Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Tabulated summary of IHC stainings indicating the number of premalignant (green) and malignant (blue) gastric lesions scored for each staining criterion. DDR/replication stress markers-53BP1, H2AX, pH2AX in red; Cell cycle regulators-p53, p16, p21 in purple. B. Summary of the stainings (DDR markers and the cell cycle regulation markers) in paired premalignant and malignant gastric lesions from gastric cancer patient8. PM= Premalignant gastric lesion, M= Malignant gastric lesion. C. Representative IHC images of paired premalignant and malignant gastric lesions from a gastric cancer patient (patient8) stained for DDR markers and cell cycle regulation markers, including H&E staining. Insets represent the zoomed area for each image.

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Summary of dysplasia grade and staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal (grey), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. B. Representative IHC images of the staining pattern of proliferation marker ki67, DDR marker pH2AX, and cell cycle regulation markers (p53 and p21) in paired normal, premalignant, and malignant gastric lesions of MNU treated mouse4 (dysplasia score 5).

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Replicative stress in gastroesophageal adenocarcinoma is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1101/2023.03.27.534412

Figure Lengend Snippet: A. Correlation plot between H2AX gene expression (log2(TPM+1)) and CIN25 score (DNA damage checkpoint expression) for the CCLE gastric cancer cell lines; R squared value and p-value calculated by simple linear correlation analysis. B. H2AX expression (log2(TPM+1)) between ‘No WGD (0)’ (grey) and ‘with WGD (1/2)’ (green) groups of gastric cancer cell lines available in the BROAD institute PRISM repurposing drug screen dataset; Difference between the H2AX expression is represented as the mean±S.D.; P-value calculated by unpaired t-test. C. Ploidy abnormalities scores for the five gastric cancer cell lines. D. Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in untreated five gastric cancer cell lines and RPE1 cells (non-neoplastic cell line) representing intrinsic replication stress/double-stranded DNA breaks (DSBs); Nucleus stained with DAPI (blue). E. Quantification of data in D., expressed as mean fluorescence intensity per cell (MFI) ± S.D from the following number of cells-RPE1-718, AGS-2080, HGC27, 1031, NUGC3-2119, GSU-2092, KE39-577. F. Quantification of pH2AX staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with ‘intrinsic low-replication stress’-RPE1, AGS (grey) and ‘intrinsic high-replication stress’-GSU, KE39 (red). Data expressed as mean fluorescence intensity (MFI) of pH2AX signal per cell ± S.D from the following number of cells-RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM-350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM-1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. G. Quantification of pKAP1 staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with ‘intrinsic low-replication stress’-RPE1, AGS (grey) and ‘intrinsic high-replication stress’-GSU, KE39 (green). Data expressed as mean fluorescence intensity of pKAP1 signal per cell ± S.D from the following number of cells-RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM-350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM-1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. H. Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in prexasertib (20nM) treated cell lines with intrinsic ‘low-replication stress’-RPE1, AGS, and intrinsic ‘high-replication stress’-GSU, KE39.

Article Snippet: Cells were then washed and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, blocked and permeabilized PBS-BSA + 0.3% Triton X-100 for 15 minutes at room temperature, and incubated with primary antibodies (rabbit anti-pTIF1β/pKAP1 (Ser824) (1:500, CST # 4127S) and Mouse anti-pH2AX (1:600, CST #80312) in PBS-BSA overnight at +4°C.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Communications Chemistry

Article Title: A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance

doi: 10.1038/s42004-022-00661-z

Figure Lengend Snippet: Cancer proliferative activity ( a ) and GSH content ( b ) as well as DNA damage in tumor ( c ) and kidney ( d ) tissues. Mice bearing CT26 allografts ( n = 4 per group) were treated twice a week with equimolar concentrations of BSO-OxMal (23.5 mg/kg) or oxaliplatin (9 mg/kg) and sacrificed 24 h after the last drug dosing. Immunohistochemical staining of Ki-67 ( a ) and the DNA damage parameter pH2AX ( c ) were quantified in 8–10 regions of interest (ROI) per tumor within random non-necrotic, viable cancer regions by ImageJ software. In ( d ) cells stained positively for pH2AX in the kidney were counted within 5 ROI per kidney section. Representative images are shown for tumor sections in Supplementary Fig. and for organ sections in Supplementary Fig. . In ( b ) tumor GSH content of the respective cancer samples was quantified by a calorimetric method as described in the methods section. Data in ( b , d ) are depicted as mean ± SEM (standard error of mean). Statistical significance was tested using one-way ANOVA ( a , c ) or Student´s t test ( b , d ). In all cases: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse-specific Ki-67 rabbit mAb (#12202) and mouse-specific pH2AX rabbit mAb (#9718) were purchased from Cell Signaling Technology (Beverly, MA, USA) and used following the manufacturer’s recommendations in a dilution of 1:200 (Ki-67) and 1:500 (pH2AX) for immunohistochemical staining.

