identified as phosphorylated by MS analysis. The phospho-motif is absent in KIND3. c , GFP immunoprecipitation (IP) from untreated and STLC-arrested HeLa cells expressing EGFP–KIND2 or EGFP only and analysed by WB for CDK1, cyclin B1 and cyclin A2. d , WB of KIND2-pS181 in untreated and STLC-arrested HeLa cells expressing endogenous KIND2 and overexpressing EGFP–KIND2-WT or EGFP–KIND2-S181A in the presence of 5 μg ml –1 non-phosphorylated KIND2 peptide (GSGSIYSSPGLYSKT). e , WB of KIND2 and KIND2-pS181 in untreated and STLC-arrested HeLa cell lysates treated with or without alkaline and lambda phosphatases (PPs). f , Representative confocal images of KIND2-pS181 (green), phalloidin (red) and DAPI (blue) in HeLa cells at indicated cell cycle phase. The area with nucleus/chromosomes is magnified in the bottom panel. g , SIM images of KIND2-pS181 (green) and phalloidin (red) in round mitotic HeLa cell bodies and retraction fibres (boxes show high magnifications of retraction fibres; 30 min after RO3306 release). h , Quantification of the KIND2-pS181 signal at indicated cell cycle phases of HeLa cells. n = 20 cells for each cell cycle phase pooled from 3 independent experiments. All WB experiments were repeated at least three times, with similar results obtained. pH3 served as an indicator for mitosis and GAPDH as the loading control. Scale bars, 2.5 μm (magnifications in g ) or 10 μm ( f , g ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Software, Immunoprecipitation, Expressing

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) Amino acid sequences from position 174 to 186 of K2-WT, K2-S181D, K2-S181A, K2-5SA and K2-SWAP. Amino acid substitutions are shown in red. (b) WB with anti-GFP antibody of untreated EGFP-K2-WT or K2-S181D expressing HeLa cells treated with MG132 (5 µM for 6 hr) or Bafilomycin A1 (1 µM for 6 hr). (c) MTT assay showing proliferation of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD, n=3 independent experiments. (d) Representative confocal images of immunostained paxillin (green), phalloidin (red) and DAPI (blue) in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Scale bar, 10 μm. (e) Quantification of paxillin-positive FA numbers of FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD; n = 11 cells for WT and S181D, 10 cells for 5SA and SWAP pooled from 3 independent experiments. (f) Quantification of paxillin-positive FA sizes in FN-seeded interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. n (FA number): 294 for K2-WT, 108 for K2-S181D, 426 for K2-5SA and 431 for K2-SWAP pooled from 3 independent experiments. (g) WB of GFP in untreated and STLC-arrested HeLa cell expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K1-5SA. (h) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K1-WT, K1-S179D, K1-S179A or K2-5SA quantified after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. (i) WB with anti-myc antibody of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-tagged Rap1-G12V (myc-Rap1-CA). (j) Montage of phase contrast recordings from FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA. Time (min) starts at RO3306 release. Scale bar, 20 µm. (k) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and retrovirally transduced with myc-Rap1-CA quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. (l) WB with anti-Rap1 antibody recognizing both Rap1A and Rap1B isoforms of mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with non-targeting control (siNTC) siRNA or siRNAs targeting Rap1A and Rap1B mRNAs (siRap1). Cells were harvested 48 hr after transfection. (m) Montage of phase contrast recordings of FN-seeded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1. 32 hr after siRap1 transfection, cells were synchronized with RO3306 for 16 hr and then released for imaging. Time (min) starts at RO3306 release. Scale bar, 20 µm. (n) Percentages of rounded mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP and transfected with siRap1, and quantified 5 min after RO3306 release. Mean ± SD; 200 cells counted for each cell line, n = 3 independent experiments. ns: not significant. P values in (e, f, k, n) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). All WB experiments have been repeated at least three times with similar results. pH3 served as an indicator for mitosis and GAPDH as loading control.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, MTT Assay, Transduction, Transfection, Imaging

