Journal: Journal of Virology

Article Title: The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G 2 Cell Cycle Arrest and Positively Regulate Viral Replication

doi: 10.1128/jvi.01575-19

Figure Lengend Snippet: Fig. 5. NSs interacts with CDK1 and inhibits the cyclin B1-CDK1 complex from 744

Article Snippet: Primary 426 antibodies to CDK1 (cat. no. 9611s) and Cyclin B1 (cat. no. 9915s) were 427 purchased from Cell Signaling Technology.

Techniques:

Journal: Journal of Virology

Article Title: The Severe Fever with Thrombocytopenia Syndrome Virus NSs Protein Interacts with CDK1 To Induce G 2 Cell Cycle Arrest and Positively Regulate Viral Replication

doi: 10.1128/jvi.01575-19

Figure Lengend Snippet: Fig. 6. The interaction between CDK1 and NSs is IB-dependent. (A) Schematic of 770

Article Snippet: Primary 426 antibodies to CDK1 (cat. no. 9611s) and Cyclin B1 (cat. no. 9915s) were 427 purchased from Cell Signaling Technology.

Techniques:

Journal: bioRxiv

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.1101/2020.04.20.050674

Figure Lengend Snippet: A, B. HeLa cells expressing GFP-CHMP7 δHelix6 were imaged live (A) and the intensity of GFP-CHMP7 δHelix6 puncta was quantified during M-phase (B). Quantification of 59 cells from 9 experiments. Fluorescence traces normalised to metaphase onset (open arrowhead); nuclear envelope breakdown indicated by closed arrowhead. C. Lysates of HeLa cells stably expressing GFP-CHMP7 and subject to the indicated synchronisations were immunoprecipitated using GFP-trap resin and resolved using normal or PhosTag SDS-PAGE. Inputs and captured fractions were examined by western blotting with antisera raised against GFP, or CDK1 substrate consensus sequences ([K/H]-[pS]-[P] or [pS]-[P]-[X]-[R/K]). Western blots representative of 6 PhosTag immunoprecipitations. D. Lysates of HeLa cells stably expressing GFP-CHMP7 or GFP-CHMP7 S3A, S441A and subject to the indicated synchronisations were immunoprecipitated using GFP-trap resin and resolved using normal or PhosTag SDS-PAGE. Inputs and captured fractions were examined by western blotting with antisera raised against GFP, Histone H3 pSer10 or GAPDH (N = 3).

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Expressing, Fluorescence, Stable Transfection, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.1101/2020.04.20.050674

Figure Lengend Snippet: A. 293T cells expressing the indicated GFP-CHMP7 constructs were cultured asynchronously or synchronised in prometaphase with STLC. GFP-tagged proteins were immunoprecipitated and examined by western blotting with antisera raised against GFP and the CDK1 substrate consensus sequence [K/H]-[pS]-[P]. N = 3. B. HeLa cells stably expressing GFP-CHMP7 393-CT or GFP-CHMP7 393-CT S441A were cultured asynchronously or synchronised prometaphase with STLC, then were lysed, resolved by normal or PhosTag SDS-PAGE and examined by western blotting with antisera raised against GFP, pSer10 Histone H3 or GAPDH (N = 3). C, D. 293T cells expressing the indicated GFP-CHMP7 NT proteins were cultured asynchronously or synchronised in prometaphase with STLC, then were lysed, resolved by normal or PhosTag SDS-PAGE and examined by western blotting with antisera raised against GFP or GAPDH (N = 3). In C, Ser/Thr containing mutants selected from a previously generated panel of alanine-scanning GFP-CHMP7 NT mutations were assessed for a mobility shift in mitosis. Mobility shift was compromised by M1, M27 and M28, although M27 and M28 were poorly expressed. M1 encodes the mutations W2A, S3A, P4A, E5A; M27 encodes the mutations R106A, E107A, S108A, D109A; M28 encodes the mutations F110A, M111A, A112G, S113A. Individual mutations of S3A, S108A and S113A from M1, M27 and M28 revealed the site of phosphorylation to be Ser 3 (D).

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Expressing, Construct, Cell Culture, Immunoprecipitation, Western Blot, Sequencing, Stable Transfection, SDS Page, Generated, Mobility Shift, Phospho-proteomics

Journal: bioRxiv

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.1101/2020.04.20.050674

Figure Lengend Snippet: A. Recombinant CHMP7 was incubated with recombinant CDK1 and CCNB1 in the presence or absence of ATP and sedimented by ultracentrifugation. CHMP7 was recovered from pellet and supernatant fractions respectively, resolved by PhosTag SDS-PAGE and analysed by western blotting using antisera raised against CHMP7. B. Data from A presented as mean ± S.D., N = 4, *** P = 0.0007 by paired 2-tailed T-test. C. Recombinant CHMP7, CHMP7 S3D/S441D or CHMP7 S3E/S441E were sedimented by ultracentrifugation, recovered from pellet and supernatant fractions, and analysed by western blotting with antisera raised against CHMP7. D. Data from C presented as mean ± S.D., N = 8, *** P = 0.0004. E. Recombinant CHMP7 was incubated with recombinant CDK1 and CCNB1 in the presence or absence of ATP. Recombinant HA-LEM2 CT was added and CHMP7 was captured on magnetic anti-CHMP7 Dynabeads. Captured and input fractions were resolved by SDS-PAGE and examined by western blotting with anti-CHMP7 or anti-HA antisera. F. Data from G are presented as mean ± S.D., N = 7, P < 0.0001 by paired 2-tailed T-test. G. Recombinant HA-LEM2 CT was incubated alone, with CHMP7 or CHMP7 S3D/S441D. CHMP7 was captured on magnetic anti-CHMP7 Dynabeads. Captured and input fractions were resolved by SDS-PAGE and examined by western blotting with anti-CHMP7 or anti-HA antisera. Please note non-specific bands detected by CHMP7 antibody (*). H. Data from G are presented as mean ± S.D., N = 6, P < 0.0001 by paired 2-tailed T-test. I. Recombinant LEM2 CT , CHMP7 or CHMP7 S3D/S441D were examined by negative stain electron microscopy, either alone or in the combinations indicated. When incubated alone, no examples of polymerisation were observed. Images representative of N = 3 independent experiments. Scale bar is 25 nm.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Recombinant, Incubation, SDS Page, Western Blot, Staining, Electron Microscopy

Journal: bioRxiv

Article Title: CDK1 controls CHMP7-dependent nuclear envelope reformation

doi: 10.1101/2020.04.20.050674

Figure Lengend Snippet: A, B. CAL-51 cells homozygously edited to express mNG-CHMP7 (A) or HeLa cells stably expressing LEM2-mCh (B) were synchronised to prometaphase with STLC and then released through addition of RO-3306. Cells were imaged live through synchronised M-exit. C. Quantification of assembly onset (assembly duration presented in ) post RO3306 release. mNG-CHMP7 +/+ (onset 16.3 ± 0.88 mins; duration 3.1 ± 0.44 mins; N = 9, n = 104) or LEM2-mCh (onset 16.2 ± 2.22 mins; duration 4.1 ± 0.44 mins; N = 3, n = 80). Additional quantification of the above parameters from HeLa cells stably expressing GFP-CHMP7 (onset 16.9 ± 1.21 mins; duration 3.3 ± 0.13 mins; N = 3, n = 97) and subject to STLC arrest and RO3306 release (from ) and CAL-51 cells homozygously edited to express mNG-CHMP7 (onset 16.1 ± 2.02 mins; duration 4.2 ± 2.19 mins; N = 7, n = 39) and subject to nocodazole arrest and RO3306 release (from ) are also presented in C. A 1-way ANOVA with Tukey’s multiple comparisons revealed no significant differences between the datasets. D. E, HeLa cells stably expressing GFP-CHMP7 NT (D) or GFP-CHMP7 (E) were either untreated or arrested in mitosis through STLC inhibition and forced out of mitosis using RO3306 for the indicated times. Lysates in D, reporting Ser3 dephosphorylation were resolved by PhosTag or normal SDS-PAGE and examined with antisera raised against GFP or GAPDH respectively. Lysates from E, reporting Ser441 dephosphorylation were immunoprecipitated using GFP-trap resin and both inputs and captured fractions were examined by western blotting with antisera raised against phosphorylated CDK1 substrates and GFP (N = 3). F-H. CHMP7-depleted HeLa cells stably expressing GFP-CHMP7 R , GFP-CHMP7 R S3A/S441A or GFP-CHMP7 R S3D/S441D. were imaged live during mitotic exit (H). In F, the fold-enrichment of GFP-signal at the reforming nuclear envelope was calculated from 3 independent experiments, presented as mean (N = 3) ± S.E.M. (WT, n = 24; S3A/S441A, n = 27, n.s. (P = 0.92), S3D/S441D, n = 52, P = 0.049; 1-way ANOVA with Dunnett’s multiple comparisons). In G, the degree of furrow ingression (midzone diameter/daughter cell diameter) at the point of maximal GFP-CHMP7 recruitment was used as a proxy of progression through M-exit and was calculated from 3 independent experiments, presented as mean (N = 3) ± S.E.M. (WT, n = 24; S3A/S441A, n = 24, n.s. (P = 0.99), S3D/S441D, n = 46, P = 0.042; 1-way ANOVA with Dunnett’s multiple comparisons). In H, for cells stably expressing GFP-CHMP7 R S3A/S441A, arrowheads depict precocious clustering of CHMP7 in the peripheral ER during anaphase that develop into larger clusters during mitotic exit. In all panels, time in minutes and a scale bar of 10 μm are reported.

