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: Science Advances

Article Title: Human DNA topoisomerase I poisoning causes R loop–mediated genome instability attenuated by transcription factor IIS

doi: 10.1126/sciadv.adm8196

Figure Lengend Snippet: ( A and B ) Enrichment analysis of observed R loop gain with and without (w/o) DSBs at early, early/mid, and late IZs (A) and at early, early/mid, and late IZs and in TAD classes (B) assessed as in . ( C ) PLA assay of PCNA with RNAPII in cells expressing (+ doxy) or not (− doxy) TFIISm. CTRL, control samples using the indicated antibody only. Fold changes of CPT-treated versus untreated and induced versus uninduced samples are reported over each bar. Mean of analyzed cells is 800. Statistical significance was calculated with one-tailed paired t test. ( D ) Experimental protocol of 1-hour CPT treatment with single (top) or dual (bottom) labeling of HeLa cells. ( E ) Representative images of the possible cases of dual labeled cells. ( F to H ) Levels of micronuclei triggered in late G 1 , late S, or early/mid S phase [as in (E)], respectively. Micronucleus levels are reported as micronuclei (MNi) per 100 cells (left) or as fold change in RNase H1–expressing (RH) versus control cells (right). Means of analyzed cells are 250 (F), 150 (G), and 400 (H). Statistical significance was calculated comparing micronucleus distribution of treated over untreated samples with two-tailed Mann-Whitney test. ( I and J ) γH2AX detected by immunofluorescence microscopy at the end of EdU administration (I) or at the end of BrdU administration (J) as in (D). Levels of nuclear fluorescence are reported as fold change over untreated cells (number of analyzed cells is 450). Statistical significance was calculated considering treated over untreated samples with one-tailed ratio paired t test. In (C) and (F) to (J), bars show means ± SEM of three biological replicates. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human cancer HCT116 cells, U2OS, and HeLa cell lines were purchased from American Type Culture Collection (LGC Standards S.r.l., Milan, Italy).

Techniques: Expressing, Control, One-tailed Test, Labeling, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Microscopy, Fluorescence

Journal: mBio

Article Title: Unconventional N-Linked Glycosylation Promotes Trimeric Autotransporter Function in Kingella kingae and Aggregatibacter aphrophilus

doi: 10.1128/mBio.01206-15

Figure Lengend Snippet: EmaA is an adhesin and involved in autoaggregation. (A) Mean adherence to human epithelial cells by A. aphrophilus with the wild-type emaA gene, an interrupted emaA gene, and the interrupted emaA gene reverted to the wild type. (B) Mean autoaggregation of A. aphrophilus with the wild-type emaA gene, an interrupted emaA gene, and the interrupted emaA gene reverted to the wild type. Each graph shows data from biological replicates performed in triplicate. Error bars represent standard errors. An asterisk denotes a P value of <0.05 obtained from a paired t test.

Article Snippet: Quantitative adherence assays were performed as previously described using Chang epithelial cells (ATCC CCL 20.2) ( ).

Techniques: