3mb pp1  (Aobious Inc)

Journal: bioRxiv

Article Title: Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis

doi: 10.1101/2025.02.12.637959

Figure Lengend Snippet: (A) Representative images of 5-EU staining corresponding to nascent transcription in prometaphase RPE1 cells with indicated drug treatment. (B) Quantification of chromosomal 5-EU fluorescent signal in prometaphase MDA-MB-231 cells treated with DMSO or 200 nM BI-2536 for 2 hours. (C) Quantification of chromosomal 5-EU fluorescent signal in prometaphase MDA-MB-453 cells treated with DMSO or 200 nM BI-2536 for 2 hours. (D) Quantification of chromosomal 5-EU fluorescent signal in prometaphase MDA-MB-468 cells treated with DMSO or 200 nM BI-2536 for 2 hours. (E) Representative images of Plk1 AS prometaphase cell spreads. Cells were synchronized in STLC and collected by shakeoff, followed by 2 hours DMSO or final concentration of 10 µM 3MB-PP1 in 1 mM 5-EU and 5 µM STLC containing media. (F) Quantification of chromosomal 5-EU fluorescent signal in Plk1 AS prometaphase cell spreads showed in (E). (G) Quantification of chromosomal 5-EU fluorescent signal in prometaphase RPE1 cells treated with DMSO or 1 µM Plk1 inhibitor GSK461364 for 2 hours. For microscopic images, scale bar: 5 µm. For 5-EU quantification, each dot represents a cell, and different colors represent different biological replicates. 3 or more biological replicates were examined.

Article Snippet: Plk1 (Santa Cruz, sc-17783, 1:500), mCherry (Takara Bio, 632496, 1:500), ACA (Immunovision, HCT-0100, 1:1000), FLAG (Sigma, F1804, 1:500), GFP (Thermo Fisher, A-11120, 1:500), Halo (Promega, G9281, 1:1000), H3 (Cell Signaling, 14269, 1:1000) 5-EU (Vector Laboratories, CCT-1261-25), AzDye 594 (Vector Laboratories, CCT-1295), Alexa Fluor 488 Azide (Thermo Fisher, A10266), BI-2536 (VWR, S1109), 3MB-PP1 (Fisher, 52-958), STLC (Santa Cruz, sc-202799), Thymidine (Millipore Sigma, 6060), GSK461364 (MedChemExpress, HY-50877), IAA (Thermo Fisher, AAA1055614), PhIAA (MedChemExpress, HY-134653), α-amanitin (Santa Cruz, sc-202440A)

Techniques: Staining, Concentration Assay

Journal: bioRxiv

Article Title: Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis

doi: 10.1101/2025.02.12.637959

Figure Lengend Snippet: (A) Representative images of anaphase RPE1 cells with erroneous chromosome segregation after 5-hour treatment of final concentration of 50 µg/mL α-amanitin. (B) Quantification of erroneous chromosome segregation in anaphase RPE1 cells treated with DMSO, 50 µg/mL α-amanitin, or 10 nM BI-2536 for 5 hours. (C) Quantification of erroneous chromosome segregation in anaphase Plk1 AS cells treated with DMSO, 50 µg/mL α-amanitin, or 200 nM 3MB-PP1 for 5 hours. (D) Representative images of anaphase DLD1 tet-OsTIR1 mClover-mAID-POLR2A (POLR2A-AID) cells with erroneous chromosome segregation after 4-hour 0.5 mM IAA treatment. (E) Quantification of erroneous chromosome segregation in anaphase POLR2A-AID cells pretreated for 23-hour 1 μg/mL doxycycline, and incubate in DMSO, 0.5 mM IAA or 10 nM BI-2536 containing media for 4 hours. (F) Illustration of the design of subcellular localization-specific Plk1 activity rescue system in Plk1 AS lines. (G) Quantification of prometaphase chromosomal 5-EU fluorescent signal in indicated cell lines treated with DMSO, 10 µM 3MB-PP1, or 10 µM 3MB-PP1+200 nM BI-2536 for 2 hours with the presence of STLC and 5-EU. (H) Quantification of erroneous anaphase chromosome segregation in indicated cell lines treated with DMSO, 200 nM 3MB-PP1, or 200 nM 3MB-PP1+200 nM BI-2536 for 4 hours. For microscopic images, scale bar: 5 µm. For 5-EU quantification, each dot represents a cell, and different colors represent different biological replicates. For mitotic error quantification, each dot represents a biological replicate (>50 anaphase cells were analyzed). 3 or more biological replicates were examined.

