Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1 controls cell proliferation and apoptosis in granule cell progenitors (GCPs) via E2F1 acetylation. (A) Murine cerebellum Western blot. Representative images of endogenous GLI1 and ARRB1 in murine cerebella at different days (2, 5, 7, 15) of cerebellar development. GAPDH: loading control. (B–G) GCPs experiments. Shh was added to cultures of murine cerebellar GCPs that had or had not undergone siRNA-mediated silencing of ARRB1 (siArrb1). After 48 h of Shh stimulation, GCPs were assayed for: (B) Gli1 mRNA and protein levels (as a read-out of Shh signaling activity), and ARRB1 protein level; ACTIN as loading control; (C) proliferation reflected by bromodeoxyuridine (BrdU) uptake; (D) left: p27 mRNA levels ( (D) right and : Overexpression data are consistent); (E) differentiation reflected by β III tubulin mRNA levels; (F) apoptosis evaluated by TUNEL assay, and (G) ARRB1, E2F1, E2F1-ac, cleaved CASPASE-3 protein expression levels. ACTIN and HISTONE H3: loading controls. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Western Blot, Control, Activity Assay, Over Expression, TUNEL Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: E2F1-ac targets’ transcripts after ARRB1 modulation. (A,B) Expression of pro-apoptotic E2F1-ac target genes ( Trp73, Casp3 and Casp7 ) in GCPs that had or had not undergone siArrb1 (black) or over-expression of Arrb1 (Arrb1-HA) (orange). (C,D) Expression of proliferative (grey) and epithelial mesenchymal transition (violet) E2f1-ac target genes ( Cdc25a, Birc5, Tyms, Zeb1, Zeb2, Vim and Fn1 ) in GCPs that had or had not undergone siArrb1. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001. (E) IHC staining for ARRB1 and P27 in representative mouse cerebellum sections at several differentiation stages (p2, p4, p7, p10, p15) (upper panels). Magnification ×10; insets ×40. Scale bar, 250 µm. CASPASE-3 and CDC25A indexes (expressed as percentage) calculated as DAB positive cells of total number of cells (bottom panels). Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.001 vs. p10. # p < 0.05; ## p < 0.01 vs. p15.

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Expressing, Over Expression, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1-E2F1 complex direct regulates the expression of E2F1-ac pro-apoptotic targets. (A,B) qPCR-ChIP assay of ARRB1 in GCPs stimulated or not with Shh. Immunoprecipitation with IgG was performed as control. Eluted DNA was amplified by qPCR using primers specific for the regulatory region of the indicated genes. Actin and Gapdh (not shown) were used as endogenous non-enriched regions. qPCR data are presented as percentage of ChIP input controls. Data represent means ± S.D., from at least three independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Expressing, Immunoprecipitation, Control, Amplification

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: ARRB1 controls NSCs apoptosis via E2F1 acetylation. (A) Effects of Arrb1-HA overexpression and mock transfection (control) of NSCs on ARRB1 and E2F1-ac protein levels (Western blot assay-left) and Trp73 and Cdc25a mRNA levels (right). * p < 0.05 vs. mock transfected cells (Mock). GAPDH: loading control for Western blot. (B) Western Blot analysis of endogenous ARRB1, E2F1, and E2F1-ac expression in NSCs cultured in SCM (0 h) and after 8–24 h culture in DFM. GAPDH: loading control. Effects of si-Arrb1 or scrambled control (siCtr) on (C) Trp73 and Cdc25a mRNA levels (left) and ARRB1 protein levels (Western blot assay-right) in NSCs cultured in SCM (0 h) and after 18 h culture in DFM. ACTIN: loading control for Western blot. For Western blot, densitometry values are shown below the blots and densitometric graphs are presented in . * p < 0.05 vs. scrambled control (siCtr).

