Journal: PLoS ONE

Article Title: Targeting Human Telomeric G-Quadruplex DNA and Inhibition of Telomerase Activity With [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+

doi: 10.1371/journal.pone.0084419

Figure Lengend Snippet: TRAP Assay with Hela cells at gradient concentrations of [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ for 24 h shown in (A), quantitative analysis of the telomerase inhibition by ELISA shown in (B). PC means positive control with HEK293 provided by the kit, NC means the negative control obtained by heating Hela cell protein for 10 min at 85 °C. Hela means Hela cells untreated with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ . 24 h, 48 h, 72 h means Hela cells respectively treated with the complex at MTT IC 50 = 120 nM for 24 h, 48 h and 72 h. Data were represented as mean +/- S.E.M. * means significant difference by GraphPad Prism5 One way ANOVA (Tukey's Multiple Comparison Test).

Article Snippet: In our experiment, we used two cancer cell lines of human cervical cancer Hela cells (RR-B51S, ATCC# PTA-5258) and chronic myelogenous leukemia cell line of K-562 (ATCC# CCL-243).

Techniques: TRAP Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Comparison

Journal: PLoS ONE

Article Title: Targeting Human Telomeric G-Quadruplex DNA and Inhibition of Telomerase Activity With [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+

doi: 10.1371/journal.pone.0084419

Figure Lengend Snippet: MTT assay was performed on human normal fibroblast cells treated with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4 A), human cervical cancer Hela cells treated with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ at gradient concentrations B), Hela cells treated with a clinical chemotherapeutic cisplatin C), K562 cells treated with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4 D) and K562 cells treated with cisplatin respectively at gradient concentrations. The inhibition ratio was collected as the average of triplicate wells relative to that of the untreated wells. Result shown here was the representative one among three independent experiments. Data were presented as mean +/- S.E.M.* means P < 0.05, ** means P < 0.005, *** means P < 0.0001 significant difference was observed by GraphPad Prism5 Two-way RM ANOVA (Bonferroni posttests).

Article Snippet: In our experiment, we used two cancer cell lines of human cervical cancer Hela cells (RR-B51S, ATCC# PTA-5258) and chronic myelogenous leukemia cell line of K-562 (ATCC# CCL-243).

Techniques: MTT Assay, Inhibition

Journal: PLoS ONE

Article Title: Targeting Human Telomeric G-Quadruplex DNA and Inhibition of Telomerase Activity With [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+

doi: 10.1371/journal.pone.0084419

Figure Lengend Snippet: A) The representative green fluorescence images taken under microscope after treatment with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ at MTT IC 50 = 120 nM at different time points, B) the percentage of green fluorescence positive cells after [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ treatment in comparison with both the negative and positive controls. NC means the untreated Hela cells used as the negative control, PC means Hela cells treated with DNase I recombinant (3000U/ml-3U/ml in 50 mM Tris-HCl, PH 7.5, 1 mg/ml BSA) for 10 min at 15-25 °C used as the positive control. 24 h, 48 h and 72 h mean Hela cells treated with [(dmb) 2 Ru(obip)Ru(dmb) 2 ] 4+ for 24 h, 48 h and 72 h respectively before labelling. Scale bar 25 um. Data were presented as mean +/- S.E.M. * means significant difference was observed by GraphPad Prism5 One way ANOVA (Tukey's Multiple Comparison Test).

Article Snippet: In our experiment, we used two cancer cell lines of human cervical cancer Hela cells (RR-B51S, ATCC# PTA-5258) and chronic myelogenous leukemia cell line of K-562 (ATCC# CCL-243).

Techniques: Fluorescence, Microscopy, Comparison, Negative Control, Recombinant, Positive Control

Journal: EBioMedicine

Article Title: A truncating MEIOB mutation responsible for familial primary ovarian insufficiency abolishes its interaction with its partner SPATA22 and their recruitment to DNA double-strand breaks

doi: 10.1016/j.ebiom.2019.03.075

Figure Lengend Snippet: The MEIOB-truncating mutation disrupts MEIOB-SPATA22 recruitment to DNA damage foci. a-b- The truncated form of MEIOB is stable when expressed in HeLa cells. a- Western blot showing the expression of the wild-type (MEIOB MF ) and the truncated forms (MEIOB MF-mut ) of MEIOB transfected in HeLa cells. The ProSieve QuadColor protein marker 4,6 kDa–300 kDa (Lonza) was run on the left (Ladder). b- MEIOB MF or MEIOB MF-mut and SPATA22 were co-expressed from a single plasmid MEIOB∧SPATA22 in HeLa cells before exposure to etoposide (25 μM). Cells were fixed in PFA without pre-permeabilisation to retain both cytoplasmic and nuclear fractions. Antibodies were used to immunostain MEIOB (red) and SPATA22 (white). Yellow arrows highlight transfected cells expressing both MEIOB and SPATA22. c-d- The truncation prevents MEIOB recruitment with SPATA22 to DSBs. MEIOB MF or MEIOB MF-mut and SPATA22 HA were expressed in HeLa cells before exposure to etoposide. Cells were permeabilised before fixation in PFA to analyse the chromatin-associated fraction of proteins. RPA foci are formed at DNA damage sites. c- MEIOB or SPATA22 alone are not recruited to DNA damage sites. d- When co-expressed, MEIOB MF and SPATA22 are recruited to DNA-damage sites and colocalise with RPA. This colocalisation is not observed for the truncated MEIOB MF-mut . Antibodies are used to immunostain MEIOB (red), SPATA22 (white), and RPA (green). Scale bar = 10 μm in all panels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: HeLa cells were used for minigene assays (CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030).

