Journal: Genes & development

Article Title: ATR inhibition induces synthetic lethality in mismatch repair-deficient cells and augments immunotherapy.

doi: 10.1101/gad.351084.123

Figure Lengend Snippet: Figure 2. ATRi preferentially induces DNA damage in MMR-d cells. (A,B) U2OS cells were transfected with control, MHL1, or MSH2 siRNA for 2 d; pulse-labeled with EdU for 20 min; and then treated with 2 µM ATRi (AZD6738) for 4 h. γ-H2AX and biotinylated EdU were analyzed by immunofluorescence. Representative images are shown in A. Frac- tions of cells positive for γ-H2AX and EdU were quantified as shown in B. Scale bar, 20 µm. (C) U2OS cells transfected with control, MLH1, or MSH2 siRNA were treated with 10 μM CDK1i (RO-3360) for 20 h followed by 2 μM ATRi (AZD6738) for 4 h. Fractions of cells positive for γ-H2AX and EdU were quantified. (D) U2OS cells were transfected with control, MLH1, MSH1, and MUS81 siRNAs as indicat- ed and then treated with 2 μM ATRi (AZD6738) for 4 h. Fractions of cells positive for γ-H2AX and EdU were quantified. Scale bar, 20 µm. (E) U2OS cells were transfected with control or MLH1 siRNA and treated with 2 μM ATRi (AZD6738) for 4 h. Cells were analyzed by PLA using γ-H2AX antibody, MSH2 antibody, or both. The numbers of PLA foci in individual cells were quantified. (Red bars) Mean PLA foci per nucleus in cell popula- tions. (F) U2OS cells were treated as in E and analyzed by PLA using γ-H2AX antibody, PCNA antibody, or both. The numbers of PLAs in individual cell foci were quantified as in E.

Article Snippet: For the γ-H2AX-PCNA PLA, U2OS cells transfected with control or MLH1 siRNA were incubated with ATRi AZD6738 for 4 h. Subsequently, cells were fixed in ice-cold methanol and permeabilized in 0.5% Triton X, and PLAwas performed using the Duolink PLA kit (Sigma) according to themanufacturer’s instructions using the γ-H2AX (Cell Signaling Technologies) and PCNA (Santa Cruz Biotechnologies) antibodies.

Techniques: Transfection, Control, Labeling, Immunofluorescence

Journal: Frontiers in Microbiology

Article Title: Intratumoral microbiota omics analysis in head and neck squamous cell carcinoma

doi: 10.3389/fmicb.2025.1711139

Figure Lengend Snippet: Construction of a 4NQO-induced HNSCC model. (A) Schematic overview of the 4NQO-induced HNSCC model (4NQO group, n = 6; CTRL group, n = 6). (B) Representative images of H&E staining in mouse tongue tissue. (C) Prevalence of normal, dysplasia and carcinoma in the tongue tissue. (D) Representative images of immunohistochemcal staining of PCNA positive cells in the tongue tissue. (E) The score for the number of PCNA positive cells in the tongue tissue ( n = 5 fields from 6mice). Data are expressed as mean ± SD. Statistical significance was determined by two-tailed Student’s t -test. 4NQO, 4-nitroquinoline-1-oxide; PCNA, proliferating cell nuclear antigen. *** p < 0.001.

Article Snippet: Sections were incubated with a primary antibody targeting Proliferating cell nuclear antigen (PCNA) (13110S, Cell Signaling Technology, Danvers, Massachusetts, United States) at a dilution of 1:4000.

Techniques: Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

doi: 10.1038/srep16922

Figure Lengend Snippet: ( A ) RT-PCR showed the expression of UCHL1, RET, GPR125, GFRA1, PLZF, MAGEA4, THY1, PCNA and SV40 in the immortalized human male germline stem cells. ACTB was used as a loading control of total RNA, whereas RNA without RT (RT-) but with PCR served as a negative control. ( B,C ) Western blots revealed the expression of SV40, PCNA, UCHL1, and RET in the human SSC line ( B ) at different passages and primary human SSCs ( C ). ACTB was utilized as a loading control of loading proteins, while replacement of primary antibodies with PBS served as negative controls (NC). Notes: passage 2 (P2); passage 5 (P5); passage 8 (P8); S1, S2, and S3 indicated 3 independent experiments of primary human SSCs.

Article Snippet: After permeabilization with 0.4% Triton X-100 and blocking with 5% donkey serum (Maibio), the sections were incubated with primary antibodies, including PCNA (Santa Cruz), UCHL1 (AbD Serotec), GPR125 (Abcam), and HumNuc (Abcam) at a 1:100 dilution overnight at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Negative Control, Western Blot

Journal: Scientific Reports

Article Title: Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

doi: 10.1038/srep16922

Figure Lengend Snippet: ( A,B ) The expression of eGFP in the control seminiferous tubules of recipient mice without cell transplantation ( A ) and with transplantation of the immortalized human male germline stem cells ( B ). Scale bars in ( A,B ) = 200 μm. ( C–H ) Immunohistochemisty illustrated the expression of PCNA ( C ), UCHL1 ( D ), GPR125 ( E ), MAGEA4 ( F ), and HumNuc ( G ), as well as co-expression of UCHL1 and HumNuc ( H ) in the seminiferous tubules of recipient mice with transplantation of the immortalized human male germline stem cells. Scale bars in ( C–E ) = 20 μm; scale bar in ( F,G ) = 30 μm; scale bar in ( H ) = 10 μm.

Article Snippet: After permeabilization with 0.4% Triton X-100 and blocking with 5% donkey serum (Maibio), the sections were incubated with primary antibodies, including PCNA (Santa Cruz), UCHL1 (AbD Serotec), GPR125 (Abcam), and HumNuc (Abcam) at a 1:100 dilution overnight at 4 °C.

Techniques: Expressing, Control, Transplantation Assay

Journal: Scientific Reports

Article Title: Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

doi: 10.1038/srep16922

Figure Lengend Snippet: The primer sequences of genes used for RT-PCR.

Article Snippet: After permeabilization with 0.4% Triton X-100 and blocking with 5% donkey serum (Maibio), the sections were incubated with primary antibodies, including PCNA (Santa Cruz), UCHL1 (AbD Serotec), GPR125 (Abcam), and HumNuc (Abcam) at a 1:100 dilution overnight at 4 °C.

Techniques: Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: ACS Omega

Article Title: Pterostilbene-Isothiocyanate Inhibits Proliferation of Human MG-63 Osteosarcoma Cells via Abrogating β-Catenin/TCF-4 Interaction—A Mechanistic Insight

doi: 10.1021/acsomega.3c02732

Figure Lengend Snippet: Effect of PTER-ITC on transcription and translation of different factors. (A) Effect of PTER-ITC on transcription level of proliferation ( PCNA ), migration ( MMP-2/9 ), metastatic ( E/N-cadherin ), and apoptotic ( Caspase-3 and PARP-1 ) markers. (B, C) Representative immunoblot images showing the effect of PTER-ITC and ZA treatments on PCNA, MMP-2/9, E/N-cadherin, active Caspase-3, and cleaved PARP-1 markers (B) along with their corresponding quantitative densitometric analysis (C). Data represented as mean ± SE of three independent experiments. Statistical significance was determined through two-way ANOVA and represented as *, **, and *** for p < 0.05, 0.01, and 0.001, respectively, when compared to respective control.

Article Snippet: Antibodies against proliferative cell nuclear antigen (PCNA) (E-AB-10010), cleaved poly [ADP-ribose] polymerase-1 (PARP-1) (E-AB-30059), matrix metalloproteinase-2 (MMP-2) (E-AB-32054), matrix metalloproteinase-9 (MMP-9) (E-AB-70247), active Caspase-3 (E-AB-22115), TCF-4 (E-AB-60206), and β-actin (E-AB-20058) were purchased from Elabscience (Texas, USA).

Techniques: Migration, Western Blot, Control

Journal: ACS Omega

Article Title: Pterostilbene-Isothiocyanate Inhibits Proliferation of Human MG-63 Osteosarcoma Cells via Abrogating β-Catenin/TCF-4 Interaction—A Mechanistic Insight

doi: 10.1021/acsomega.3c02732

Figure Lengend Snippet: List of Primers Used in This Study

Article Snippet: Antibodies against proliferative cell nuclear antigen (PCNA) (E-AB-10010), cleaved poly [ADP-ribose] polymerase-1 (PARP-1) (E-AB-30059), matrix metalloproteinase-2 (MMP-2) (E-AB-32054), matrix metalloproteinase-9 (MMP-9) (E-AB-70247), active Caspase-3 (E-AB-22115), TCF-4 (E-AB-60206), and β-actin (E-AB-20058) were purchased from Elabscience (Texas, USA).

Techniques: Sequencing