Journal: Nature Communications

Article Title: Patient-specific cancer genes contribute to recurrently perturbed pathways and establish therapeutic vulnerabilities in esophageal adenocarcinoma

doi: 10.1038/s41467-019-10898-3

Figure Lengend Snippet: Cancer helper role of E2F1 and MCM7 . a E2F1 and MCM7 expression in EACs with amplification ( n = 11 samples each) compared to copy number neutral EACs ( n = 81 samples each). Significance was assessed by Wilcoxon rank-sum test. b E2F1 and MCM7 mRNA expression in FLO-1 cells. Lower and upper hinges and middle line of boxplots correspond to 25th, 75th, and 50th percentiles. Upper and lower whiskers extend less than 1.5 times the interquartile range. c Proliferation of FLO-1 cells overexpressing E2F1 or MCM7 compared to control. d . EdU (5-ethynyl-2′-deoxyuridine) incorporation by flow cytometry in E2F1 overexpressing cells compared to control. Cells were separated into G1, S and G2 phases. S phase cells were subdivided into 4 gates (S1-S4, Supplementary Fig. ). Differences in EdU geometric mean fluorescence intensity were assessed by Mann-Whitney U test. MCM complex loading onto chromatin in MCM7 overexpressing or control cells through MCM7 e or MCM3 f staining. Cells were pulsed with EdU and chromatin was fractioned before staining for MCM7 or MCM3 to detect chromatin-bound MCM complex. Cells were separated into cell cycle phases using EdU and DAPI intensity. MCM7 or MCM3 fluorescence intensity during S phase illustrates MCM complex unloading from chromatin. Differences in geometric mean fluorescence intensity of MCM staining were assessed using Mann-Whitney U test. For (d), (e), (f) one representative of n = 3 biological replicates is shown. Corresponding pseudocolour plots are in Supplementary Fig. . g MCM7 mRNA expression in MFD-1 and FLO-1 cells. h qRT-PCR MCM7 expression in MFD-1 cells after transduction with a MCM7 -shRNA inducible lentiviral vector. Expression was assessed with or without 96 h doxycycline treatment. i Proliferation curve of MFD-1 cells with or without doxycycline-induced MCM7 knockdown. For all qRT-PCR experiments, expression was relativised to β-2-microglobulin and normalised to FLO-1 cells. N = 2 biological replicates were performed, each in technical triplicate. For all proliferation assays, n = 2 biological replicates were performed, each with four technical replicates. Proliferation was assessed every 24 h and normalised to time zero. Mean values at 72 h were compared by two-tailed Student’s t -test. Error bars show standard deviation. Source data are available in the Source Data file

Article Snippet: The vectors pCMVHA E2F1 (Item ID 24225, Addgene), pLX_TRC317 (TRCN0000481188, Sigma-Aldrich) and pcDNA3.1 + /C-(K)-DYK (Clone ID: OHu19407D, Genscript) were used to induce E2F1 , MCM7 , and PAK1 overexpression, respectively.

Techniques: Expressing, Amplification, Control, Flow Cytometry, Fluorescence, MANN-WHITNEY, Staining, Quantitative RT-PCR, Transduction, shRNA, Plasmid Preparation, Knockdown, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: To investigate how α1 affects platelet GPCR signaling function and its interaction with certain platelet GPCRs, we transfected COS-7 cells (ATCC, CRL-1651) with plasmids encoding P2Y12 (item#66471, Addgene), F2RL3 (PAR4) (item #66279), TP (item #66517), or A2BR (item#37202) using LipofectamineTM 3000 Transfection Reagent (L3000015, ThermoFisher Scientific).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control