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , b , WB of GFP ( a ) and quantification ( b ) of EGFP–KIND2 stability in HeLa cells expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP treated with 10 μg ml –1 cycloheximide (CHX). Mean ± s.d.; n = 3 independent experiments. c , d , WB of GFP ( c ) and ratio ( d ) of EGFP–KIND2 levels in untreated and STLC-arrested HeLa cells expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-S181A, KIND2-5SA or KIND2-SWAP. Mean ± s.d.; n = 3 independent experiments. e , DNA contents of propidium-iodide-loaded untreated fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP. Percentage of aneuploid cells is indicated. f – h , Montage of phase-contrast recordings ( f ), percentage of rounded fibroblasts ( g ) and M-phase duration ( h ) of fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP after RO3306 release. Dashed outlines in f indicate cell margins. For g , data are the mean ± s.d.; 200 cells counted for each cell line, n = 3 independent experiments. For h , the time spans from cell margin retraction to completion of cytokinesis. n (cell number) = 61 for WT, 51 for S181D, 53 for 5SA and 54 for SWAP pooled from 3 independent experiments. i , Percentage of apoptotic fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP quantified 3 h after RO3306 release by staining for caspase 3 and caspase 7. Mean ± s.d.; 100 cells counted for each cell line, n = 3 independent experiments. j , k , Representative confocal images of immunostained paxillin (green), phalloidin (red) and DAPI (blue) ( j ) and quantification ( k ) of paxillin-positive FAs in fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP at the indicated times after RO3306 release. Mean ± s.d.; n = 10 cells of each cell line quantified at each time point pooled from 2 independent experiments ( k ). l , Circularity of unsynchronized fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP. Mean ± s.d.; interphase, n = 10 cells for each cell line; mitosis, n = 20 for WT and S181D, 10 for 5SA and 12 for SWAP pooled from 2 independent experiments. P values in d , h and i were calculated by one-way analysis of variance (ANOVA) Dunnett’s multiple comparison test (95% confidence interval (CI)). P values in l were calculated by unpaired Student’s t -test (two-tailed). All WB experiments were repeated three times, with similar results obtained. GAPDH served as loading control and pH3 as an indicator for mitosis. Scale bars, 10 μm ( j ) or 30 μm ( f ).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, Staining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , Representative confocal images of phalloidin (red) and DAPI (blue) in interphase and mitotic (30 min after RO3306 release) mouse fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP seeded on FN-coated L-shaped micropatterns. Boxes indicate areas of chromosomes shown at high magnifications. Scale bar, 10 μm. Scale bar of magnifications showing chromosomes, 5 μm. b , Quantification of retraction fibre length in mitotic mouse fibroblasts engineered and cultured as described in a . n (number of retraction fibres) = 164 for WT, 21 for S181D, 128 for 5SA and 116 for SWAP pooled from 3 independent experiments. P values calculated by Kruskal–Wallis Dunnett’s multiple comparison test. c , Quantification of retraction fibres in mitotic mouse fibroblasts engineered and cultured as described in a . Mean ± s.d.; n = 10 cells for each cell line pooled from 3 independent experiments. P values calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI). d , Representative confocal images of α-tubulin (red), γ-tubulin (green) and DAPI (blue) in mitotic (30 min after RO3306 release) mouse fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP. Maximal projections of Z -stack images beginning with Z -stacks capturing the first spindle from the bottom, the centrosomes and the last spindle at the top. Scale bar, 5 μm. e , Cartoon showing how spindle angles were calculated. The spindle angle ( α °) was defined as the angle between the spindle axis and substrate surface. Lengths of a and b represent the vertical and horizontal distances, respectively, between two spindle poles. The spindle angle ( α °) was calculated based on the equation shown on the top. f , Spindle angle distribution in mitotic mouse fibroblasts expressing EGFP-tagged KIND2-WT, KIND2-S181D, KIND2-5SA or KIND2-SWAP; n = 50 cells analysed for KIND2-WT and each mutant, pooled from 3 independent experiments.

