Journal: Molecular Therapy. Nucleic Acids

Article Title: JAK2V617F + single-stranded DNA modulates exosome complex activity and DNA-sensing pathways in myeloproliferative neoplasms

doi: 10.1016/j.omtn.2025.102743

Figure Lengend Snippet: Experimental design The cfDNA isolation was performed from HEL, SET2, and K562 cell lines culture supernatants, and cord blood was used as a healthy control. Then ssDNA was isolated. Allele-specific nested PCR, ELISA for DNA methylation, and fragment analysis were performed on the ssDNA. Allele-specific nested PCR, RT-qPCR, and apoptosis assays were performed on cord blood MNCs cultured for 48 h with HEL and SET2 ssDNA. The TLR9 inhibitor E6446 was applied to MNCs cultured with HEL, SET2, or CB-ssDNA. HEL and SET2 cells were incubated with ruxolitinib and CEP33779 , whereas K562 cells were incubated with imatinib to observe the effects of tyrosine kinase inhibitors on the exosome complex. After the administration of tyrosine kinase inhibitors, intracytoplasmic staining was performed to detect EXOSC1 protein expression. In addition, the effect of the combination of a TLR9 inhibitor and ruxolitinib on the exosome complex in HEL and SET2 cells was evaluated.

Article Snippet: TLR9 inhibitor, E6446 dihydrochloride (Selleckchem, Germany) (300 nM), was applied 30 min before ssDNA application to MNC culture and incubated for 48 h. Ruxolitinib (300 nM) was applied during the last 4 h, and CEP33779 (300 nM) (Selleck Biotechnology GmbH, Germany) was applied at the last hour of culture.

Techniques: Isolation, Control, Nested PCR, Enzyme-linked Immunosorbent Assay, DNA Methylation Assay, Quantitative RT-PCR, Cell Culture, Incubation, Staining, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: JAK2V617F + single-stranded DNA modulates exosome complex activity and DNA-sensing pathways in myeloproliferative neoplasms

doi: 10.1016/j.omtn.2025.102743

Figure Lengend Snippet: Treatment with TLR9 inhibitor Expression of (i) EXOSC1 ; (ii) EXOSC10 , DIS3 , and EXOSC8 ; (iii) cGAS , STING , and TLR9 ; (iv) NF-κB , IRF3 , IRF7 , and IFNG ; and (v) HMGB1 and TREX1 genes in MNCs treated with 300 nM TLR9 inhibitor E6446 30 min before culture with (A) HEL, (B) SET2, and (C) CB-ssDNA. (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: TLR9 inhibitor, E6446 dihydrochloride (Selleckchem, Germany) (300 nM), was applied 30 min before ssDNA application to MNC culture and incubated for 48 h. Ruxolitinib (300 nM) was applied during the last 4 h, and CEP33779 (300 nM) (Selleck Biotechnology GmbH, Germany) was applied at the last hour of culture.

Techniques: Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: JAK2V617F + single-stranded DNA modulates exosome complex activity and DNA-sensing pathways in myeloproliferative neoplasms

doi: 10.1016/j.omtn.2025.102743

Figure Lengend Snippet: Application of TLR9 inhibitor with JAK2 kinase inhibitor Expressions of (i) EXOSC1, (ii) EXOSC10, (iii) DIS3, and (iv) EXOSC8 in (A) HEL and (B) SET2 cells cultured with 300 nM TLR9 inhibitor E6446 30 min before culture, CB-ssDNA, and ruksolitinib. (C) Flow cytometry analysis of AnnexinV and 7-AAD labeling of HEL, SET2, and K562 cells cultured with 2 ng CB-ssDNA ( p < 0.0001). (D) Flow cytometry analysis of AnnexinV and 7-AAD labeling of MNCs cultured with HEL-, SET2-, and CB-derived ssDNA (2 ng) after 48 h incubation with TLR9 inhibitor (300 nM) for 30 min (UT, untreated). (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: TLR9 inhibitor, E6446 dihydrochloride (Selleckchem, Germany) (300 nM), was applied 30 min before ssDNA application to MNC culture and incubated for 48 h. Ruxolitinib (300 nM) was applied during the last 4 h, and CEP33779 (300 nM) (Selleck Biotechnology GmbH, Germany) was applied at the last hour of culture.

Techniques: Cell Culture, Flow Cytometry, Labeling, Derivative Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Role of Toll-like Receptors and Viral Infections in the Pathogenesis and Progression of Pulmonary Arterial Hypertension—A Narrative Review

doi: 10.3390/ijms262211143

Figure Lengend Snippet: TLR9 in pulmonary arterial hypertension (PAH). (based on [ , , , , , , , , , , , , , , , , , , , , , , , ]). Abbreviations: PAH—Pulmonary arterial hypertension, NF-κB—Nuclear factor kappa-light-chain-enhancer of activated B cells, IL-6—Interleukin-6, DAMP—Danger-associated molecular pattern, PAMP—Pathogen-associated molecular pattern, mtDNA—Mitochondrial DNA, CpG DNA—Cytosine-phosphate-guanine DNA motifs (unmethylated bacterial/viral DNA), MyD88—Myeloid differentiation primary response 88, AP-1—Activator protein 1, MCT-PAH—Monocrotaline-induced pulmonary arterial hypertension, RVSP—Right ventricular systolic pressure, TPVR—Total pulmonary vascular resistance, E6446—Selective TLR9 antagonist, Hb—Hemoglobin, EC—Endothelial cells, PASMC—Pulmonary artery smooth muscle cells, LDH—Lactate dehydrogenase, ↑ means increase, ↓ means decrease, → denotes a relationship, a causal connection, or a transition to the next piece of information.

Article Snippet: The tested rats were divided into two groups: one randomly administered a selective TLR9 antagonist, E6446 (provided by Eisai Inc., São Paulo, Brazil), and the other a non-selective quinoline agonist, most closely related to TLR9 among all Toll-like receptors, chloroquine (Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: