Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: DOT1L facilitates PARPi resistance via H3K79 methylation. A . Heatmaps of H3K79me2 levels detected by CUT&Tag around gene body regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. 3 kb windows spanning the TSS to TES of all genes were plotted. Genes were arranged by their enrichment of H3K79me2 in shNC and shDOT1L cells. B . The distributions of H3K79me2-binding regions are shown in the pie charts. C . Venn diagram showing the overlap between RNA-seq data and CUT&Tag data. The KEEP analysis revealed the significantly enriched items based on H3K79me2 signature. D . IGV tracks showing the enrichment of H3K79me2 in ABCB1 and PLCG2 gene regions in control (shNC) and DOT1L knockdown (shDOT1L) SKOV-3 cells treated with Olaparib 10µM for 48 h. E - F . ChIP–qPCR showing the level of the indicated proteins recruited to the PLCG2 ( E ) and ABCB1 ( F ) promoter regions in DOT1L-overexpressed OVCAR8 cells. The data represent the means ± SD ( n = 3). * p < 0.05. three independent sets of PLCG2 and ABCB1 primers were used. G . RT-qPCR was performed in DOT1L overexpressed OVCAR8 cells to determine PLCG2 and ABCB1 mRNA levels. H. PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L overexpressed OVCAR8 cells. I . An H3K79me2-ChIP assay was performed in DOT1L knockdown SKOV-3 cells to examine H3K79me2 occupancy at PLCG2 and ABCB1. J . RT-qPCR was performed in DOT1L knockdown SKOV-3 cells to determine PLCG2 and ABCB1 mRNA levels. K . PLCG2 and ABCB1 (P-gly) expression was measured by western blotting in DOT1L knockdown SKOV-3 cells

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Methylation, Control, Knockdown, Binding Assay, RNA Sequencing, ChIP-qPCR, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: PARP1-DOT1L transcription axis drives acquired resistance to PARP inhibitor in ovarian cancer

doi: 10.1186/s12943-024-02025-8

Figure Lengend Snippet: PARP1-DOT1L-PLCG2/ABCB1 axis contributes to PARPi resistance. A . H3K79me2-ChIP assay was performed with OVCAR8 Olaparib-resistant and parent OVCAR8 cell lines to determine H3K79me2 occupancy at PLCG2 and ABCB1. B . PLCG2 and ABCB1 mRNA levels were determined in R8 OlaR and parent OVCAR8 cells by RT-qPCR. C . Western blotting was performed in Olaparib-resistant OVCAR8 and parent OVCAR8 cell lines to examine PLCG2 and ABCB1 (P-gly) protein expression levels. D . Western blotting was performed in R8 OlaR and parent OVCAR8 cells which were transfected with shNC and DOT1L shRNA respectively with the indicated antibodies. E . R8 OlaR and parent OVCAR8 cells were transfected with shNC, ABCB1 shRNA and PLCG2 shRNA. After 72 h of transfection, cells were collected and analyzed by western blotting with the indicated antibodies. F – G . Colony formation ( F ) and cell apoptosis assay ( G ) were performed in R8 OlaR and parent OVCAR8 stably transfected cell lines. H . Immunohistochemistry (IHC) staining of DOT1L, PARP1, ABCB1 (P-gly), and PLCG2 in PARP inhibitor-resistant human ovarian carcinomas (OC) tissues and sensitive tissues. Representative images are shown. Scale bars: 400 μm (upper); 160 μm (lower). I – K . Correlation analysis between PARP1 and DOT1L(I), DOT1L and P-gly ( J ), and DOT1L and PLCG2 ( K ) were analyzed. L . Quantification of P-gly (right) and PLCG2 (left) expression in PARP inhibitor-resistant OC tissues ( n = 6) and sensitive tissues ( n = 9), ** p < 0.01