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, Cell Culture, Mutagenesis

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: ( a-d ) Representative confocal images of α-tubulin (red), γ-tubulin (green), DAPI (blue) and EGFP-K2 (cyan) in (a) mitotic (30 min after RO3306 release) and (c) interphase mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Boxes indicate areas of centrosomes shown at high magnifications. Quantifications of cells (mean ± SD; 100 cells were measured for each cell line, n = 3 independent experiments) with bipolar (b) and supernumerary centrosomes (d) . Scale bar, 10 µm. Scale bar of magnifications showing centrosomes, 2.5 µm. (e) Representative confocal images of immunostained Mad2 (green), α-tubulin (red) and DAPI (blue) in mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP at indicated times after RO3306 release. Scale bar, 5 µm. (f) Quantification of the Mad2 fluorescence intensity in mouse fibroblasts expressing EGFP-tagged K2-WT K2-S181D, K2-5SA or K2-SWAP at indicated time points after RO3306 release. Mean ± SD; n (cell number) = 20 for WT, 13 for S181D, 10 for 5SA and 11 for SWAP at 15 min; 12 for WT, 16 for S181D, 10 for 5SA and 12 for SWAP at 45 min; 10 for WT, 12 for S181D, 10 for 5SA and 11 for SWAP at 60 min pooled from 3 independent experiment. (g) Representative confocal images of γH2AX (red), phallodin (green) and DAPI (blue) in mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Boxes indicate nuclear areas shown at high magnifications. Scale bar: 10 µm (left), 5 µm (right). Note that nuclei of K2-S181D-expressing fibroblasts are smaller. (h) Percentages of γH2AX-positive cells with more than 5 foci in mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD; 200 cells quantified for each cell line, n = 3 independent experiments. (i) Quantification of the number of γH2AX-foci in foci-positive mouse fibroblasts expressing EGFP-tagged K2-WT, K2-S181D, K2-5SA or K2-SWAP. Mean ± SD, n = 50 cells pooled from 3 independent experiments. (j) Representative confocal images of phalloidin (red) and DAPI (blue) in mouse fibroblasts expressing EGFP-tagged K2-WT K2-S181D, K2-5SA or K2-SWAP. Boxes indicate areas of polyploid cells shown as DAPI at high magnifications. Scale bar, 10 µm. Scale bar of magnifications showing polyploid cells, 5 µm. (k) Percentages of polyploid mouse fibroblasts expressing EGFP-tagged K2-WT K2-S181D, K2-5SA or K2-SWAP. Mean ± SD; 200 cells were counted in each cell line, n = 3 independent experiments. P values in (b, d, f, h, i, k) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Expressing, Fluorescence

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) Scatter plot based on fold change of EGFP fluorescent intensity in individual transfections of the siRNA screening. EGFP intensity of EGFP-K2-S181D expressing HeLa cells transfected with siNTC was set as 1 and used as reference for quantifications. 800–1200 cells were quantified in each well. CUL9 and FBXL10 genes are indicated. (b) WB with anti-GFP antibody of untreated and STLC-arrested mitotic HeLa cells expressing EGFP-tagged K2 and transfected with indicated siRNAs. pH3 served as an indicator for mitosis and GAPDH as loading control. The experiment has been repeated three times with similar results. (c) Montage of phase contrast recordings of FN-seeded mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Cells were synchronized 32 hr after transfection with RO3306 for 16 hr and then released for imaging. Time (in min) starts at RO3306 release. Dashed outlines indicate cell margins. Scale bar, 20 µm. (d) Representative confocal images of α-tubulin (red), γ-tubulin (green) and DAPI (blue) in mitotic (30 min after RO3306 release) mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10, or siC+F. Scale bar, 5 µm. (e) Percentages of mitotic (30 min after RO3306 release) mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F with normal chromosome alignments. Mean ± SD; 50 cells were measured in each transfection, n = 3 independent experiments. (f) Representative confocal images of γH2AX (red), phallodin (green) and DAPI (blue) in interphase mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Scale bar, 10 µm. (g) Percentages of γH2AX-positive cells with more than 5 foci in mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. Mean ± SD; 200 cells were quantified in each transfection, n = 3 independent experiments. (h) Quantification of number of γH2AX-foci in foci-positive mouse fibroblasts transfected with siNTC, siCUL9, siFBXL10 or siC+F. n = 40 cells for each transfection pooled from 3 independent experiments. ( i,j ) SIM images of EGFP-K2 (green), phalloidin (magenta) and (i) CUL9 (red) or (j) FBXL10 (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Localization of K2 in mitotic retraction fibers was visualized by increasing the exposure time. Boxes indicate areas of retraction fibers shown at high magnifications. Scale bar, 10 μm; retraction fibers, 1 μm. P values in (e, g, h) calculated by one-way ANOVA Dunnett’s multiple comparison test (95% CI).

Article Snippet: The following chemicals were used: Alexa Fluor 647 phalloidin (A22287, Thermo Fisher; 1:1,000 for IF); phalloidin-TRITC (P1591, Sigma, 1:2,000 for IF); thymidine (sc-296542, Santa Cruz); STLC (164739, Sigma); RO3306 (sc-358700, Santa Cruz); BioTracker NucView 530 Red Caspase 3/7 Dye (SCT103, Sigma); N-ethylmaleimide (E-3876, Sigma); protease inhibitor cocktail (4693159001, Roche); phosphatase inhibitor cocktail (P5726 and P0044, Sigma); MG132 (474787, Sigma); bafilomycin A1 (BVT-0252, Adipogen); and cycloheximide (sc-3508A, Santa Cruz).

Techniques: Transfection, Expressing, Imaging