Journal: Molecular cell

Article Title: Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.

doi: 10.1016/j.molcel.2022.07.011

Figure Lengend Snippet: Figure 5. MiDAS events induced by BRCA2 inactivation map to regions of R-loop formation (A) Representative genomic regions of DRIP-seq and qDRIP-seq analyses performed in asynchronous HeLa cells (Crossley et al., 2020; Hamperl et al., 2017), RFD analysis performed in HeLa cells (Petryk et al., 2016), and EU-seq and MiDAS-seq analysis performed in BRCA2-deficient (BRCA2) H1299 cells. EU-seq was performed 100 min after release from thymidine block. (B) Average qDRIP-seq signal centered across MiDAS peaks identified in BRCA2-deficient (n = 150; BRCA2; pink) or aphidicolin-treated BRCA2-proficient (n = 346; +BRCA2 +APH; blue) H1299 cells. Span of genomic region, 300 kb. (C) Transcript levels measured by EU-seq 100 min after release from single thymidine block in BRCA2-deficient (BRCA2) H1299 cells for genes found in the vicinity (±50 kb) of MiDAS sites identified in BRCA2-deficient (BRCA2) cells or at 150 randomly selected early-replicating, R-loop-prone sites. (D) G-quadruplex (G4) density at MiDAS sites identified in BRCA2-deficient (BRCA2) cells or at 150 randomly selected early-replicating, R-loop-prone sites. (E) Quantification of RNA-DNA hybrids measured by QIBC of mean chromatin-bound GFP-RNaseH1D210N signal. Graph shows the median values obtained for each of the indicated cell-cycle stages and is representative of n = 3 independent experiments. (F) Quantification of mitotic EdU foci in HeLa expressing a DOX-inducible, FLAG-tagged RNaseH1 (RNH1-FLAG), treated as indicated and collected 9 h after release from single thymidine block with 20 mM EdU added during the final hour. Graph and error bars represent the mean and SEM of a total of 156 mitotic cells per conditions from n = 3 independent experiments. Representative images are shown. Scale bars, 16 mm. (G) Replication fork directionality (RFD) measured by OK-seq (Petryk et al., 2016) at MiDAS sites identified in BRCA2-deficient H1299 cells, which contain a single gene. 22 genes transcribed in the forward direction (green) and 32 genes transcribed in the reverse direction (red) were analyzed. Span of genomic region, 150 kb. p values were calculated using an unpaired two-tailed t test (C and D) or using one-way ANOVA followed by a Tukey test (F). **p % 0.01; ****p % 0.0001. See also Figures S4 and S5 and Tables S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ProLong Gold Antifade Mountant ThermoFisher Cat# P36930 DAPI ThermoFisher Cat# D1306 Mowiol 4-88 Sigma-Aldrich Cat# 475904 Propidium iodide solution Sigma-Aldrich Cat# P4864 PureLink RNase A ThermoFisher Cat# 12091021 Critical commercial assays Click-iT EdU Alexa Fluor 647 Cell Proliferation Kit for Imaging ThermoFisher Cat# C10340 Click-iT Nascent RNA Capture Kit ThermoFisher Cat# C10365 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit ThermoFisher Cat# C10424 TruSeq ChIP Library Preparation Kit Illumina Cat# IP-202-1012 TruSeq Stranded Total RNA with RiboZero Gold Illumina Cat# RS-122-2301 Deposited data DRIP-seq data (HeLa cells) Hamperl et al., 2017 GEO: GSM2452072 qDRIP-seq data (HeLa cells) Crossley et al., 2020 GEO: GSE134084 OK-seq data (RFD in HeLa cells) Petryk et al., 2016 https://github.com/CL-CHEN-Lab/OK- Seq/tree/master/published_results/HeLa EdU-seq BRCA2-deficient H1299 Dagg et al., 2021 GEO: GSM4650337 MiDAS-seq data This paper GEO: GSE196163 EU-seq data This paper GEO: GSE196163 Experimental models: Cell lines H1299 +shBRCA2DOX cells (male origin) Zimmer et al., 2016 N/A HeLa cells (female origin) ATCC Cat# CCL-2 HeLa +RNaseH1-FLAGDOX (female origin) Sollier et al., 2014 N/A U2OS CyclinE (tet OFF) RAD52+/+ (female origin) Sotiriou et al., 2016 N/A U2OS CyclinE (tet OFF) RAD52-/- (female origin) Sotiriou et al., 2016 N/A U2OS T-REx GFP-RnaseH1D210N (female origin) Teloni et al., 2019 N/A Oligonucleotides siGENOME BRCA2 siRNA (SMARTpool) Horizon Discovery Cat# M-003462-01 ON-TARGETplus RAD52 siRNA (SMARTpool) Horizon Discovery Cat# L-011760-00-0005 BRCA2 siRNA Qiagen Cat# SI02653434 AllStar negative control Qiagen Cat# 1027281 TP53 siRNA Dharmacon Cat# GEHCU-000146 ON-TARGETplus non-targeting control pool Horizon Discovery Cat# D-001810-10-05 Software and algorithms GraphPad Prism GraphPad https://www.graphpad.com/ Olympus ScanR Image Analysis Software 3.0.0 Olympus https://www.olympus-lifescience.com/en/ microscopes/inverted/scanr/ TIBCO Spotfire TIBCO Software https://www.tibco.com/products/tibco- spotfire Fiji Schindelin et al., 2012 https://fiji.sc/ Bedtools Quinlan and Hall, 2010 https://bedtools.readthedocs.io/en/latest/ (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 e2