Article Snippet: The antibodies in this study included: H3K79me2 (ab3594, Abcam), Histone H3 (ab1971, Abcam), DOT1L (A300-953 A, Bethyl; sc-390,879, Santa Cruz), PARP1 (13371-1-AP, Proteintech), Flag (F1804, Sigma), P glycoprotein (22336-1-AP, Proteintech), PLCG2 (PTM-6859, PTMBIO), β-tubulin (10068-1-AP, Proteintech), and γ-H2AX (#2577, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, shRNA, Apoptosis Assay, Stable Transfection, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Human Placental Mesenchymal Stem/Stromal cells (pMSCs) inhibit agonist‐induced platelet functions reducing atherosclerosis and thrombosis phenotypes

doi: 10.1111/jcmm.16848

Figure Lengend Snippet: pMSCs demonstrate differential expression of genes involved in coagulation and thrombosis: Real‐time PCR analysis (RT‐PCR) analysis of pro‐ and anti‐thrombotic genes expressed in pMSCs in comparison to HUVECs. (A) Genes inhibiting thrombosis and coagulation. TFPAI (Tissue Factor Pathway Inhibitor), PROC1 (Vitamin K‐dependent protein C1), PROS1 (Protein S1), SERPINC1 (Serpin Family C Member 1) and THBS4 (Thrombospondin 4). (B) Genes promoting thrombosis and coagulation. vWF (Von Willebrand Factor), GP6 (Glycoprotein VI platelet), PLCG2 (Phospholipase C Gamma 2), GRK5 (G Protein‐Coupled Receptor Kinase 5) and PLEK (Pleckstrin). Three independent experiments were performed for each cell type isolated from three different placentae. The data are expressed as fold change difference calculated from the ΔΔ −2 values. Bars represent mean ± SE. * p ≤ 0.05

Article Snippet: Primary antibodies to Human GP VI (cat# AF3627); Human/Mouse PLC‐gamma 2 (cat# MAB3716); Human TFPAI (cat#AF2974) and Human Protein S/PROS1 (cat# MAB4036) were purchased from R&D Systems, whereas antibodies to PROC1 (catalog# 25382‐1‐AP) and Low Density Lipoprotein from Human Plasma, oxidized (OxLDL) kit (cat# L34357) were purchased from Thermo Fisher Scientific.

Techniques: Quantitative Proteomics, Coagulation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Comparison, Isolation

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: SMR discovery and experimental validation of NETs-related biomarkers in osteoporosis. A, Forest plot summarizing SMR results for VDAC1, PLCG2 and NCF2 (β, SE, P, HEIDI P, and OR ± 95% CI). B, Representative IHC images of distal femur (brown = positive DAB; hematoxylin counter-stain) showing increased staining for the three proteins in OVX rats versus Sham; scale bar = 50 µm. C, Western blots of neutrophil lysates (Control vs PMA-induced NETs) probed for NCF2 (65 kDa), VDAC1 (31 kDa) and PLCG2 (148 kDa); GAPDH (36 kDa) serves as loading control. D–F, Quantification of IHC-positive area (% of marrow) for each protein (mean ± SD). G–I, Densitometric ratios of target proteins to GAPDH in western blots (mean ± SD). P < 0.05*, < 0.01 **, < 0.001 ***, < 0.0001 **** (two-tailed t-test).

Article Snippet: The sections were incubated in a blocking solution to reduce non-specific binding, followed by overnight incubation at 4°C with primary antibodies: VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), and NCF2 (Novus Biologicals, NBP3-16257).

Techniques: Biomarker Discovery, Staining, Western Blot, Control, Two Tailed Test

Journal: Redox Report : Communications in Free Radical Research

Article Title: Neutrophil extracellular traps drive osteoporosis via NCF2-dependent signaling: integrated transcriptomics with mechanistic validation

doi: 10.1080/13510002.2025.2534745

Figure Lengend Snippet: Docking results of available proteins small molecules. (A) NCF2 docking Diltiazem. (B) VDAC1 docking Diltiazem. (C) PLCG2 docking Exemestane. (D) NCF2 docking Cimetidine. (E) VDAC1 docking Cimetidine. (F) NCF2 docking Desipramine. (G) NCF2 docking Profenamine. (H) VDAC1 docking Emodin. (I) VDAC1 docking Tolazoline. (J) VDAC1 docking Zuclopenthixol. (K) VDAC1 docking Ethynyl estradiol. (L) PLCG2 docking Simvastatin.

