Journal: Biomolecules and Biomedicine

Article Title: Methylene blue mitigates lung injury in HCA rats by regulating macrophage pyroptosis via Nrf2/HO-1 and NLRP3 pathways

doi: 10.17305/bb.2025.11851

Figure Lengend Snippet: MB inhibits pyroptosis in AMs caused by NLRP3 inflammasome in HCA rats. (A) The pyroptosis level of rat AMs was detected by Yo-Pro-1 staining (green) and Hoechst 33342 staining (nuclear blue) (20×, bar ═ 100 µm); (B–F) Western blot bands and relative expression levels of NLRP3, caspase-1, pro-caspase-1, ASC, GSDMD-N, and GSDMD in AMs. n ═ 6, * P < 0.05. MB: Methylene blue; HCA: Hypothermic circulatory arrest; AMs: Alveolar macrophages; NLRP3: Nod-like receptor protein 3; GSDMD: Gasdermin-D.

Article Snippet: The samples were then subjected to electrophoresis on 12% SDS-PAGE gels (Invitrogen), transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen), and blocked with 5% BSA for 3 h. After washing, the membranes were incubated overnight at 4 ∘ C with the following primary antibodies: Nrf2 (PA5-27882, 1:500, Invitrogen), HO-1 (PA5-77833, 1:1000, Invitrogen), NLRP3 (MA5-23919, 1:100, Invitrogen), pro-caspase-1 (ZRB1233, 1:1000, Sigma-Aldrich), caspase-1 (MA5-16215, 1:200, Invitrogen), ASC (HY- P80548 , 1:500, MedChemExpress), GSDMD-N (ab215203, 1:1000, Abcam), or GSDMD (PA5-116815, 1:1000, Invitrogen).

Techniques: Staining, Western Blot, Expressing

Journal: Biomolecules and Biomedicine

Article Title: Methylene blue mitigates lung injury in HCA rats by regulating macrophage pyroptosis via Nrf2/HO-1 and NLRP3 pathways

doi: 10.17305/bb.2025.11851

Figure Lengend Snippet: ML385 and Nigericin can partially restore MB to inhibit HCA-induced pyroptosis of rat lung macrophages and improve lung injury. (A–E) Western blot bands and relative levels of NLRP3, caspase-1, pro-caspase-1, ASC, GSDMD-N, and GSDMD in Ams; (F) The pyroptosis level of rat AMs was detected by Yo-Pro-1 staining (green) and Hoechst 33342 staining (nuclear blue) (20×, bar ═ 100 µm); (G) Utilizing HE staining to evaluate the pathological changes in rat lung tissue, and to score lung injury (20×, bar ═ 100 µm); (H) The ROS level was measured utilizing DHE staining (20×, bar ═ 100 µm); (I–L) TNF-α, IL-6, ICAM-1, and IL-10 levels in BALF were assessed using ELISA kits. n ═ 6, * P < 0.05. MB: Methylene blue; HCA: Hypothermic circulatory arrest; BALF: Bronchoalveolar lavage fluid; AMs: Alveolar macrophages; NLRP3: Nod-like receptor protein 3; ROS: Reactive oxygen species; GSDMD: Gasdermin-D; ASC: Associated speck-like protein; HE: Hematoxylin and eosin; IL: Interleukin; DHE: Dihydroethidium.

Article Snippet: The samples were then subjected to electrophoresis on 12% SDS-PAGE gels (Invitrogen), transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen), and blocked with 5% BSA for 3 h. After washing, the membranes were incubated overnight at 4 ∘ C with the following primary antibodies: Nrf2 (PA5-27882, 1:500, Invitrogen), HO-1 (PA5-77833, 1:1000, Invitrogen), NLRP3 (MA5-23919, 1:100, Invitrogen), pro-caspase-1 (ZRB1233, 1:1000, Sigma-Aldrich), caspase-1 (MA5-16215, 1:200, Invitrogen), ASC (HY- P80548 , 1:500, MedChemExpress), GSDMD-N (ab215203, 1:1000, Abcam), or GSDMD (PA5-116815, 1:1000, Invitrogen).

Techniques: Western Blot, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Sequences of primers.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques:

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Ang II infusion induces activation of NLRP3 inflammasomes. Mice were infused with Ang II as described in Materials and Methods. After 4 weeks of infusion, hearts from each group of animals were collected. The expression levels of NLRP3 ( A ), Casp1 ( B ), and ASC ( C ) were quantified by qRT-PCR. Data are presented as the mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 Ang II-infused vs. saline-infused animals. @@@@ p < 0.0001 Ang II-infused vehicle-treated vs. Ang II-infused WFA-treated animals.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Saline

Journal: Cells

Article Title: Withaferin A as a Potential Therapeutic Target for the Treatment of Angiotensin II-Induced Cardiac Cachexia

doi: 10.3390/cells13090783

Figure Lengend Snippet: Ang II infusion induces expression of NLRP3, ASC, and Caspase 1 proteins. Mice were infused with Ang II as described in Materials and Methods. After 4 weeks of infusion, hearts from each group of animals were collected. The expression levels of NLRP3, ASC, and Caspase 1 proteins in cardiac tissues were evaluated by immunofluorescence technique. Data are presented as the mean ± SD (n = 5). * p < 0.05, *** p < 0.001, and **** p < 0.0001 Ang II-infused vs. saline-infused groups. Scale Bar = 50 µm.

Article Snippet: Antibody used were for NLRP3 (cat MAB7578-SB from R&D Systems), ASC (cat # NBP1-78978 from Novus), and Casp1 (Cat # ab74279), respectively, with appropriate dilution according to instructions from the suppliers.

Techniques: Expressing, Immunofluorescence, Saline