Techniques: Blocking Assay, Expressing, Two Tailed Test

Journal: Chemosensors

Article Title: Increased Autonomous Bioluminescence Emission from Mammalian Cells by Enhanced Cofactor Synthesis

doi: 10.3390/chemosensors12110223

Figure Lengend Snippet: Figure 2. The effect of overexpression of different proteins on the bioluminescence emission in mammalian cell lines: (A) LiveLight HEK293; (B) HEK293; (C) HeLa. Cells grown in 24-well plates were transfected with a mixture of 0.4 µg lux plasmids and 0.1 µg of the indicated genes (all constructs in pcDNA3.1(+)). The signal was normalized to the bioluminescence emission of cells transfected with 0.4 µg lux plasmids and 0.1 µg of the empty pcDNA3.1(+) vector (−). For LiveLight HEK293, 0.5 µg of the indicated gene in pcDNA3.1(+) was transfected. Error bars represent standard deviation from 5 wells.

Article Snippet: HeLa cells were obtained from LGC Standards (Wesel, Germany, cat. no. ATCC-CCL-2).

Techniques: Over Expression, Transfection, Construct, Plasmid Preparation, Standard Deviation

Journal: Chemosensors

Article Title: Increased Autonomous Bioluminescence Emission from Mammalian Cells by Enhanced Cofactor Synthesis

doi: 10.3390/chemosensors12110223

Figure Lengend Snippet: Figure 3. Fluorescence ratio of iNap1 in Lux-expressing cells with and without Akt2CA. (A) LiveLight HEK293; (B) HEK293; (C) HeLa cells grown on coverslips were transfected with a mixture of 0.6 µg lux plasmids, 0.2 µg iNap1 pcDNA3.1 Hygro(+) and 0.2 µg Akt2CA pcDNA3.1(+) or the empty pcDNA3.1(+) vector (–). For LiveLight HEK293, 0.2 µg iNap1 pcDNA3.1 Hygro(+) and 0.8 µg Akt2CA pcDNA3.1(+) or the empty pcDNA3.1(+) vector were transfected. iNap1 fluorescence excited at 405 (F405) and 491 nm (F491) was recorded with a custom-built microscope. Error bars represent standard deviation from at least 50 cells. ** and *** represent p values of <0.01 and <0.001, respectively, calculated by a 2-tailed Student’s t test.

Article Snippet: HeLa cells were obtained from LGC Standards (Wesel, Germany, cat. no. ATCC-CCL-2).

Techniques: Fluorescence, Expressing, Transfection, Plasmid Preparation, Microscopy, Standard Deviation

Journal: Chemosensors

Article Title: Increased Autonomous Bioluminescence Emission from Mammalian Cells by Enhanced Cofactor Synthesis

doi: 10.3390/chemosensors12110223

Figure Lengend Snippet: Figure 5. MTT assay of cells transfected with Akt2CA-P2A-RFK. (A) LiveLight HEK293; (B) HEK293; (C) HeLa cells grown in 24-well plates were transfected with a mixture of 0.1 µg Akt2CA-P2A-RFK and 0.4 µg of the empty vector or 0.5 µg of the empty vector (−) (all constructs in pcDNA3.1(+)). For LiveLight HEK293, 0.5 µg of the empty vector or the Akt2CA-P2A-RFK plasmid was transfected. Absorbance of cell lysates was measured at 570 nm. Error bars represent standard deviation from 10 wells.

Article Snippet: HeLa cells were obtained from LGC Standards (Wesel, Germany, cat. no. ATCC-CCL-2).

Techniques: MTT Assay, Transfection, Plasmid Preparation, Construct, Standard Deviation

Journal: Chemosensors

Article Title: Increased Autonomous Bioluminescence Emission from Mammalian Cells by Enhanced Cofactor Synthesis

doi: 10.3390/chemosensors12110223

Figure Lengend Snippet: Figure 4. Effect of combined expression of RFK and Akt2CA on the bioluminescence emission in different mammalian cell lines: (A) LiveLight HEK293; (B) HEK293; (C) HeLa. Cells were grown in 24-well plates and transfected with a mixture of 0.4 µg lux plasmids and 0.1 µg of the indicated constructs (all in pcDNA3.1(+)). For RFK + Akt2CA, two separate plasmids containing RFK and Akt2CA were cotransfected (0.05 µg each). The signal was normalized to cells transfected with 0.4 µg lux plasmids and 0.1 µg of the empty pcDNA3.1(+) vector (−). For LiveLight HEK293, 0.5 µg of the indicated constructs was transfected. Error bars represent standard deviation from 5 wells.

Article Snippet: HeLa cells were obtained from LGC Standards (Wesel, Germany, cat. no. ATCC-CCL-2).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Standard Deviation

Journal: Chemosensors

Article Title: Increased Autonomous Bioluminescence Emission from Mammalian Cells by Enhanced Cofactor Synthesis

doi: 10.3390/chemosensors12110223

Figure Lengend Snippet: Figure 6. Bioluminescence of HeLa cells with and without Akt2CA-P2A-RFK. Cells grown on coverslips were transfected with a mixture of 0.8 µg lux plasmids and (A) 0.2 µg empty pcDNA3.1(+) vector or (B) 0.2 µg Akt2CA-P2A-RFK (all constructs in pcDNA3.1(+)). Bioluminescence emission was recorded using the indicated exposure times. The colormap was adjusted to the minimum and maximum pixel value of each image.

Article Snippet: HeLa cells were obtained from LGC Standards (Wesel, Germany, cat. no. ATCC-CCL-2).

Techniques: Transfection, Plasmid Preparation, Construct

Journal: EMBO Molecular Medicine

Article Title: ENOblock synergizes with colistin to treat Acinetobacter baumannii infections

doi: 10.1038/s44321-025-00331-2

Figure Lengend Snippet: ( A ) Time-kill curves of E. coli Ec MCR1 + and Kp10 strains in the presence of 2xMIC ENOblock for 24 h. ( B ) Membrane permeabilization of E. coli Ec MCR1 + and Kp10 strains in the presence of 0.5xMIC ENOblock, incubated for 10 min, was quantified by Typhon Scanner. Data are represented as mean ± SEM from three independent replicates and experiments. * P = 0.001: Ec MCR1 + vs Ec MCR1 + + ENOblock(two-tailed Student’s t test). ( C ) Analysis of E. coli Ec MCR1 + and Kp10 strains adhesion into HeLa cells with (1xMIC) and without ENOblock treatment. The data are presented as means ± SEM, * P = 0.015: Ec MCR1 + vs Ec MCR1 + + ENOblock and * P = 0.02: Kp10 vs Kp10 + ENOblock (two-tailed Student’s t test). Analysis of E. coli Ec MCR1 + and Kp10 strains invasion into HeLa cells with (1xMIC) and without ENOblock treatment. The data are presented as means ± SEM, * P = 0.002: Ec MCR1 + vs Ec MCR1 + + ENOblock and * P = 0.037: Kp10 vs Kp10 + ENOblock treatment (two-tailed Student’s t test).

