Journal: STAR Protocols

Article Title: Optimized lentiviral vector transduction of adherent cells and analysis in sulforhodamine B proliferation and chromatin immunoprecipitation assays

doi: 10.1016/j.xpro.2023.102109

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Article Snippet: Rabbit polyclonal anti-E2F1 , Cell Signaling Technology , CAT#3742S.

Techniques: Western Blot, Virus, Bacteria, Recombinant, Protease Inhibitor, Magnetic Beads, Plasmid Preparation, Control, Software, Blocking Assay, Inverted Microscopy, Transferring, Spectrophotometry, Hood

Journal: The Journal of Cell Biology

Article Title: Active RB causes visible changes in nuclear organization

doi: 10.1083/jcb.202102144

Figure Lengend Snippet: Investigation of the role of DP1, EZH2, HDAC, TOPIIα, and TOPIIβ in RB - mediated dispersion. (A) Western blots of WT and ΔCDK-RB RPE cells expressing GFP or DNDP1. MW, molecular weight. (B) E2F ChIP-qPCR and RB1 ChIP-qPCR showing that expression of DNDP1 decreased E2F1 and RB1 binding to sites in the MCM3, MCM4 promoters, compared with cells expressing only GFP. Average scores from three technical replicates were calculated per sample and per epitope. Holm–Sidak multiple t test was performed, and asterisks denote P values. Scale bar = 25 µm. (C) Effect of DNDP1 expression on cell cycle profile of the cells in A. Profile shows G1, S, and G2 cells in the different samples. DNDP1 expression in ΔCDK-RB cells interfered with G1 arrest and increased the percentage of cells in S and G2. (D) Effect of DNDP1 expression on dispersion. Quantitation of mean skeleton dot lengths after DNDP1 expression in WT RPE cells. Expression of DNDP1 in WT RPE does not cause a significant increase in mean skeleton dot length when compared with WT RPE cells or GFP-expressing WT RPE cells. (E) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with EZH2 inhibitor. (F) Western blot for WT RPE cells treated with DMSO or EZH2 inhibitor (Inh.). Note that treatment with EZH2 inhibitor reduces H3K27 trimethylation levels. (G) Western blot for WT contact inhibited (C.I.) RPE and ΔCDK-RB treated with TSA for 72 h. H4 acetylation in both WT C.I. and ΔCDK-RB cells increases after TSA treatment. (H and I) Western blot for WT RPE and ΔCDK-RB transfected with control and TOPIIα (H) or TOPIIβ (I) siRNAs. siRNA-mediated knockdown of TOPIIα reduced the levels of the appropriate endogenous protein in WT and ΔCDK-RB cells. We note that TOPIIα was expressed at lower levels in cells expressing ΔCDK-RB compared with WT RPE cells. TOPIIβ siRNA-mediated knockdown causes complete loss of endogenous TOPIIβ in both WT and ΔCDK-RB cells. It was also observed that TOPIIβ levels were lower in ΔCDK-RB, compared with WT. (J) Mean skeleton dot lengths (chromosome 7 α-satellite probe) after WT and ΔCDK-RB RPE cells were treated with control and TOPIIα siRNAs. Numbers of foci quantified for each sample ( n ) are as follows (in the order they appear on the bar graphs): D, n = 29, 48, 40, 29; E, n = 37, 82, 19, 58; J, n = 103, 75, 75, 56. Error bars are SEM. Nonparametric two-tailed Mann–Whitney U test was performed for pairs of samples indicated on graphs, and asterisks denote P values. ns, P > 0.05; ***, P ≤ 0.001. Source data are available for this figure: . Dashed lines indicate the cutoffs for defining the categories compact, diffused, and dispersed for chromosome 7 α-satellite.

Article Snippet: Solubilized chromatin was immunoprecipitated with FLAG rabbit monoclonal antibody (14793; Cell Signaling Technologies) or E2F1 rabbit polyclonal antibody (3742; Cell Signaling Technologies) overnight at 4°C.

Techniques: Dispersion, Western Blot, Expressing, Molecular Weight, ChIP-qPCR, Binding Assay, Quantitation Assay, Transfection, Control, Knockdown, Two Tailed Test, MANN-WHITNEY