Journal:

Article Title: Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector

doi: 10.1128/JVI.76.20.10497-10502.2002

Figure Lengend Snippet: Estimation of vector gene copy number in AAV-transduced livers by quantitative PCR (3 months after vector administration). A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems. Template for PCR was as follows: 100 ng of genomic DNA extracted from several random pieces of liver and subsequently combined for each individual animal. Each sample column represents an individual animal. standards, linearized plasmid pAAV-EF1α-hF.IX (0.01, 0.1, or 1 pg) mixed with 100 ng of genomic mouse DNA (extracted from untransduced animal); NC, negative control (template, genomic DNA from untransduced animal). Bands were analyzed by densitometric scanning, and intensities were quantitated with NIH Image 6.16 software. Shown is one representative blot. Ratios of band intensities (hF.IX band/mAAT band) are for the blot shown. Gene copy number estimates are average for two experiments.

Article Snippet: A 350-bp fragment of the hF.IX cDNA as present in the AAV-EF1α-hF.IX vector was coamplified with a 1.1-kb fragment from the endogenous murine HPRT gene using biotinylated primers (20 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min, separated on a 2% agarose gel, transferred to a nylon membrane, and visualized with the Southern light detection system from Applied Biosystems.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

doi: 10.1136/jitc-2023-007716

Figure Lengend Snippet: PARG inhibition (PARGi) decreases basal and IL-6-induced phosphorylated STAT3 (pSTAT3) in immune cells and stimulates immune activation in vitro. (A) Splenocytes from tumor-free mice were isolated and cultured in the presence of vehicle (DMSO), 10 µM olaparib (PARPi), or 10 µM COH34 (PARGi) overnight. The levels of pSTAT3 and total STAT3 were analyzed by immunoblotting. (B) Immunoblot comparing pSTAT3 in healthy donor-derived PBMCs and ovarian cancer patient ascites-derived CD3 + T cells after overnight incubation with either DMSO or 5 µM PARGi. (C) Immunoblotting of pSTAT3 in mouse naïve CD19 + B cells, CD4 + and CD8 + T cells pretreated with vehicle control or PARGi (5 µM, overnight) and stimulated with 20 ng/mL IL-6 for 30 min. (D) Analysis of IL-6-induced pSTAT3 similar to (C) in human immune cells from (B) after overnight pretreatment with either DMSO or 5 µM PARGi. (E) Real-time PCR of Ifng gene expression in mouse splenocytes cultured in the presence or absence of PARGi for 24 hours. Gene expression data are shown after normalization to Actb expression and are presented as the mean-fold induction (mean±SD) relative to unstimulated samples. (F) ELISA measuring IFN-γ levels in the supernatants from cocultures of mouse CD8 + T cells with murine ID8 tumor cells in the presence of vehicle or 10 µM PARGi for 24 hours. Data are presented as the mean-fold induction (mean±SD) relative to vehicle-treated samples. (G) Levels of IFN-γ and granzyme B in the supernatants from ovarian cancer patient ascites-derived CD3 + T cells after treatment with either 5 µM PARGi or DMSO for 24 hours as determined by ELISA. The protein levels of pSTAT3 shown in the immunoblotting images were quantified by band intensity using ImageJ software and normalized with the levels of GAPDH. DMSO, Dimethyl sulfoxide; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PARG, poly(ADP-ribose) glycohydrolase; PBMCs, peripheral blood mononuclear cells; STAT3, signal transducer and activator of transcription 3.

Article Snippet: In this study, we used anti-mouse CD8α (clone 53-6.72) monoclonal antibody (mAb) and isotype control rat IgG 2b (BioXcell) for in vivo depletion of CD8 + T cell experiments.

Techniques: Inhibition, Activation Assay, In Vitro, Isolation, Cell Culture, Western Blot, Derivative Assay, Incubation, Control, Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

doi: 10.1136/jitc-2023-007716

Figure Lengend Snippet: PARG inhibition induces immune activation in ovarian tumor organoids. (A) Study design for (B,C) from ovarian cancer patient-derived tumor organoid/PBMC cocultures in the presence of 0.1% DMSO and 10 µM COH34 (96 hours), or 20 µg/mL PS-IgG and PS-PARG antibodies (48 hours). (B) Immunoblotting of phosphorylated STAT3 (pSTAT3) in ovarian cancer PDOs and patient-matched PBMCs after the PDO/PBMC cocultures were treated by DMSO, COH34, PS-IgG or PS-PARG antibodies. Data are representative of three independent experiments. (C) IFN-γ, granzyme B, and IL-10 levels in the PDO/PBMC coculture supernatants as measured by ELISA. Expression data are presented as mean-fold induction (mean±SD) relative to control samples from three different patients. (D) Study design for (E,F). PDOs or OVCAR8 spheroids and healthy donor PBMCs were prepared separately before coculturing. Tumor organoids (E) or OVCAR8 tumor cells (F) were cocultured with healthy donor PBMCs for 72 hours in the presence of 0.1% DMSO, 10 µM COH34, PS-IgG, or PS-PARG antibodies. Single-cell suspensions prepared from the cocultures of tumor organoids or OVCAR8 tumor cells and healthy donor PBMCs were analyzed by flow cytometry for activated T cells (GzmB + or CD107a + ) in CD8 + T cell populations. For tumor organoids, n=4; and for OVCAR8 tumor cells, n=3 (n represents the number of PBMC donors). The protein levels of pSTAT3 in the immunoblotting images were quantified by band intensity using ImageJ software and normalized with the levels of GAPDH. DMSO, Dimethyl sulfoxide; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PARG, poly(ADP-ribose) glycohydrolase; PBMCs, peripheral blood mononuclear cells; PDO, patient-derived organoid; STAT3, signal transducer and activator of transcription 3.

Article Snippet: In this study, we used anti-mouse CD8α (clone 53-6.72) monoclonal antibody (mAb) and isotype control rat IgG 2b (BioXcell) for in vivo depletion of CD8 + T cell experiments.

Techniques: Inhibition, Activation Assay, Derivative Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Control, Flow Cytometry, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer

doi: 10.1136/jitc-2023-007716

Figure Lengend Snippet: In vivo PARG inhibition induces antitumor immune responses, and the antitumor effects are partially mediated by CD8 + T cells. 5×10 6 Brca1 -null ID8 tumor cells were injected subcutaneously into female C57BL/6 mice aged 7–10 weeks old. The tumor-bearing mice were treated with vehicle or COH34 (20 mg/kg) every day for 7 days. Single-cell suspensions prepared from the tumors were analyzed by flow cytometry to detect: (A) phosphorylated STAT3 (pSTAT3) in CD45 + immune cells. (B) FoxP3 + Tregs in CD4 + T cells and; (C, D, E) activated CD8 + T cells (CD69 + cells, IFN-γ + , and GZMB + ). Data are shown as means±SD (n=4–5, each sample was pooled from 3 to 4 mice). (F) In vivo study design for (G). To deplete CD8 + T cells, C57BL/6 mice were injected intraperitoneally with rat anti-CD8 antibody or rat IgG2b (isotype control) on days −3 to –2, −1, and 0 relative to subcutaneous injection of Brca1 -null ID8 tumor cells (day 0). When the tumors reached an average size of 100 mm³ on day 5, vehicle or COH34 (20 mg/kg) were administered by intraperitoneal injections every day for 7 days. (G) On day 18, mice were euthanized and tumor weight was measured. Data are shown as means±SD (n=5–7 mice per group). PARG, poly(ADP-ribose) glycohydrolase; STAT3, signal transducer and activator of transcription 3.

Article Snippet: In this study, we used anti-mouse CD8α (clone 53-6.72) monoclonal antibody (mAb) and isotype control rat IgG 2b (BioXcell) for in vivo depletion of CD8 + T cell experiments.

Techniques: In Vivo, Inhibition, Injection, Flow Cytometry, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 suppresses Lcn2 expression and secretion via the inhibition of Ccl2-evoked NF-κB activation. BMMSCs were isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and WT controls. a Gene expression of Ccr2 in BMMSCs, as determined by real-time PCR. b–g Recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) was added to the adipogenic and osteogenic induction medium during BMMSC differentiation. The expression levels of osteogenic genes ( b–d ) and adipogenic genes ( e–g ) were determined via real-time qPCR. h–n WT and KO BMMSCs were treated with rmCcl2 (100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) for 24 h. h Sonicated BMMSCs were subjected to chromatin immunoprecipitation (ChIP) using an anti-p65 antibody. The ChIP DNA samples were then subjected to qPCR amplification using specific primers against the promoter region of Lcn2, as shown in supplementary Fig. . Representative gel images of end-point ChIP-qPCR products are shown. i Quantitative results are shown as the percentage of input DNA: % of Input = 2^(CT Input −CT IP )/dilution factor × 100%. j Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Lcn2 promoter (Lcn2-WT) or the mutated Lcn2 promoter with mutations in the NF-κB binding motif (Lcn2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. k Schematic diagram illustrating the actions of rmCcl2, the CCR2 antagonist, and the NF-κB-specific inhibitor QNZ on NF-κB translocation and Lcn2 expression; created with BioRender ( https://BioRender.com ). l ELISA analysis of total p65 in the nucleus and cytoplasm of BMMSCs. m Representative immunofluorescence images of intracellular p65 localization. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. n P65 nuclear translocation was quantified by ImageJ software via Pearson’s correlation coefficient (PCC) between the p65 immunofluorescence signal and DAPI staining across segmented nuclear regions. o , p WT and KO BMMSCs were treated with the NF-κB-specific inhibitor QNZ (10 nM) for another 2 h after 24-h treatment of rmCcl2 (100 ng/mL) and INCB3344 (10 nM). o The mRNA level of Lcn2 . p Lcn2 levels in the culture medium were determined by ELISA. q Illustration of how the AP-1 inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific inhibitor T-5224 (10 μM) for 24 h. r Sonicated BMMSCs were subjected to ChIP assay using an anti-c-Jun antibody. Representative gel images of end-point ChIP-qPCR products are shown. s Quantitative results are shown as the percentage of input DNA. t The mRNA level of Ccl2 . u Ccl2 levels in the culture medium were determined by ELISA. v Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Ccl2 promoter (Ccl2-WT) or the mutated Ccl2 promoter with mutations in the c-Jun binding motif (Ccl2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. –

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Expressing, Inhibition, Activation Assay, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Sonication, Chromatin Immunoprecipitation, Amplification, ChIP-qPCR, Luciferase, Reporter Assay, Transfection, Construct, Binding Assay, Sequencing, Activity Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Software