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Over Expression, Transfection, Control, Western Blot, Expressing, Cell Culture

Journal: Frontiers in Cell and Developmental Biology

Article Title: β-arrestin1-E2F1-ac axis regulates physiological apoptosis and cell cycle exit in cellular models of early postnatal cerebellum

doi: 10.3389/fcell.2023.990711

Figure Lengend Snippet: Schematic model of ARRB1/E2F1-ac functions in GCPs and NSCs. Overview of roles played by ARRB1/E2F1-ac in normal cerebellar development. (A) : Committed neuronal precursors (i.e., NSCs grown in DFM, GCPs). In our previous works, we identified miR-326 as a miRNA necessary for maturation of granule cell progenitors (GCPs) into mature granule cells . Moreover, this miRNA is integrated into the first intron of the Arrb1 gene and shares the same regulatory regions as its host gene. miR-326 also contributes to ARRB1 functions by blunting proliferative signals mediated by E2F1, Hedgehog, and Notch, and by promoting cell differentiation ( ; ; ; ). Committed neuronal precursors express ARRB1 and mir-326, which regulate their development at multiple levels. Shh signaling upregulates ARRB1 levels and promotes its translocation to the nucleus. There ARRB1, in complex with P300, induces acetylation of E2F1 (E2F1-ac), redirecting the transcription factors activity from survival/proliferative gene targets towards those that promote apoptosis ( Trp73, Caspases 3 and 7 ). Interacting with CREB and P300, ARRB1 upregulates the expression and nuclear accumulation of P27, which eventually blocks cell cycle progression. miR-326 favors neuronal cell differentiations by inhibiting multiple survival/proliferative signaling: E2F1, Hedgehog (Hh) and Notch via direct binding of the 3′-UTRs of E2f1, Smo, Gli2, Notch1 and Notch2 . (B) : In NSCs, non-expression of ARRB1 and miR-326 promotes cell proliferation, survival, and stemness by favoring non-acetylated E2F1 activity and active Hedgehog (Hh) and Notch signaling.

Article Snippet: The following antibodies were used: anti-GLI1 (L42B10, Cell Signaling), anti-CASPASE-3 (D3R6Y, Cell Signaling), anti-β-ARRESTIN1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-ACTIN I-19 (sc-1616; Santa Cruz Biotechnology), anti-E2F1 C-20 (sc-193; Santa Cruz Biotechnology), anti-E2F1 (acetyl K120/K125) (AP10555SU-N, Acris Antibodies); anti-ZIC1 (ab72694; Abcam); anti-PARP p85 Fragment (G7342; Promega), anti-SP1 1C6 (sc-420X; Santa Cruz Biotechnology), anti-PCNA (2586; Cell signaling), anti-H3 (ab 1971, Abcam), and anti-GAPDH (ab8245; Abcam).

Techniques: Cell Differentiation, Translocation Assay, Activity Assay, Expressing, Binding Assay

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

Figure Lengend Snippet:

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

Journal: Cancer letters

Article Title: Identification of a novel catalytic inhibitor of topoisomerase II alpha that engages distinct mechanisms in p53 wt or p53 -/- cells to trigger G2/M arrest and senescence.

doi: 10.1016/j.canlet.2021.11.025

Figure Lengend Snippet: Fig. 7. E2F1 is stabilized in response to MPO. (A) HCT116 p53WT and (B) p53−/−cells were treated with MPO (20 μM) for 24–96 h and lysates from the nuclear and cytosolic fractions were subjected to Western blotting analysis using anti-E2F1 and anti-β actin antibodies. (C) HCT p53WT cells were treated with MPO (20 μM) for 24 h in the presence or absence of cycloheximide (CHX) (2.5–5 μg/ml) or actinomycin D (Act. D) (0.5 μg/ml) and cell lysates were subjected to Western blotting analysis and probed with anti-E2F1 or anti-β actin antibodies.

Article Snippet: The following antibodies were used in the study: Mouse monoclonal β-actin (Sigma Aldrich Co., St. Louis, MO), mouse monoclonal p53, p21, topo IIα, (BD Pharmingen, San Diego, CA, USA), mouse monoclonal phospho-p53(ser15), phospho-ATM(ser1981) (Cell Signaling Technology Inc., Danvers, MA), mouse monoclonal ATM, Chk2, Chk1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), mouse monoclonal phosphohistone H2AX(ser139), clone JBW301 (Upstate Biotechnology Inc., Lake Placid, NY), rabbit monoclonal phospho-Chk2(thr68), phospho-Chk1 (ser345) (Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal E2F1 (Cell Signaling Technology Inc., Danvers, MA), rabbit polyclonal topo IIα (TopoGEN, Inc., Columbus, OH), rabbit polyclonal ATR (Calbiochem, San Diego, CA), goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated secondary antibodies (Pierce Chemical Co., Rockford, IL).

Techniques: Western Blot