Article Snippet: Plk1 (Santa Cruz, sc-17783, 1:500), mCherry (Takara Bio, 632496, 1:500), ACA (Immunovision, HCT-0100, 1:1000), FLAG (Sigma, F1804, 1:500), GFP (Thermo Fisher, A-11120, 1:500), Halo (Promega, G9281, 1:1000), H3 (Cell Signaling, 14269, 1:1000) 5-EU (Vector Laboratories, CCT-1261-25), AzDye 594 (Vector Laboratories, CCT-1295), Alexa Fluor 488 Azide (Thermo Fisher, A10266), BI-2536 (VWR, S1109), 3MB-PP1 (Fisher, 52-958), STLC (Santa Cruz, sc-202799), Thymidine (Millipore Sigma, 6060), GSK461364 (MedChemExpress, HY-50877), IAA (Thermo Fisher, AAA1055614), PhIAA (MedChemExpress, HY-134653), α-amanitin (Santa Cruz, sc-202440A)

Techniques: Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Polo-like kinase 1 maintains transcription and chromosomal accessibility during mitosis

doi: 10.1101/2025.02.12.637959

Figure Lengend Snippet: (A) Schematic of ATAC-seq in prometaphase arrested Plk1 AS cells after 2-hour DMSO or 10 µM 3MB-PP1 treatment. (B) IGV screenshot showed representative ATAC-seq peaks in control and Plk1 inhibited prometaphase Plk1 AS cells. Consensus, differential downregulated and upregulated peaks were shown. (C) Overlay of total mapped ATAC-seq peak profiles in control and Plk1 inhibited prometaphase Plk1 AS cells. (D) Venn diagram showed the merged peaks in control and Plk1 inhibited prometaphase Plk1 AS cells and the consensus peaks. (E) The volcano plot showed the differential expression of consensus peaks. Adjusted p-values was calculated using Benjamini-Hochberg method. Red line marked padj = 0.05. (F) Annotation profile of consensus and differential expressed peaks in control and Plk1 inhibited prometaphase Plk1 AS cells. (G) Representative ATAC-seq profiles of total peaks subsetted by gene annotation in control and Plk1 inhibited prometaphase Plk1 AS cells. (H) List of top 30 motif matched transcription factor records with enriched HOMER motif on differential peaks after Plk1 inhibition in prometaphase Plk1 AS cells. Adjusted p-value < 0.0001 (Benjamini-Hochberg method).

Article Snippet: Plk1 (Santa Cruz, sc-17783, 1:500), mCherry (Takara Bio, 632496, 1:500), ACA (Immunovision, HCT-0100, 1:1000), FLAG (Sigma, F1804, 1:500), GFP (Thermo Fisher, A-11120, 1:500), Halo (Promega, G9281, 1:1000), H3 (Cell Signaling, 14269, 1:1000) 5-EU (Vector Laboratories, CCT-1261-25), AzDye 594 (Vector Laboratories, CCT-1295), Alexa Fluor 488 Azide (Thermo Fisher, A10266), BI-2536 (VWR, S1109), 3MB-PP1 (Fisher, 52-958), STLC (Santa Cruz, sc-202799), Thymidine (Millipore Sigma, 6060), GSK461364 (MedChemExpress, HY-50877), IAA (Thermo Fisher, AAA1055614), PhIAA (MedChemExpress, HY-134653), α-amanitin (Santa Cruz, sc-202440A)

Techniques: Control, Quantitative Proteomics, Inhibition