Article Snippet: Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S).

Techniques: Stable Transfection, Expressing, Inhibition, De-Phosphorylation Assay, SDS Page, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis

doi: 10.1038/s41467-024-48189-1

Figure Lengend Snippet: A FMNL1 schematic ( B ) MSMS scan of residues 1024–1034 (1 experiment). C Representative GFP-FMNL1 IP from cells treated with vehicle ( ~ 2 h DMSO) or adavosertib ( ~ 6.5 h 300 nM). Probed with anti-GFP and anti-phoso-CDK1 substrate sequence. Repeated 3X. D Effects of ~4 h adavosertib at 300 nM on wave. Unequal contrasting between groups for ease of viewing. E Effects of CDK1 inhibitors on wave. Asterisk denotes contrast adjustment between conditions, performed as acquisition parameters varied throughout experiment. F CDK1 siRNA treatment. G Effects of adavosertib on wave speed; bars represent means with SEM. Two-sided Student’s t-test. N = 20 and 23 cells across 4 experiments for DMSO and Wee1 inhibitor, respectively. H Effects of CDK1 inhibitors on wave area. CDK1 inhibitors tested in a larger screen; DMSO data run in parallel previously published in Extended figure 8 A of ref. . Analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons was run. n = 151 and 212 cells across 3 experiments for Ro3306 and CGP74 conditions, respectively. I Effects of CDK1 knockdown on wave area. Kruskal-Wallis test with Dunn’s multiple comparisons. n = 60 cells across 3 experiments. J CDK1 knockdown (see Figure ). Means from 3 experiments. K Effects of GFP-FMNL1 WT and S1031A on wave; staining TOMM20 and phalloidin. GFP signal for ‘GFP alone’ condition was differentially scaled. L Wave areas analyzed by Kruskal-Wallis test, Dunn’s multiple comparisons; 40 cells across 4 experiments. M Western blot of GFP-FMNL1 WT and S1031A. N Mean fluorescence: GFP-FMNL1 WT/S1031A. Bars indicate means of triangles, which represent means of 4 biological replicates. O Effects of S1031E on wave; staining for TOMM20 and phalloidin. P Effects on wave area; two-sided Mann-Whitney test, 60 cells across 3 experiments. Q Western of GFP-FMNL1 S1031E. R Mean fluorescence: GFP-FMNL1 WT/S1031E. A – R Scale bars: 10 μm for cell images, 2 μm for insets. For blots, antibody/total protein approximately aligned/scaled the same; kDa, unit of molecular weight. Box plots: center lines indicate medians, plus signs indicate means, error bars = 10–90th percentile. Source data provided. *, **, and *** indicate p -values of < 0.05, ≤ 0.005, and ≤ 0.0005 respectively.

Article Snippet: For western blots and immunofluorescence the following primary antibodies were used: TOMM20 (Santa Cruz, sc-17764), FMNL1 (Abcam, ab97456), FMNL2 (Santa Cruz, sc-390298), GFP (abcam, ab1218), GFP (Aves, 1020), phospho-CDK1 substrate consensus sequence (CST, 2325), CDK1 (abcam, ab131450), ARP3 (Proteintech, 13822-1-AP), WHAMM (abcam, ab122572).

Techniques: Sequencing, Knockdown, Staining, Western Blot, Fluorescence, MANN-WHITNEY, Molecular Weight