Techniques: Mutagenesis, Western Blot, Expressing, Transfection, Marker, Plasmid Preparation

Journal: iScience

Article Title: Heat-induced SIRT1-mediated H4K16ac deacetylation impairs resection and SMARCAD1 recruitment to double strand breaks

doi: 10.1016/j.isci.2022.104142

Figure Lengend Snippet:

Article Snippet: Hela human cell line , ATCC , CRL-12401; RRID: CVCL_W341.

Techniques: Virus, Generated, Control, Recombinant, Protease Inhibitor, Extraction, cDNA Synthesis, Plasmid Preparation, Software

Journal: Journal of Biological Chemistry

Article Title: Interaction of E2F7 Transcription Factor with E2F1 and C-terminal-binding Protein (CtBP) Provides a Mechanism for E2F7-dependent Transcription Repression

doi: 10.1074/jbc.m113.467506

Figure Lengend Snippet: FIGURE 1. E2F1–3 interact with E2F7 and E2F8. A, E2F1 interacts with E2F7 or E2F8. Lysate from 293 cells transfected with both HA-E2F1 and Myc-E2F7 or Myc-E2F8 was immunoprecipitated (IP) followed by immunoblotting with anti-HA and anti-Myc antibodies as indicated. Immunoprecipitation with normal mouse IgG was used as negative control. B, endogenous E2F1 interacts with E2F7. Lysate from HeLa cells was immunoprecipitated (IP) with anti-E2F1 antibody (KH95, Santa Cruz Biotechnology) followed by immunoblotting with anti-E2F7 antibody (N-20, Santa Cruz Biotechnology). C, E2F2 and E2F3 interact with E2F7 and E2F8. HA-E2F2 or HA-E2F3 was co-transfected with Myc-E2F7 or Myc-E2F8 into 293 cells. Cell lysate was immunoprecipitated (IP) and immunoblotted with anti-HA and anti-Myc antibodies as indicated. D, GST pulldown assay. Recombinant GST or GST-E2F1 (shown by Ponceau staining) was bound to the glutathi- one-Sepharose beads, incubated with in vitro-translated Myc-E2F7 or Myc-E2F8, washed with PBS buffer, and boiled in SDS sample buffer followed by immunoblotting (IB) using antibodies as indicated.

Article Snippet: Lysate from HeLa cells was immunoprecipitated (IP) with anti-E2F1 antibody (KH95, Santa Cruz Biotechnology) followed by immunoblotting with anti-E2F7 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, GST Pulldown Assay, Recombinant, Staining, Incubation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Interaction of E2F7 Transcription Factor with E2F1 and C-terminal-binding Protein (CtBP) Provides a Mechanism for E2F7-dependent Transcription Repression

doi: 10.1074/jbc.m113.467506

Figure Lengend Snippet: FIGURE 4. E2F7 mediates transcription repression through CtBPs. A, illustration of potential CtBP-binding motif (PIDLS) located at the N terminus of E2F7. B, E2F7 interacts with CtBP1 and CtBP2. 293 cells were transfected with overexpression plasmids encoding Myc-tagged CtBPs and HA-E2F7 as indicated. Cell lysate was immunoprecipitated (IP) with anti-Myc antibody followed by immunoblotting with anti-HA or anti-Myc antibodies as indicated. Immunoprecipita- tion with normal mouse IgG was used as negative control. C, endogenous E2F7 interacts with CtBPs. Lysate from HeLa cells was immunoprecipitated (IP) with anti-E2F7 antibody followed by immunoblotting with anti-CtBP1 or anti-CtBP2 antibody as indicated. Note that the lower band of CtBP1 blot is nonspecific (NS). D, ChIP-reChIP assays. 293 cells were co-transfected with HA-E2F7 and Myc-CtBP2. Cells were cross-linked after 24 h, lysed, sonicated, precleared, and immunoprecipitated with the indicated antibodies overnight followed by incubation with protein A/G beads for 2 h. After stringent washes, chromatin-bound proteins were eluted with 1 mM dithiothreitol followed by a second immunoprecipitation with the indicated antibodies. Chromatin was eluted from beads, uncross-linked,purified,andsubjectedtorealtimePCRforE2F1promoter.Two-tailedttestwasperformedforstatisticalanalysis.Theasteriskindicatesthatthe difference is significant (p 0.05). E, CtBP2 is required for E2F7-mediated repression. 293T cells (5 105 cells) were infected with lentivirus harboring short hairpin RNAs (shRNA) targeting CtBP2 (two different hairpin sequences per gene were chosen). Cells were selected with 2 g/ml puromycin for 48 h, transfected with 500 ng of HA-E2F7 for 14 h, and harvested for assays. Western blot was conducted to confirm the depletion of CtBP2 proteins (upper panel). RNA was isolated and reverse-transcribed, and the E2F1 level was measured by real time PCR. Cell line integrated with a nonsilencing shRNA was used as a control (Ctrl). All values across samples were normalized with glyceraldehyde phosphate dehydrogenase level.

Article Snippet: Lysate from HeLa cells was immunoprecipitated (IP) with anti-E2F1 antibody (KH95, Santa Cruz Biotechnology) followed by immunoblotting with anti-E2F7 antibody (N-20, Santa Cruz Biotechnology).

Techniques: Binding Assay, Transfection, Over Expression, Immunoprecipitation, Western Blot, Negative Control, Sonication, Incubation, Purification, Infection, shRNA, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control