rabbit anti phospho t288 aurka  (Cell Signaling Technology Inc)

Journal: Oncogenesis

Article Title: Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

doi: 10.1038/s41389-021-00329-8

Figure Lengend Snippet: A A2058 EV and AURKA overexpressing cells were treated with either 5 μM ICRF193 or DMSO control. 200 cells were counted for each experimental group. Cumulative mitotic index was assessed as an indicator of G2 phase delay. The bar graph shows G2 phase delay quantified as the slope of the cumulative mitotic index curve using ICRF193 and 1 μM AURKA inhibitor (AURKAi) MK5801, as single and combination treatments, or DMSO. B Immunoblot of the level of AURKA and pAURKA in a panel of melanoma cell line (MM603 is ATM checkpoint functional, SKMEL13, D20 are ATM checkpoint defective, C0125 and D35 are PP6C mutants). α-tubulin was used as a loading control. C Bar graphs of the G2 phase delay of C0125 cells treated with ICRF193, AURKAi alone, or in combination. Bars represent means of three independent experiments (±SD); ** p < 0.01; * p < 0.05.

Article Snippet: Membranes were probed with antibodies against phospho-AURKA T288 #3079, phospho-CHK2 T68 #2661, phospho-CHK1 Ser317 #2344, phospho-H2AX Ser139 #2577, B55α #2290, Histone 3 #9715 (Cell Signalling), AURKA (BD Bioscience #610938), PLK1 (Millipore) #05-844, Rad51 (14B4) # NB100-148 (Novus) and α-tubulin (Abcam).

Techniques: Control, Western Blot, Functional Assay

Journal: Oncogenesis

Article Title: Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

doi: 10.1038/s41389-021-00329-8

Figure Lengend Snippet: A Levels of pPLK T210 in S/G2 phase cells in a panel of asynchronously growing melanoma cell line (A2058 and A375 are ATM functional, SKMEL13 and D20 are ATM checkpoint defective, CO25 and D35 are PP6C mutants) were quantified on the basis of DNA content (DAPI) using high content imaging. B Box and whisker plot shows the level of pPLK1 in A2058 and A375 (EV and AURKA over-expressing) cell lines by high-content imaging. Cells were treated with or without ICRF193 for 6 h before fixing. Experiment was performed in triplicate. C G2 phase delay assessed by time lase imaging of A2058 EV, AURKA-overexpressing cells treated with ICRF193 or 100 nM PLK1 inhibitor (PLK1i) BI2536 alone, or in combination. Bars represent means (±SD) of three independent experiments. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Membranes were probed with antibodies against phospho-AURKA T288 #3079, phospho-CHK2 T68 #2661, phospho-CHK1 Ser317 #2344, phospho-H2AX Ser139 #2577, B55α #2290, Histone 3 #9715 (Cell Signalling), AURKA (BD Bioscience #610938), PLK1 (Millipore) #05-844, Rad51 (14B4) # NB100-148 (Novus) and α-tubulin (Abcam).

Techniques: Functional Assay, Imaging, Whisker Assay, Expressing

Journal: Oncogenesis

Article Title: Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

doi: 10.1038/s41389-021-00329-8

Figure Lengend Snippet: A G2 phase delay assessed by time lase imaging of PPP6C mutant C0125 cells treated with ICRF193 or 100 nM PLK1 inhibitor (PLK1i) BI2536 alone, or in combination. Bars represent means (±SD) of three independent experiments. ** p < 0.01. B Representative line graphs demonstrate the rate of cells entering into the mitosis. A375 EV and AURKA overexpressing cells were treated with 4 μM of etoposide or 5 μM of ATM inhibitor (ATMi) KU55933 or 0.5 μM of CHK1 inhibitor (CHK1i) GNE323 alone, or in combination. Statistical analysis was performed using one-way ANOVA. Bars represent means (±SD); * p < 0.05.

