Journal: bioRxiv

Article Title: Reliability of high-quantity human brain organoids for modeling microcephaly, glioma invasion, and drug screening

doi: 10.1101/2023.10.29.564523

Figure Lengend Snippet: A. Comparison of healthy control (IMR90) and Hi-Q brain organoids derived from mutant patients harboring mutations in CDK5RAP2 and Cockayne syndrome B gene (CSB 739) (i-iii). CDK5RAP2 brain organoids (ii) are microcephalic as they are significantly smaller than healthy organoids. On the other hand, CSB organoids are significantly larger than healthy controls. The panel shows the scale bar. At least twenty (n=20) randomly chosen day 20 organoids were analyzed across three independent batches (N=3). Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, ***P < 0.001. Data presented as mean ± SEM. B. Tissue sections of healthy control (IMR90), CDK5RAP2, and CSB 739 brain organoids (i-iii) . The magnified panel under each variety shows VZ. SOX2 labels NPCs, and TUJ-1 marks primitive cortical plate (CP). Compared to healthy organoids (IMR90), CDK5RAP2 organoids have slightly disorganized and smaller VZ. Bar graphs at the bottom quantify the average number of VZs and their diameter in each kind. Panels show the scale bar. At least twenty sections from three different organoids of each group were analyzed. Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, ***P < 0.001. Data presented as mean ± SEM. C. CSB 739 (iii) but not healthy (i) or CDK5RAP2 (ii) organoids display a significantly increased level of pH2AX-positive nuclei (magenta) indicative of DNA double-stranded breaks. The bar graph at the right quantifies the relative proportions of pH2AX-positive nuclei. Panels show the scale bar. An average of at least twenty sections from three different organoids of each group was considered. Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, ***P < 0.001. Data presented as mean ± SEM. D. CSB 739 (iii) but not healthy (i) or CDK5RAP2 (ii) organoids display a significantly increased level of TUNNEL-positive cells (magenta) indicative of dead cells. The bar graph quantifies the relative proportions of TUNNEL-positive nuclei. An average of at least twenty sections from three different organoids of each group was considered. Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, ***P < 0.001. Data presented as mean ± SEM. Panels show the scale bar. E. Hi-Q brain organoids reveal the kinetics of the apical progenitor division plane. P-Vimentin selectively labels the dividing apical progenitors at the VZ’s apical side. Healthy brain organoids (IMR90) predominantly display dividing progenitors whose division plane is horizontal to the VZ lumen (i) . In contrast, CDK5RAP2 brain organoids (ii) harbor apical progenitors whose division plane is mainly vertical to the VZ lumen. The bar diagram quantifies the distribution of the division plane. H, horizontal. V, vertical. A schematic at the right shows horizontal and vertical division planes. An average of at least fifteen sections from three different organoids of each group was considered. Statistical analysis was carried out by One-way ANOVA, followed by Tukey’s multiple comparisons test, *P < 0.1. Data presented as mean ± SEM. Panels show the scale bar.

Article Snippet: Mouse anti-Nestin (1:100, Novus Biologicals), mouse anti-SOX2 (1:50, Abcam), rabbit anti-Arl13b (1:100, Proteintech), rabbit anti-Doublecortin (1:100, Synaptic Systems), rabbit anti-MAP2 (1:200, Proteintech), rabbit anti-synapsin-1 (1:200, Cell Signalling),Rabbit anti-TUJ1(1:400, Sigma - Aldrich), Mouse anti-acetylated tubulin (1:400, Sigma-Aldrich), Mouse anti-Tau (1:100, DSHB), Rat anti-CTIP2 (1:300, Abcam), Rabbit anti-PCP4 (1:100, Proteintech), Rabbit anti-pH2AX (1:400, Cell signalling), Mouse anti-Actin (1:100, R&D systems), rabbit anti-PSD95 (1:100, Proteintech), mouse anti-PAX6 (1:100, Proteintech), mouse anti-p-vimentin (1:200, Abcam) and TUNEL staining kit (Thermo Fischer).

Techniques: Comparison, Derivative Assay, Mutagenesis

Journal: Cell Death Discovery

Article Title: The involvement of caspases in the process of nuclear removal during lens fiber cell differentiation

doi: 10.1038/s41420-023-01680-y

Figure Lengend Snippet: Ai–Ci E12 and Aii–Cii E15 lens cryosections were labeled by TUNEL assay (red), and co-immunolabeled with antibody to pH2AX (green), and the nuclear dye DAPI (blue). A – C Images are 40× tiles acquired by confocal microscopy across the lens fiber cell differentiation region, with data shown as A TUNEL, B pH2AX, and C TUNEL/pH2AX/DAPI overlay. The insets in Aii-Cii show a higher magnification of the nuclei denoted by an asterisk, highlighting that pH2AX, but not TUNEL, labels nuclei prior to their condensation. Since TUNEL assay labels double stranded DNA breaks and pH2AX can label both single and double stranded breaks, these results suggest that single-stranded breaks are executed in lens fiber cell chromatin prior to more significant fragmentation of lens DNA that accompanies nuclear elimination. Scale bar, 50 μm. Results are representative of three independent studies.

Article Snippet: Antibodies to lamin B (13435) and pH2AX (2577) were from Cell Signaling Technology.

Techniques: Labeling, TUNEL Assay, Immunolabeling, Confocal Microscopy, Cell Differentiation

Journal: Cell Death Discovery

Article Title: The involvement of caspases in the process of nuclear removal during lens fiber cell differentiation

doi: 10.1038/s41420-023-01680-y

Figure Lengend Snippet: A Model depicting the process of nuclear loss in the embryonic chick lens at E15, with the red boxed-in area the region of interest imaged as a 40× confocal tile in B i. ( B i-iv) E15 lens cryosections co- immunolabeled for CAD (green) and ICAD (red), with chromatin labeled using DAPI (blue). ( B i) CAD/ICAD overlay with the boxed in area in Bi shown at higher magnification for each label in ( B ii) DAPI, ( B iii) CAD, and ( B iv) ICAD. ( B ii- B iv) The dashed line boxed-in region where fiber cell nuclei have a highly elongated morphology (single asterisk) and in the region just prior to nuclear condensation (double asterisk) are shown at higher magnification in the panels to the right. The loss of ICAD in the nuclei of fiber cells bordering the central fiber zone where nuclei become condensed suggests CAD is activated prior to nuclear condensation. C , D E10 lens cryosection co-immunolabeled for CAD (green) and pH2AX (red) and co-stained with DAPI (blue), images acquired as a 40× tile by confocal microscopy across the central region of lens fiber cells; C CAD/pH2AX overlay, D DAPI/pH2AX overlay. Region denoted by asterisk is shown at higher magnification in the inset. The pH2AX labeling of elongated central fiber cell nuclei at E10 demonstrates that chromatin cleavage occurs at this early stage of fiber cell differentiation, prior to nuclear condensation. Scale bar, 50 μm. Results representative of 3 independent studies.

Article Snippet: Antibodies to lamin B (13435) and pH2AX (2577) were from Cell Signaling Technology.

Techniques: Immunolabeling, Labeling, Staining, Confocal Microscopy, Cell Differentiation

Journal: Cell Death Discovery

Article Title: The involvement of caspases in the process of nuclear removal during lens fiber cell differentiation

doi: 10.1038/s41420-023-01680-y

Figure Lengend Snippet: Cryosections of A – C E10 or D – F E13 lenses exposed for 48 h in organ culture to A , D DMSO, vehicle control, B , E the caspase-3 inhibitor Z-DEVD-FMK, or C , F the pan-caspase inhibitor Z-VAD-FMK, immunolabeled for pH2AX (red) and co-labeled with DAPI (blue). Insets are a higher magnification of DAPI staining alone of the boxed-in region at the center. Blocking caspases at D10 for 48 h suppressed both DNA cleavage and nuclear condensation. Blocking caspases at D13 for 48 h suppressed DNA cleavage and nuclear elimination. Scale bar, 50 μm. Results representative of 3 independent studies.

Article Snippet: Antibodies to lamin B (13435) and pH2AX (2577) were from Cell Signaling Technology.

Techniques: Organ Culture, Immunolabeling, Labeling, Staining, Blocking Assay

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR and cell cycle markers in matched normal, premalignant, and malignant human gastric specimens (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, p53 in red; Cell cycle regulators- 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. Scale bar: 100 μM.

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: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR markers linked with dsDNA breaks and cell cycle regulation markers in the carcinogen-induced mouse model of gastric premalignancy (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 (gray), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. (B) Representative IHC images of 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). Scale bar: 100 μM.

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: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score 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)” (gray) 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 and p53 status 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); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines 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 (gray) 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 (gray) 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; Scale bar: 50μm.

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, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet:

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: Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR and cell cycle markers in matched normal, premalignant, and malignant human gastric specimens (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, p53 in red; Cell cycle regulators- 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. Scale bar: 100 μM.

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-p16INK4 (1:1000, Abcam, #ab54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #ab1431), rabbit anti-p21 (1:1000, Abcam, #ab188224).

Techniques: Staining

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR markers linked with dsDNA breaks and cell cycle regulation markers in the carcinogen-induced mouse model of gastric premalignancy (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 (gray), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. (B) Representative IHC images of 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). Scale bar: 100 μM.

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-p16INK4 (1:1000, Abcam, #ab54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #ab1431), rabbit anti-p21 (1:1000, Abcam, #ab188224).

Techniques: Staining, Marker

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score 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)” (gray) 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 and p53 status 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); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines 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 (gray) 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 (gray) 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; Scale bar: 50μm.

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-p16INK4 (1:1000, Abcam, #ab54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #ab1431), rabbit anti-p21 (1:1000, Abcam, #ab188224).

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet:

Article Snippet: The primary antibodies used for immunohistochemistry were: Human tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-H2AX (1:1000, CST, #7631), rabbit anti-pH2AX ser139 (1:1000, CST, #9718), mouse anti-53BP1 (1:1000, Millipore, #3802), mouse anti-p53 (1:1000, CST, #48818), mouse anti-p16INK4 (1:1000, Abcam, #ab54210), rabbit anti-p21 (1:1000, CST, #2947), Murine tissue- rabbit anti-ki67 (1:1000, Biocare Medical, #CRM325-5b6), rabbit anti-pH2AX ser139, (1:1000, CST, #9718), rabbit anti-p53 (1:1000, Abcam, #ab1431), rabbit anti-p21 (1:1000, Abcam, #ab188224).

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR and cell cycle markers in matched normal, premalignant, and malignant human gastric specimens (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, p53 in red; Cell cycle regulators- 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. Scale bar: 100 μM.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Staining

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Evaluation of DDR markers linked with dsDNA breaks and cell cycle regulation markers in the carcinogen-induced mouse model of gastric premalignancy (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 (gray), premalignant (green), and malignant (blue) gastric lesions of four MNU treated mice. (B) Representative IHC images of 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). Scale bar: 100 μM.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Staining, Marker

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score 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)” (gray) 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 and p53 status 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); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines 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 (gray) 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 (gray) 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; Scale bar: 50μm.

Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence

Journal: iScience

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

doi: 10.1016/j.isci.2023.108169

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Article Snippet: Mouse anti-pH2AX ser139 , Cell Signaling Technology , Cat# 80312; RRID: AB_2799949.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Software

Journal: Molecular Therapy. Nucleic Acids

Article Title: Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

doi: 10.1016/j.omtn.2023.102035

Figure Lengend Snippet: Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of pH2AX (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.

Article Snippet: Then, the embryos were incubated overnight at 4°C in a 1:200 solution of the primary pH2AX antibody (CST, 9718) or Cas9 antibody (Huabio, ET1703-85) in an antibody diluent buffer.

Techniques: Injection, Fluorescence