plk1 fret sensor c jun substrate plasmid 32  (Addgene inc)

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Top, experimental setup. Following 4-h treatment with 100 ng/mL nocodazole (NOC), mitotic cells were collected via shake off and treated for 1 h with PLK1i (1 μM BI-2536), Chk2i (10 μM BML-277), or DMSO, then collected and immunoblotted. Left, representative blot of A549 cells. Right, quantification of N = 3 (A549) or N = 4 (RPE-1) biological replicates. * p < 0.05, two-tailed t -test. B Following 4-hour arrest in 10 μM S-trityl- l -cysteine (STLC), inhibitors or DMSO were added for 1 h. Cells were then fixed and stained for p-TCTP. Scale bar, 5 μm. Points are individual cells ( n = 172 DMSO, 115 PLK1i, 168, Chk2i) combined from 3 experiments. * p < 0.05, two-tailed t -test of normalized replicate means. C A549 wild-type (WT) or Chk2 −/ − cells treated as in ( A ). Right, quantification of N = 3 (DMSO, Chk2i) or N = 2 (PLK1i) experimental replicates * p < 0.05, unpaired one-way ANOVA with Bonferroni multiple comparison correction. ns, not significant. D Quantification of normalized CFP/FRET change for each condition in RPE-1 and U2OS cells. Error bars SD of individual cell values combined from N = 3 (U2OS) or N = 5 (RPE-1) experimental replicates. Individual cells n = 24, RPE-1 DMSO; n = 18, RPE-1 PLK1i; n = 16, RPE-1 Chk2i; n = 56, U2OS DMSO; n = 28 U2OS PLK1i; n = 28 U2OS Chk2i. * p < 0.05, two-tailed t -test of individual cell values. DMSO values are the same as in Fig. . E Top, protein sequence alignment of kinase ATP-binding pocket residues. Putative gatekeeper residues are red. Bottom, cartoon of analog-sensitive (as)Chk2 allele. F ADP-Glo quantification of kinase activity with DMSO or 10 μM 3-MB-PP1. Points denote an experimental replicate. Error bars SD ( N = 6 WT and asChk2 DMSO; N = 3 WT and asChk2 3-MB-PP1). ns not significant, * p < 0.05, two-tailed t -test. G Quantification of relative change in CFP/FRET ratio 1 h after addition of inhibitors or DMSO in cells co-expressing PLK1 FRET and asChk2-mCherry. Each point represents one cell normalized to the average value in the DMSO condition for each replicate. Error bars SD of individual cell values combined from ≥3 experimental replicates, n = 26 DMSO, n = 16 PLK1i, n = 19 Chk2i. * p < 0.05, two-tailed t -test of individual cell values. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Two Tailed Test, Staining, Comparison, Sequencing, Binding Assay, Activity Assay, Expressing

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Representative western blot (left) and quantification (right) of U2OS cells treated with MG132 (20 μM) in combination with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), CDK1i (5 μM RO-3306), CDK1i Chk2i combination, or a DMSO vehicle control. Each point represents one experimental replicate * p < 0.05, paired 1-way ANOVA with Bonferroni multiple comparisons correction. ns not significant ( p > 0.05). Error bars SD. B Representative western blot (left) and quantification (right) of U2OS cells treated with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), AurAi (50 nM MLN-8237), AurAi Chk2i combination, or a DMSO vehicle control. * p < 0.05, one-way ANOVA with Bonferroni multiple comparisons correction. ns, not significant ( p > 0.05). Error bars SD. C Left, immunoblot of mitotic HeLa cells treated with a vehicle control or Chk2 inhibitor for 1 h. Right, immunoblot of the same samples on a PhosTag gel. The putative phosphorylated specifies of PLK1 is indicated. *, uncharacterized bands recognized by PLK1 antibody. Right, quantification of phosphorylated PLK1 (slower-migrating PLK1 band on PhosTag gel) normalized to the total PLK1 protein abundance (68 kDa band on SDS-Page gel). Each point represents one biological replicate. * p < 0.05, two-tailed t -test. Error bars SD. D Experimental setup and representative western blot of PLK1 immunoprecipitated from mitotic HeLa cells treated with DMSO or Chk2i. E Quantification of phospho-antibody signal normalized to total PLK1 abundance from ( D ). Each point represents the value from one experimental replicate. * p < 0.05, two-tailed t -test. Error bars SD. F In vitro kinase assay with recombinant HIS-PLK1, MBP-Chk2 incubated with DMSO, PLK1i, or Chk2i as marked. Recombinant proteins were incubated in kinase assay buffer in a 1:1 molar ratio with 500 μM ATP for 30 minutes at 30 °C with gentle agitation. The reaction was quenched with 5 mM EDTA before being combined with denaturing sample buffer and analyzed via western blotting. p-PLK1 T210 antibody shown is Cell Signaling Technology D5H7. *, nonspecific recognition of MBP-Chk2. This experiment has been replicated 5 times with similar results. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Western Blot, Control, Quantitative Proteomics, SDS Page, Two Tailed Test, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Incubation, Gentle

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Top, experimental setup. Bottom, relative cell viability (DMSO = 1) measured by crystal violet following 3-day treatment with PLK1 inhibitor BI-2536. Points represent average of N = 4 experimental replicate values averaged from technical triplicates. Error bars SEM of replicate averages. B Representative blots of cells treated for 3 days with either a nontargeting siRNA (control) or siRNA against Chk2 in Hela, U2OS, and A549 cells. C Quantification of cell viability following siRNA treatment and 2.27 nM BI-2536. Each point marks the mean of N = 3 HeLa, N = 4 U2OS and A549 experimental replicates, each averages from technical triplicates and normalized to DMSO-treated cells. Error bars SD of experimental replicates. p = 0.049 HeLa, p = 0.065 U2OS, p = 0.0176 A549 ratio paired two-tailed t -test of replicate mean values. D Map of Chk2 protein with indicated domains including S/T-Q cluster, FHA2, kinase, and nuclear localization sequence (NLS). Top, trace file of Chek2 locus from HCT-116 cells. Gray shading indicates the heterozygous L355P mutation. E Top, experimental setup. Bottom, representative western blot of HCT-116 cells with or without Chk2-mCherry challenged with etoposide (Etop, 10 μM) or in combination with Chk2i (10 μM BML-277) for 6 h. Numbers are quantification of p-Y15 signal normalized to GAPDH loading control and to DMSO-treated condition. This experiment has been repeated 1 other time with similar results. F Left, quantification of p-TCTP abundance in STLC-arrested HCT-116 cells transduced with a doxycycline (DOX)-inducible 3xFLAG-Chk2 construct. Cells were either treated with 0.1 μM DOX or untreated for 48 h prior to STLC addition. Each point represents one cell normalized to the average value in the untreated condition for each replicate. Error bars SD of individual cell values combined from N = 3 experimental replicates, n = 131 -DOX, n = 141 + DOX. * p < 0.05, two-tailed t -test of replicate mean values. Right, representative immunofluorescence image. Scale bar, 5 μm. G Representative western blot of HCT-116 cells with or without expression of the Chk2-mCherry rescue construct. H Quantification of relative cell viability in HCT-116 cells following 3-day treatment with BI-2536 titration. Points represent average of 3–4 experimental replicate values averaged from technical triplicates. Error bars SEM of replicate averages. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Control, Two Tailed Test, Sequencing, Mutagenesis, Western Blot, Transduction, Construct, Immunofluorescence, Expressing, Titration