Article Snippet: Membranes were probed with antibodies against phospho-AURKA T288 #3079, phospho-CHK2 T68 #2661, phospho-CHK1 Ser317 #2344, phospho-H2AX Ser139 #2577, B55α #2290, Histone 3 #9715 (Cell Signalling), AURKA (BD Bioscience #610938), PLK1 (Millipore) #05-844, Rad51 (14B4) # NB100-148 (Novus) and α-tubulin (Abcam).

Techniques: Imaging, Mutagenesis

Journal: Oncogenesis

Article Title: Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

doi: 10.1038/s41389-021-00329-8

Figure Lengend Snippet: A Time dependent immunoblot analysis for γH2AX levels and quantification. Asynchronous A2058 EV and AURKA overexpressing cells were exposed to γ radiation (10 Gy), harvested at indicated hours after exposure, and immunoblotted for γH2AX. α-Tubulin was used as a loading control. [B] DNA content (DAPI intensity) of cells determined by high content imaging. γ-H2AX intensities were plotted against DNA content. Using the DNA profile, G1 and S/G2 populations were identified. B Box and whisker plot shows γH2AX intensities in G1 and S/G2 cell cycle phases. A2058 EV and AURKA overexpressing cells were either fixed as unirradiated controls (Con) or irradiated with 6 Gy and fixed immediately after irradiation (0 h), or after 8 h either without or with AURKAi MK5108 (1 μM). DNA content (DAPI) and γ-H2AX intensities were quantified in G1 and S/G2 cell cycle phases using high content imaging. C Both EV and AURKA over-expressing lines were irradiated with 6 Gy radiation then cells were fixed 24 h later and stained for DNA. Inset shows the micronuclei formed. The percentage of cells with micronuclei was in triplicate samples for each condition and cell line. 300–1000 cells were counted for each replicate. D A375 EV and AURKA overexpressing cells were exposed to γ radiation (10 Gy), treated without or with CHK1i then followed by time lapse microscopy. The cumulative mitotic index for each culture was assessed.

Article Snippet: Membranes were probed with antibodies against phospho-AURKA T288 #3079, phospho-CHK2 T68 #2661, phospho-CHK1 Ser317 #2344, phospho-H2AX Ser139 #2577, B55α #2290, Histone 3 #9715 (Cell Signalling), AURKA (BD Bioscience #610938), PLK1 (Millipore) #05-844, Rad51 (14B4) # NB100-148 (Novus) and α-tubulin (Abcam).

Techniques: Western Blot, Control, Imaging, Whisker Assay, Irradiation, Expressing, Staining, Time-lapse Microscopy

Journal: Oncogenesis

Article Title: Dysregulated G2 phase checkpoint recovery pathway reduces DNA repair efficiency and increases chromosomal instability in a wide range of tumours

doi: 10.1038/s41389-021-00329-8

Figure Lengend Snippet: A Histograms of the numbers of chromosomes in the isogenic A375 and A2058 pairs, counted in 50 mitotic chromosome spreads for each line. B The bars represent the cumulative dysregulation of PPP6C , AURKA , PLK1 and PPP2R2A /B55α as a percentage of total cases in TCGA pan cancer data set. BLCA, bladder cancer; BRCA, breast cancer; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; CESC, cervical squamous cell carcinoma; COAD, colorectal adenocarcinoma; ESCA, oesophageal adenocarcinoma; HNSC, head and neck SCC; Liver, Liver hepatocellular carcinoma; LUSC, lung SCC; OV, ovarian cancer; STAD, stomach adenocarcinoma; SKCM, melanoma; UCS, Uterine carcinoma; SARC, sarcomas. C Correlation of the cumulative pathway score for PPP6C - AURKA - PLK1 - PPP2R2A and CIN70 and HRD proliferation adjusted (prolif adj) scores for the indicated tumour types from TCGA.

Article Snippet: Membranes were probed with antibodies against phospho-AURKA T288 #3079, phospho-CHK2 T68 #2661, phospho-CHK1 Ser317 #2344, phospho-H2AX Ser139 #2577, B55α #2290, Histone 3 #9715 (Cell Signalling), AURKA (BD Bioscience #610938), PLK1 (Millipore) #05-844, Rad51 (14B4) # NB100-148 (Novus) and α-tubulin (Abcam).

Techniques: