Journal: BMC Pulmonary Medicine

Article Title: Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway

doi: 10.1186/s12890-023-02660-9

Figure Lengend Snippet: AS-IV reduced pyroptosis and fibrosis development in hypoxia-treated PASMCs. PASMCs were divided into 3 groups: Nor, Hyp and AS, cultured with normoxia or hypoxia, and treated with or without AS-IV. A - D , Cropped blots of GSDMD-N, NLRP3, cleaved Caspase-1, Fibronectin and Collagen1 and quantification of grayscale value (the samples derived from the same experiment and that gels/blots were processed in parallel). E , IL-1β and IL-18 levels in cell supernatant were measured by ELISA. F , Cell death was detected by PI/Hoechst double fluorescent staining. Scale bar = 50 μm. G, LDH activity was detected by a LDH release agent. H - I , Proteins related to extracellular matrix metabolism, including MMP2/9 and TIMP4 were detected by ELISA method. * P <0.05, ** P <0.01, *** P <0.001, comparison with Nor group. # P <0.05, ## P <0.01, ### P <0.001, comparison with Hyp group

Article Snippet: The nuclei were counterstained with Hoechst 33,258 (Abcam, #ab228550), and the image was visualized under a microscope and analyzed by the image J software.

Techniques: Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Comparison

Journal: BMC Pulmonary Medicine

Article Title: Astragaloside IV restrains pyroptosis and fibrotic development of pulmonary artery smooth muscle cells to ameliorate pulmonary artery hypertension through the PHD2/HIF1α signaling pathway

doi: 10.1186/s12890-023-02660-9

Figure Lengend Snippet: AS-IV depressed pyroptosis and the production of fibrosis markers induced by hypoxia in PASMCs through the PHD2-HIF1α axis. A - D , PASMCs were divided into 5 groups: Nor, Hyp, AS, AS + si-PHD2 and LW6. Cropped blots of GSDMD-N, NLRP3, cleaved Caspase-1, Fibronectin and Collagen1 (the samples derived from the same experiment and that gels/blots were processed in parallel), and quantitation of the pyroptosis markers and the fibrosis markers. E , Cell death was detected by PI/Hoechst double fluorescent staining. Scale bar = 50 μm. F , IL-1β and IL-18 levels in cell supernatant were measured by ELISA. G, LDH activity was detected by a LDH release agent. H - I , Proteins of MMP2/9 and TIMP4 were detected by ELISA method. * P <0.05, ** P <0.01, *** P <0.001, comparison with Nor group. # P <0.05, ## P <0.01, ### P <0.001, comparison with Hyp group. ^ P <0.05, ^^ P <0.01, ^^^ P <0.001, comparison with AS group

Article Snippet: The nuclei were counterstained with Hoechst 33,258 (Abcam, #ab228550), and the image was visualized under a microscope and analyzed by the image J software.

Techniques: Derivative Assay, Quantitation Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: Combined TMZ+TTF treatment produces greater growth suppression, clonogenic inhibition, and reduction in migratory capacity in glioblastoma cells. (A, B) ATP‐based luminescence viability assays showing relative ATP activity in U251 and SHG140 cells at 24, 48, and 72 h under different treatment conditions. (C, D) CCK‐8 viability assays of U251 and SHG140 cells at 72 h. (E) Representative colony formation images of cells subjected to different treatments; scale bar = 5 mm. (F) Quantitative analysis of colony numbers across groups. (G) Transwell migration assays showing migrated cells under different treatment conditions using crystal violet staining; scale bar = 500 μm. (H) Quantification of migrated cells. Vehicle control indicates complete culture medium containing 0.2% DMSO, matching the final DMSO concentration in TMZ‐containing groups. Pairwise comparisons among treatment groups were performed for the quantitative analyses, including TMZ versus TMZ+TTF and TTF versus TMZ+TTF comparisons in the Transwell migration assay. For ATP‐based and CCK‐8 assays, five replicate wells were averaged within each independent experiment. For Transwell analysis, three random fields per insert were averaged to generate one value per experiment. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis for ATP‐based time‐course viability assays was performed using two‐way ANOVA followed by Tukey's multiple comparisons test. Statistical analysis for CCK‐8, colony formation, and Transwell migration assays was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For CCK‐8 assays, cells were seeded in 96‐well plates at 8 × 10 3 –1 × 10 4 cells/well, treated as indicated, and incubated with 10 μL CCK‐8 reagent (Targetmol, USA; Cat. No. C0005) for 1–2 h at 37°C.

Techniques: Inhibition, Activity Assay, CCK-8 Assay, Colony Assay, Analysis, Migration, Staining, Control, Concentration Assay, Transwell Migration Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: ALK5/TGFβRI inhibition partially attenuates TTF‐associated SMAD phosphorylation, growth suppression, and cleaved caspase‐3 induction. (A) Western blot analysis of phosphorylated SMAD2 (p‐SMAD2), phosphorylated SMAD3 (p‐SMAD3), and total SMAD2 in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334. (B) Quantification of p‐SMAD2, p‐SMAD3, and total SMAD2 protein expression levels. Densitometric values for p‐SMAD2, p‐SMAD3, and SMAD2 were normalized to GAPDH. (C, D) CCK‐8 assays measuring cell viability in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334 under the same 72 h assay conditions used in Figure . SB525334 was used at 10 μM as a selective ALK5/TGFβRI inhibitor. (E) Immunofluorescence staining of cleaved caspase‐3 (red) and nuclei (blue, DAPI) in U251 and SHG140 cells under the indicated treatment conditions; merged images are shown. (F) Quantification of cleaved caspase‐3 fluorescence intensity. For cleaved caspase‐3 quantification, three random 20× fields were averaged within each independent experiment. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For CCK‐8 assays, cells were seeded in 96‐well plates at 8 × 10 3 –1 × 10 4 cells/well, treated as indicated, and incubated with 10 μL CCK‐8 reagent (Targetmol, USA; Cat. No. C0005) for 1–2 h at 37°C.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Analysis, Control, Expressing, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence