Journal: PLoS ONE

Article Title: The Transcription Factor NFAT5 Is Required for Cyclin Expression and Cell Cycle Progression in Cells Exposed to Hypertonic Stress

doi: 10.1371/journal.pone.0005245

Figure Lengend Snippet: A) Dot plots representing γH2AX (H2AX phosphorylated in Ser 139) and DNA content in live NFAT5 +/+ and NFAT5 −/− lymphocytes after 24 hours in isotonic or hypertonic conditions. Bars on the right represent the percentage of γH2AX + cells after 24 hours in isotonic or hypertonic medium (values are the mean±SEM of seven independent experiments; * = p<0.05). B) T cells grown in isotonic or hypertonic conditions during 24 hours were stained with the DNA dye Hoechst 33342 and annexin-V-Fluos, and analyzed by flow cytometry. The experiment shown is representative of three independently performed. C) Single-cell alkaline gel electrophoresis assay (comet assay) done on the population of live cells isolated from cultures of NFAT5 −/− and wild-type cells after 6 or 24 hours in isotonic or hypertonic conditions. Etoposide-treated wild-type cells are included as a positive control. The experiment shown is representative of three independently performed. D) Cell cycle distribution of viable, γH2AX-negative cells after 24 hours in hypertonic medium (representative of seven independent experiments).

Article Snippet: Rabbit polyclonal anti-phospho-p53 (Ser15) (Cat. 9284), mouse monoclonal anti-p53 (Cat. 2524) and mouse monoclonal anti-cyclin D3 (Cat. 2936) were from Cell Signaling Technology (Danvers, MA, USA); mouse monoclonal anti-phospho-histone H2AX (Ser139, γH2AX) (Cat. 05-636) was purchased from Upstate Technologies (Lake Placid, NY, USA); mouse monoclonal anti-BrdU antibody (Cat. 555627) was purchased from BD Pharmingen (San Diego, CA, USA).

Techniques: Staining, Flow Cytometry, Nucleic Acid Electrophoresis, Single Cell Gel Electrophoresis, Isolation, Positive Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of XIST knockdown on cell proliferation and radiosensitivity of NPC cells. ( A ) Transfection efficiency of si-XIST-1 and si-XIST-2 in CNE1 and CNE2 cells was detected by qRT-PCR. ( B ) CCK-8 assay was performed to determine cell proliferation at 24 h, 48 h, and 72 h in si-XIST- or si-con-transfected CNE1 and CNE2 cells. ( C ) Colony formation assay was used to measure colony survival rate 2 weeks after CNE1 and CNE2 cells transfected with si-XIST or si-con were exposed to the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy). ( D ) CNE1 and CNE2 cells transfected with si-XIST or si-con were irradiated with 4-Gy X-rays and then subjected to immunofluorescent staining for γ-H2AX expression. ( E ) Western blot analysis of γ-H2AX expression level in CNE1 and CNE2 cells transfected with si-XIST or si-con under 4 Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Transfection, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Irradiation, Staining, Expressing, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Downregulation of lncRNA X Inactive Specific Transcript (XIST) Suppresses Cell Proliferation and Enhances Radiosensitivity by Upregulating mir-29c in Nasopharyngeal Carcinoma Cells

doi: 10.12659/MSM.905370

Figure Lengend Snippet: Effect of miR-29c overexpression on cell proliferation and radiosensitivity of NPC cells. ( A ) CCK-8 assay was carried out to evaluate cell proliferation at 24 h, 48 h, and 72 h in CNE1 and CNE2 cells transfected with miR-29c or miR-con. ( B ) The clonogenic survival curves were compared in CNE1 and CNE2 cells transfected with miR-29c or miR-con with the indicated single doses of irradiation (0, 2, 4, 6, or 8 Gy) treatment. ( C ) Immunofluorescent staining for γ-H2AX expression in CNE1 and CNE2 cells transfected with miR-29c or miR-con under 4-Gy irradiation. ( D ) Western blot analysis of γ-H2AX level in CNE1 and CNE2 cells transfected with miR-29c or miR-con with 4-Gy irradiation. * P <0.05.