Article Snippet: HeLa Cell Line , LGC Standards , Cat# ATCC-CCL-2.

Techniques: Membrane, Incubation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: ENOblock synergizes with colistin to treat Acinetobacter baumannii infections

doi: 10.1038/s44321-025-00331-2

Figure Lengend Snippet: ( A ) Schematic of the bacterial adhesion/invasion assay. ( B ) Analysis of Ab ATCC 17978 and Ab CR17 strains adhesion into HeLa and macrophage cells with (1xMIC) and without ENOblock treatment. The data are presented as means of three biological replicates ± SEM, For HeLa cells, * P = 0.023: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.048: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). For macrophage cells, * P = 0.018: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.035: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). ( C ) Analysis of Ab ATCC 17978 and Ab CR17 strains invasion into HeLa and macrophage cells with (1×MIC) and without ENOblock treatment. The data are presented as means of three biological replicates ± SEM, For HeLa cells, * P = 0.001: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.011: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). For macrophage cells, * P = 0.001: Ab ATCC 17978 vs Ab ATCC 17978 + ENOblock and * P = 0.002: Ab CR17 vs Ab CR17 + ENOblock (two-tailed Student’s t test). ( D ) Immunostaining of fibronectin of HeLa cells (magenta) and Ab ATCC 17978 and Ab CR17 strains (green) pretreated with ENOblock (0× and 1×MIC), after bacterial adherence for 2 h, was performed by specific primary antibodies against both strains and their respective secondary antibodies. Blue staining shows the location of HeLa cell nuclei. A representative image out of three biological replicates is shown. .

Article Snippet: HeLa Cell Line , LGC Standards , Cat# ATCC-CCL-2.

Techniques: Invasion Assay, Two Tailed Test, Immunostaining, Staining

Journal: EMBO Molecular Medicine

Article Title: ENOblock synergizes with colistin to treat Acinetobacter baumannii infections

doi: 10.1038/s44321-025-00331-2

Figure Lengend Snippet: ( A ) PCA plot of TMT data from Ab infected macrophages and epithelial cells of three biological replicates each group. ( B ) Venn diagrams showing the overlap of DEGs between the two comparisons: Ab infected macrophages vs Ab infected epithelial cells. The upper panel shows overlaps of overexpressed proteins, while the lower panel shows overlaps of downexpressed proteins. A total of six proteins were found to be commonly overexpressed in Ab upon exposure to both epithelial and macrophage cells: enolase, tyrosine–tRNA ligase, malonyl-CoA:acyl carrier protein transacylase, N-succinylarginine dihydrolase, carbonic anhydrase, and biotin carboxylase. Additionally, one protein, isocitrate dehydrogenase, was consistently downregulated in response to both cell types. ( C ) COG category distribution of DEPs. The overexpressed and downexpressed proteins in each comparison are categorized by their COG functional groups, with blue representing downexpressed proteins and orange representing overexpressed proteins. ( D ) Volcano plots depicting the DEPs for the two comparisons. Proteins significantly overexpressed are shown in red, while significantly downexpressed proteins are shown in blue. Enolase overexpression is shown in green. Non-significant proteins are indicated in black. Three biological replicates from each group were compared. ( E ) A. baumannii Ab ATCC 17978 and Δ eno strains, with or without supplementation of 4 mM phosphoenolpyruvate, adhesion to HeLa cells. The data are presented as means of three biological replicates ± SEM, and Student t test was used for statistical analysis. * P = 0.006: Ab ATCC 17978 vs Δ eno and * P < 0.001: Ab ATCC 17978 vs Δ eno + PEP (two-tailed Student’s t test). MΦ macrophage cells, Epit epithelial cells. .

Article Snippet: HeLa Cell Line , LGC Standards , Cat# ATCC-CCL-2.

Techniques: Infection, Comparison, Functional Assay, Over Expression, Two Tailed Test