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

Journal: Science Advances

Article Title: Chemo-metallo nanodrivers for spatiotemporally controlled in situ vaccination

doi: 10.1126/sciadv.aee4577

Figure Lengend Snippet: ( A ) Volcano plot of differentially expressed genes (DEGs) in MFC cells (Control versus DDP + Zn 2+ ). ( B ) KEGG pathway enrichment of DEGs. ( C ) Quantification of extracellular dsDNA. ( D ) JC-1 staining assessed by fluorescence microscopy and flow cytometry. ( E ) γ-H2AX immunofluorescence staining. Scale bar, 20 μm. ( F ) ASC speck colocalization with AIM2, Caspase-1, Caspase-8, and RIPK1. Scale bars, 5 μm (overview) and 1 μm (enlarged). ( G ) Western blot analysis of necroptosis markers (RIPK1, p-RIPK1, MLKL, and p-MLKL) and PANoptosis-related proteins (AIM2 and ZBP1). ( H ) Western blot analysis of apoptosis executors (Caspase-8, Caspase-3, and cleaved forms). ( I ) Western blot analysis of pyroptosis markers (Caspase-1, GSDMD, and cleaved forms). ( J ) Cell viability after DDP + Zn 2+ combined with VX-765, Ac-DEVD-CHO, or Nec-1. ( K ) PANoptosis-related proteins in si AIM2 cells. ( L and M ) cGAS activity (L) and expression (M) under Zn 2+ treatment. ( N ) Violin plot of cGAS expression. ( O ) Western blot analysis of STING pathway (STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3). ( P ) CIBERSORT analysis of immune cell infiltration. ( Q ) BMDC maturation (CD80 + CD86 + ) after different treatments. ( R ) Schematic of the DDP + Zn 2+ –induced ICD. Data in (C), (J), and (L) are mean ± SD. Statistical analysis for (C), (J), (L), (N), and (P) used a two-tailed unpaired Student’s t test. (R) was created in BioRender. Fei, G. (2026) https://BioRender.com/8ri399r . * P < 0.05.

Article Snippet: Cell Signaling Technology (CST) provided antibodies against cleaved caspase-1 (no. 89332), caspase-3 (no. 9661), caspase-8 (no. 4790), cleaved caspase-8 (no. 8592), cleaved GSDMD (no. 10137), ASC-Alexa Fluor 488 (no. 17507), ZBP1 (no. 33402), cGAS (no. 31659), STING (no. 13647), phospho-STING (no. 72971), TBK1 (no. 3504), phospho-TBK1 (no. 5483), phospho-IRF3 (no. 4302), IRF3 (no. 4947), and RIPK3 (no. 95702).

Techniques: Control, Staining, Fluorescence, Microscopy, Flow Cytometry, Immunofluorescence, Western Blot, Analysis, Activity Assay, Expressing, Immune Cell, Two Tailed Test

Journal: Science Advances

Article Title: Chemo-metallo nanodrivers for spatiotemporally controlled in situ vaccination

doi: 10.1126/sciadv.aee4577

Figure Lengend Snippet: ( A ) Fluorescence images of ZHDG internalization in MFC cells. ( B ) Quantification of the intracellular ZHDG fluorescence intensity. ( C ) Viability of MFC cells across treatment groups. ( D ) Annexin V–FITC/PI staining (fluorescence and flow cytometry). ( E ) TEM images of ultrastructural damage in MFC cells. ( F ) Relative LDH release. ( G ) Intracellular ATP levels. ( H ) Immunofluorescence analysis of CRT exposure and HMGB1 release. ( I ) Zinpyr-1 staining in different treatment groups. ( J ) Quantification of JC-1 aggregates and monomers. ( K ) JC-1 staining (flow cytometry). ( L ) Extracellular dsDNA release. ( M ) Western blot analysis of necroptosis markers (RIPK1, p-RIPK1, MLKL, and p-MLKL) and AIM2. ( N ) Western blot analysis of pyroptosis markers (Caspase-1, GSDMD, and cleaved forms). ( O ) Western blot analysis of apoptosis executors (Caspase-8, Caspase-3, and cleaved forms). ( P ) Western blot analysis of PANoptosis-related proteins in si AIM2 MFC cells treated with ZHDG + NIR. ( Q ) Schematic of ZHDG under NIR photoactivation inducing AIM2-PANoptosome formation and ICD. Data in (B), (C), (F), (G), and (L) are mean ± SD. Statistical analysis for (B), (C), (F), (G), and (L) was performed using a two-tailed unpaired Student’s t test. (Q) was created in BioRender. Fei, G. (2026) https://BioRender.com/wcbo7ma .

Article Snippet: Cell Signaling Technology (CST) provided antibodies against cleaved caspase-1 (no. 89332), caspase-3 (no. 9661), caspase-8 (no. 4790), cleaved caspase-8 (no. 8592), cleaved GSDMD (no. 10137), ASC-Alexa Fluor 488 (no. 17507), ZBP1 (no. 33402), cGAS (no. 31659), STING (no. 13647), phospho-STING (no. 72971), TBK1 (no. 3504), phospho-TBK1 (no. 5483), phospho-IRF3 (no. 4302), IRF3 (no. 4947), and RIPK3 (no. 95702).

Techniques: Fluorescence, Staining, Flow Cytometry, Immunofluorescence, Analysis, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: Chemo-metallo nanodrivers for spatiotemporally controlled in situ vaccination

doi: 10.1126/sciadv.aee4577

Figure Lengend Snippet: ( A ) Volcano plot of DEGs in MFC cells (Control versus ZHDG + NIR). ( B ) KEGG pathway analysis. ( C ) Immunofluorescence showing ASC speck colocalization with AIM2, ZBP1, Caspase-1, Caspase-8, and RIPK1. Scale bars, 5 μm (overview) and 1 μm (enlarged panels). ( D ) Violin plot of cGAS expression across treatment groups. ( E ) Western blot analysis of cGAS-STING pathway proteins (cGAS, STING, p-STING, TBK1, p-TBK1, IRF3, and p-IRF3). ( F ) CIBERSORT analysis of immune cell infiltration. ( G ) BMDC maturation assessed by flow cytometry of CD80 + CD86 + cells. ( H ) Schematic of NIR-activated ZHDG initiating the STING-AIM2-PANoptosome cascade to potentiate antitumor immunity. Statistical analysis for (D) and (F) was performed using a two-tailed unpaired Student’s t test. (H) was created in BioRender. Fei, G. (2026) https://BioRender.com/m2dnb22 . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cell Signaling Technology (CST) provided antibodies against cleaved caspase-1 (no. 89332), caspase-3 (no. 9661), caspase-8 (no. 4790), cleaved caspase-8 (no. 8592), cleaved GSDMD (no. 10137), ASC-Alexa Fluor 488 (no. 17507), ZBP1 (no. 33402), cGAS (no. 31659), STING (no. 13647), phospho-STING (no. 72971), TBK1 (no. 3504), phospho-TBK1 (no. 5483), phospho-IRF3 (no. 4302), IRF3 (no. 4947), and RIPK3 (no. 95702).

Techniques: Control, Analysis, Immunofluorescence, Expressing, Western Blot, Immune Cell, Flow Cytometry, Two Tailed Test