Techniques: Activity Assay, Immunohistochemical staining, Staining, Software

Journal: PLoS Pathogens

Article Title: Uropathogenic E. coli induces DNA damage in the bladder

doi: 10.1371/journal.ppat.1009310

Figure Lengend Snippet: a . LC-MS/MS quantification of the C14-Asn cleavage product released following colibactin maturation by the ClbP peptidase in bacterial pellets of UPEC strains UTI89, CFT073 and their respective Δ clbN mutants (which are not able to synthesise C14-Asn), and four clinical UPEC strains S7, S238, S245, S257. Quantifications were performed in triplicate and represented as a mean ± standard error of the mean (SEM). nd: not detected b. Formation of DNA interstrand cross-links upon exposure of linear double stranded DNA to UTI89, NC101 and their respective Δ clbP mutants as well as seven representative clinical human UPEC isolates (S7, S45, S122, S228, S238, S254, S257), visualised by electrophoresis under denaturing conditions. DNA with interstrand cross-links (red arrow) migrates at an apparent molecular weight of twice that of DNA without cross-links. In the absence of any crosslinking DNA migrates as a single denatured strand. The gel shown is representative of three independent experiments. c. S33p-RPA32 and pH2AX immunofluorescence staining of HeLa cells, 16 hours after infection with the reference NC101 strain, its Δ clbP mutant or human UPEC strains S45, S122, S228. pH2AX and S33p-RPA32 = pRPA: grayscale. Merged: green = pH2AX; magenta = S33p-RPA32; blue = DAPI; scale bar = 10 μm. Human UPEC strains from AS = asymptomatic bacteriuria; IU = cystitis; P = pyelonephritis.

Article Snippet: Slices were stained with anti-pH2AX (Rabbit, Cell Signalling S9718; or Mouse, Millipore #O5-636) at a dilution of 1:200, anti-Krt14 antibodies (Chicken, Ozyme BLE906001) at a dilution of 1:250 and anti-Upk III (Rabbit, Abcam, ab157801) at a dilution of 1:800 in MaxBlock, 0.3% Triton X100.

Techniques: Liquid Chromatography with Mass Spectroscopy, Electrophoresis, Molecular Weight, Immunofluorescence, Staining, Infection, Mutagenesis

Journal: PLoS Pathogens

Article Title: Uropathogenic E. coli induces DNA damage in the bladder

doi: 10.1371/journal.ppat.1009310

Figure Lengend Snippet: a-d . Confocal microscopic detection of ClbI-GFP expression and pH2AX in frozen bladder sections, 6 hours post infection with UTI89, hosting the p- clbI - gfp fusion plasmid. The individual channel images are shown in grayscale, the umbrella cell containing the IBC is circled with a yellow dashed line. In the merged (d) image: blue = DNA, green = ClbI-GFP, magenta = pH2AX, grey = phase contrast. The immunofluorescence staining was repeated three times on different slides, other representative images are shown in . e. Bladder tissue E . coli counts, 6 h and 24 h after infection with wild-type E . coli UTI89 (circles) or Δ clbP (squares). Each data point corresponds to one mouse (n = 6), with mean ± standard error of the mean (SEM) shown for each group. Mann-Whitney U test. f-l. Bladder sections 6 hours post inoculation with wild-type UTI89 (f, g) or the isogenic Δ clbP mutant (h-l) stained with haematoxylin-eosin (f,h,j), FISH (g,i,k) or DAPI (l, grayscale) all of which detect IBCs (arrows). In the FISH images: blue = DAPI stain DNA; green = FITC-conjugated wheat germ agglutinin (WGA) stained polysaccharides; red = bacterial cells, labelled with the universal 16S FISH probe. The images are representative of the 5 animals per group observed.

Article Snippet: Slices were stained with anti-pH2AX (Rabbit, Cell Signalling S9718; or Mouse, Millipore #O5-636) at a dilution of 1:200, anti-Krt14 antibodies (Chicken, Ozyme BLE906001) at a dilution of 1:250 and anti-Upk III (Rabbit, Abcam, ab157801) at a dilution of 1:800 in MaxBlock, 0.3% Triton X100.

Techniques: Expressing, Infection, Plasmid Preparation, Immunofluorescence, Staining, MANN-WHITNEY, Mutagenesis

Journal: PLoS Pathogens

Article Title: Uropathogenic E. coli induces DNA damage in the bladder

doi: 10.1371/journal.ppat.1009310

Figure Lengend Snippet: a-i. Immunofluorescence staining of pH2AX on paraffin-embedded bladder sections 24 hours after PBS inoculation (a-c), or UTI89 wild-type (d-f) or UTI89Δ clbP (g-i) infection. The individual fluorescence channel images are shown in grayscale. Merged images: blue = DNA, magenta = pH2AX, grey = phase contrast. Yellow arrows highlight bacteria, white dotted lines represent the basal membrane of the urothelium. Scale bar = 10 μm. The experiment was repeated twice on 5 to 8 mice, other representative images are shown in . j-l. pH2AX positive nuclei (j), Krt14 positive cells (k) and Krt14 and pH2AX positive cells (l) were counted objectively using Fiji and CellProfiler softwares on the confocal images from large mosaic of bladder sections. Each dot represents one mouse bladder. Mann Whitney U-test performed between UTI89 wild-type and UTI89 Δ clbP . m-t. Paraffin-embedded bladders sections were immuno-stained for pH2AX and Krt14 24 hours post infection with UTI89 wild-type (m-p) or UTI89Δ clbP (q-t). The individual fluorescence channel images are shown in grayscale. Merged images: blue = DNA, magenta = pH2AX, green = Krt14. Cells positive for both Krt14 and pH2AX are circled in pink. White dotted lines highlight the basal membrane of the urothelium. Scale bar = 10 μm. Other representative images are shown in .

Article Snippet: Slices were stained with anti-pH2AX (Rabbit, Cell Signalling S9718; or Mouse, Millipore #O5-636) at a dilution of 1:200, anti-Krt14 antibodies (Chicken, Ozyme BLE906001) at a dilution of 1:250 and anti-Upk III (Rabbit, Abcam, ab157801) at a dilution of 1:800 in MaxBlock, 0.3% Triton X100.

Techniques: Immunofluorescence, Staining, Infection, Fluorescence, MANN-WHITNEY