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: TMZ+TTF treatment enhances apoptotic responses in glioblastoma cells. (A) Immunofluorescence staining of cleaved caspase‐3 (red) and nuclei (blue, DAPI) in U251 and SHG140 cells under different treatment conditions. (B) Quantification of cleaved caspase‐3 fluorescence intensity. (C, E) Representative Annexin V‐FITC/PI flow cytometry plots showing the distribution of viable and apoptotic cell populations at 24 and 48 h in U251 and SHG140 cells, respectively. Lower left: Viable cells; lower right: Early apoptotic cells; upper right: Late apoptotic cells; upper left: PI‐positive non‐apoptotic/dead cells. (D, F) Quantitative analysis of viable‐cell fractions derived from Annexin V‐FITC/PI flow cytometry in U251 and SHG140 cells at 24 and 48 h. For cleaved caspase‐3 quantification, three random 20× fields were averaged within each independent experiment. At least 10,000 events were acquired per flow‐cytometry sample. 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 within each time point. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For cleaved caspase‐3 immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, blocked with 5% BSA, and incubated overnight at 4°C with anti‐cleaved caspase‐3 antibody (Proteintech, USA; Cat. No. 25546–1‐AP; 1:200).

Techniques: Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Analysis, Derivative 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 cleaved caspase‐3 immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, blocked with 5% BSA, and incubated overnight at 4°C with anti‐cleaved caspase‐3 antibody (Proteintech, USA; Cat. No. 25546–1‐AP; 1:200).

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