Article Snippet: After being blocked with 5% nonfat milk in TBST for 1 h at room temperature, the membranes were incubated with primary antibodies, including mouse monoclonal anti-γ-H2AX (No. 2577; 1: 1000 dilution, Cell Signaling Technology, Beverly, MA) and mouse monoclonal anti-GAPDH (No. 97166; 1: 2000 dilution, Cell Signaling Technology) at 4°C overnight, and then by probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (sc-2379; 1: 1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. Reactive protein expression was visualized and detected using a chemiluminescence kit (Millipore).

Techniques: Over Expression, CCK-8 Assay, Transfection, Irradiation, Staining, Expressing, Western Blot

Journal: Cancer cell

Article Title: De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

doi: 10.1016/j.ccell.2022.07.011

Figure Lengend Snippet: (A and B) Immunoblot analysis of HOG-EV and HOG-R132H cells (A) or GSC lines (B) treated with BAY 2402234 (HOGs: 1, 3, or 10 nM; GSCs: 3 or 10 nM) or DMSO. (C) Immunoblot analysis of HOG-EV and HOG-R132H cells treated with 10 nM BAY 2402234, 5 μM lometrexol, or DMSO. (D) Quantification of 53BP1 foci formation in HOG-EV and HOG-R132H cells treated with BAY 2402234 (n ≥ 325 cells per condition per time point) and representative images. Scale bar, 5 μm; dashed lines show individual nuclei. (E–H) Immunoblot analysis and cell death assays in HOG-EV and HOG-R132H cells treated with BAY 2402234, DMSO, 15 μM dC, and/or 15 μM dT (n = 3) (E and F), or pre-treated with 1 μM palbociclib or DMSO (G and H), then treated with BAY 2402234 or DMSO (n = 3). (I and J) Representative hematoxylin and eosin, anti-γH2A.X IHC staining (I), and γH2A.X quantification (J) of MGG152 orthotopic xenografts treated with BAY 2402234 (n = 5) or vehicle (n = 2). Scale bar, 100 μm. (K and L) MALDI-MSI ion images (K) and quantified relative changes (L) of brains from mice with MGG152 orthotopic glioma xenografts treated with BAY 2402234 or vehicle. Scale bar, 3 mm. (M) Schema depicting model by which BAY 2402234 (BAY) treatment preferentially kills IDH-mutant glioma cells. For (D), data are means and variance is not displayed. For (L), Tukey plots are shown. For all other panels, data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Two-tailed p values were determined by unpaired t test.

Article Snippet: Mouse monoclonal anti-γH2A.X , Cell Signaling Technology , Cat# 9718S, RRID:AB_2118009.

Techniques: Western Blot, Immunohistochemistry, Mutagenesis, Two Tailed Test

Journal: Cancer cell

Article Title: De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

doi: 10.1016/j.ccell.2022.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-γH2A.X , Cell Signaling Technology , Cat# 9718S, RRID:AB_2118009.

Techniques: Recombinant, Gel Extraction, Irradiation, Mutagenesis, Plasmid Preparation, Software, Lysis

Journal: bioRxiv

Article Title: Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

doi: 10.1101/2022.08.16.503984

Figure Lengend Snippet: Replication stress links nucleotide depletion to differentiation. A. Percent viability (blue) and differentiation, as assessed by CD11b and GFP expression (green), for ER-Hoxa9 cells after treatment with the indicated doses of HU or APH for 96 hours (n=2). B. Log fold change of intracellular levels of dATP, dCTP, or TTP (top) and ATP, CTP, GTP, or UTP (bottom) in ER-Hoxa9 following 24h of treatment with DMSO, 1 μM BRQ, 50 μM HU, or 1 μM APH (n=4). (ns) not significant, (*) p < 0.05, (**) p < 0.005, (***) p < 0.0005, and (****) p < 0.00005, and adjusted p-values were calculated using a two-way ANOVA with the Šidák correction for multiple comparisons. C. Representative flow cytometry plots (EdU-AF647 vs. DAPI) in ER-Hoxa9 cells cultured in DMSO, 1 μM BRQ, 50 μM HU, or 1 μM APH for 4 hours then pulsed with EdU for the last 30 minutes. D. Median fluorescence intensity (MFI) of EdU incorporation during S phase in ER-Hoxa9 cells cultured in DMSO, 1 μM BRQ, 50 μM HU, or 1 μM APH for 4 hours (n=3-4). (***) p < 0.0005, (****) p < 0.00005, and p-values were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. E. Immunofluorescence images of ER-Hoxa9 cells treated with DMSO, 1 μM BRQ, or 25 μM HU for 24h then stained for DAPI (blue), RPA (yellow), and γH2AX (green). Scale bars represent 10 μm. F. Number of γH2AX foci, number of RPA foci, and RPA coefficient of variation per cell after treatment of ER-Hoxa9 cells with DMSO, 1 μM BRQ, or 25 μM HU for 24h. (****) p < 0.00005, Mann-Whitney U test. G. Schematic showing experiment to assess response to brequinar in AML cells in mice. Five million THP-1 cells expressing BFP were injected subcutaneously into the flanks of nude mice, and once tumors formed a measurable size (∼11 days) mice were treated with vehicle (PEG) or 50 mg/kg BRQ IP for 3 days, followed by tumor harvest and analysis of BFP+ cells by flow cytometry. H. Assessment of tumor size before and after vehicle (PEG) or BRQ treatment (n=4). P values from Student’s two sample t-test. I. Percentage of CD11b+ CD14-, CD11b+ CD14+, or CD11b-CD14+ THP-1 cells in tumors as assessed by flow cytometry (n=4). P values from Student’s two sample t-test. J. Percentage of pChk1-positive THP-1 cells in tumors as assessed by flow cytometry (n=4). P values from Student’s two sample t-test. K. Percentage of γH2AX-positive THP-1 cells in tumors as assessed by flow cytometry (n=4). P values from Student’s two sample t-test.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in PBS with 0.1% Tween and 1% BSA: rabbit anti-mouse RPA2 (Abcam; ab76420; 1:50), rat anti-mouse RPA2 (CST; 2208S; 1:200), and rabbit anti-mouse γH2AX (CST; 9718; 1:1000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Fluorescence, Immunofluorescence, Staining, MANN-WHITNEY, Injection

Journal: bioRxiv

Article Title: Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

doi: 10.1101/2022.08.16.503984

Figure Lengend Snippet: Replication stress drives differentiation during S phase and is independent of the replication stress response. A. (Top left) Schematic showing ER-Hoxa9 Geminin-mCherry cells that express mCherry during S/G2 and GFP as a differentiation marker. (Top right) Representative flow cytometry plots (20,000 cells) of cells treated with 1 μM BRQ for different amounts of time. (Bottom) Fold change in the percentage of GFP+ cells in G1 or S/G2 phases after treatment with DMSO, 1 μM BRQ, 50 μM HU, or 10 μM FULV for different amounts of time. All values are normalized to the median percentage of GFP+ cells in G1 or S/G2 phases at the start of the experiment. (ns) not significant, (*) p < 0.05, (**) p < 0.005, (***) p < 0.0005, and (****) p < 0.00005, and adjusted p-values were calculated using a two-way ANOVA with the Šidák correction for multiple comparisons. BRQ, brequinar. HU, hydroxyurea. FULV, fulvestrant. B. (Left) Flow cytometry plots assessing EdU incorporation versus DNA content (DAPI) for ER-Hoxa9 treated with DMSO for 24 hours or BRQ for 16, 20, or 24 hours, with CD11b+ cells colored in red. (Right) Quantification of the percentage of CD11b+ cells in S phase for cells treated with DMSO, BRQ, or HU for 0, 16, 20, or 24 hours (n=3). (***) p < 0.0005, (****) p < 0.00005, (ns) p > 0.05, and p-values are calculated using a Student’s t-test compared to all data values for DMSO treatment. C. Relative scRNAseq expression of Ung (a G1 marker), Rrm2 (S phase), and Aurka (G2/M phase), scaled from 0 to 1, for ER-Hoxa9 cells treated with 1 μM BRQ (top) or estrogen withdrawal (-E2, bottom) for 0, 12, 24, and 48 hours. Cells are plotted in UMAP space calculated from a principal components analysis conducted using only cell cycle-related genes. D. Granulocyte score for BRQ-treated (top) or E2 withdrawn (bottom) cells treated at 0, 12, 24, and 48h, plotted in the same coordinates as in (C). E. Percentage of ER-Hoxa9 cells in each cell cycle phase after 24h of growth in media with 100 ng/ml or 2 ng/ml SCF as determined by flow cytometry. F. Percentage of CD11b-expressing ER-Hoxa9 cells following 48h of treatment with DMSO, 1 μM BRQ, or 10 μM FULV in 100 or 2 ng/ml SCF media. G. Single-guide CD11b scores for negative control guides or knockdown of ITGAM (CD11b; negative control), KAT2A (a gene whose knockdown induces significant differentiation), ATR , ATM , CDKN1A , CHEK1 , and CHEK2 in THP-1 dCas9-KRAB-mCherry cells from the CRISPRi screen in . P-values were calculated using the Mann-Whitney U test compared to scores for negative control guides. H. Percentage of CD11b-expressing ER-Hoxa9 cells following 24h of treatment with DMSO, 1 μM BRQ, or 1 μM APH combined with either vehicle, ATRi (20 nM AZ20), ATRi + ATMi (80 nM AZD0156), or Chk1i (100 nM rabusertib). APH, aphidicolin. I. Percentage of CD11b-expressing ER-Hoxa9 dCas9-KRAB cells following knockdown of p21 (sgCdkn1a) or control guide expression (sgNTC) and treatment with DMSO, 1 μM BRQ, or 1 μM APH for 24 hours. J. Schematic of experiments testing the effects of compounds in G1-arrested ER-Hoxa9 cells. K. Median percentage of ER-Hoxa9 cells with at least 5 γH2AX foci after 24h of treatment with DMSO, 1 μM BRQ, 2.5 μM CIS, or 2.5 μg/ml NCS during asynchronous cycling or following G1 arrest in 2 ng/ml SCF. CIS, cisplatin. NCS, neocarzinostatin. L. Percentage of CD11b-expressing ER-Hoxa9 cells after 24h of treatment with DMSO, 1 μM BRQ, 2.5 μM CIS, or 2.5 μg/ml NCS during asynchronous cycling or following G1 arrest in 2 ng/ml SCF.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in PBS with 0.1% Tween and 1% BSA: rabbit anti-mouse RPA2 (Abcam; ab76420; 1:50), rat anti-mouse RPA2 (CST; 2208S; 1:200), and rabbit anti-mouse γH2AX (CST; 9718; 1:1000).

Techniques: Marker, Flow Cytometry, Expressing, Negative Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Characteristic DNA methylation profiles of chorionic villi in recurrent miscarriage

doi: 10.1038/s41598-022-15656-y

Figure Lengend Snippet: SPATS2L expression in chorionic villi samples. ( a ) and ( b ) Immunohistochemical analysis of SPATS2L in chorionic villi of artificial abortion (AA), and recurrent miscarriage (RM) samples. Scale bar indicates 100 μm. Arrows indicate representative cytotrophoblast cells. ( c ) Percentage of SPATS2L-positive cytotrophoblast cells (n = 14 AA, 12 RM). Error bars indicate the SD. **, P value < 0.01. ( d ) Expression of SPATS2L after treatment of HTR-8/SVneo cells with the si-RNA negative control (siNC) or si SPATS2L #1 and #2. Relative mRNA expression levels were normalized to GAPDH mRNA levels and are indicated on the y-axis. Error bars indicate the SD. *, P value < 0.05. ( e ) Protein expression by Western blotting. GAPDH protein was used as a loading control. Band densities were calculated and are indicated below the Western blots as relative values (SPATS2L/GAPDH). ( f ) Effect of si SPATS2L on the invasive ability of HTR-8/SVneo cells. Controls indicate wells with no matrigel basement membrane, while matrigel indicates wells with matrigel basement membranes. Scale bar indicates 200 μm. ( g ) Cell invasion rate of HTR-8/SVneo cells. Error bars indicate the SD. *, P value < 0.05. ( h ) Effect of si SPATS2L on migration of HTR-8/SVneo cells. The images are at 0 h (immediately after scratch) and 24 h. The black lines show the areas without cells. Scale bar indicates 200 μm. ( i ) The migration rate of HTR-8/SVneo cells. Error bars indicate the SD. *, P value < 0.05.

Article Snippet: A total of 20 μg of protein was separated by 10% SDS/PAGE gels, transferred to nitrocellulose membranes and incubated with the following primary antibodies: rabbit polyclonal anti-SPATS2L (16,938-AP, Proteintech, Rosemont, USA) and mouse monoclonal anti-GAPDH (#97,196, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Immunohistochemical staining, Negative Control, Western Blot, Migration