Article Snippet: The sections were incubated in a blocking solution to reduce non-specific binding, followed by overnight incubation at 4°C with primary antibodies: VDAC1 (Novus Biologicals, NBP3-15871), PLCG2 (Novus Biologicals, NBP1-86030), and NCF2 (Novus Biologicals, NBP3-16257).

Techniques:

Journal: Biomedicines

Article Title: Targeting the PTN/PTPRZ1-ROS Pathway to Promote Bone Regeneration

doi: 10.3390/biomedicines13030695

Figure Lengend Snippet: PTN-induced PLCG1 phosphorylation and NCOA3 nucleus translocation. ( A , B ) High-throughput mass spectrometry was performed to explore the phosphate homeostasis changes after PTN induction. PTN significantly induced phosphorylation profile changes, as shown in the histogram ( A ) and volcano diagram ( B ). (n = 3). ( C , D ) GO ( C ) and InterPro ( D ) enrichment analysis showed that rBMSCs were significantly enriched in kinase activities, phosphorylation modification functions, and epigenetic regulations after PTN stimulation. ( E ) Differential analysis of phosphorylation modifications revealed that PTN significantly promoted PLCG1 (Tyr1253) and NCOA3 (Ser214) phosphorylation in rBMSCs, as shown in the heat map. ( F , G ) PLCG1 (Tyr1253) phosphorylation conditions were detected by immunofluorescence staining after PTN induction ( F ) or anti-PTPRZ1 blocking ( G ). Scale bar: 20 μm. ( H , I ) DAG levels in rBMSCs were detected after PTN induction ( H ) or anti-PTPRZ1 blocking ( I ). ( J , K ) PTN promoted phosphorylated NCOA3 nucleus translocation detected through immunofluorescence staining ( J ), and this effect was blocked by anti-PTPRZ1 treatment ( K ). Scale bar: 20 μm. Each group contained at least three biological replicates ( H , I ). The significance of differences between the groups was analyzed using Student’s t -test ( H ) or one-way ANOVA followed by Tukey’s post hoc test ( I ). * p < 0.05.

Article Snippet: The antibodies used in this study were as follows: mouse anti-BMP2 (1:500, Proteintech, 66383-1-Ig); rabbit anti-OCN (1:500, Proteintech, 23418-1-AP); rabbit anti-Caspase-3 (1:400, Proteintech, 19677-1-AP); rabbit anti-Caspase-8 (1:400, Proteintech, 13423-1-AP); rabbit anti-GLRX (1:500, Proteintech, 15804-1-AP); rabbit anti-GPX3 (1:500, Proteintech, 13947-1-AP); rabbit anti-PRDX6 (1:500, Proteintech, 13585-1-AP); rabbit Phospho-PLCG1 (Tyr1253) antibody (1:500, GeneTex, Irvine, CA, USA, GTX32238); rabbit Phospho-NCOA3 (Ser214) antibody (customized from QYAOBIO, Shanghai, China); goat Anti-Mouse (Alexa Fluor ® 488) secondary antibody (1:400, Abcam, Cambridge, UK, ab150113); and goat Anti-Rabbit (Alexa Fluor ® 488) secondary antibody (1:400, Abcam, Cambridge, UK, ab150077).

Techniques: Phospho-proteomics, Translocation Assay, High Throughput Screening Assay, Mass Spectrometry, Modification, Immunofluorescence, Staining, Blocking Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Follicular lymphoma-associated BTK mutations are inactivating resulting in augmented AKT activation

doi: 10.1158/1078-0432.CCR-20-3741

Figure Lengend Snippet: HEK293T cells were co-transfected with BTK WT or BTK mutant plasmids and PLCγ2. Some cells were pre-treated with ibrutinib for 1 h followed by anti-Ig treatment for 10’. Detergent cell lysates were made, fractioned by SDS-Page and prepared for immunoblotting with antibodies targeting the indicated epitopes. Displayed are composite data from three independent experiments per cell line. Lane 1: no plasmid transfection control; lanes 2 and 3: PLCγ2 encoding plasmid only. Vector: cells transfected with PLCγ2 plasmid only (paired two-sample t-test; * p<0.05; ** p<0.01).

Article Snippet: Anti-p-PLCγ2-Y759 (MAB7377) was from R&D System.

Techniques: Transfection, Mutagenesis, SDS Page, Western Blot, Plasmid Preparation