comet assay kit  (Beyotime)

Journal: bioRxiv

Article Title: Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

doi: 10.64898/2026.07.22.740157

Figure Lengend Snippet: (A) Pan-cancer ranking of mean GSDME expression across 30 solid tumor types from TCGA, ordered by decreasing GSDME expression (left). Effect sizes and statistical significance for tissue-level comparisons of gasdermin family member expression between GBM tumor tissue and normal brain tissue from TCGA are shown (right). (B) UMAP visualization of scRNA-seq profiles from 12 GBM PDCLs, including six matched primary-recurrent pairs. CME037/CME038 and CME014/CME016 represent two matched primary-recurrent pairs. Bar plots show the composition of Neftel et al. GBM cellular states in these four PDCLs. (C) Immunoblot analysis of GSDME and GSDMD expression in CME037, CME038, and CME014. (D) Representative immunohistochemistry images of GSDME in normal cortex, low-grade glioma, and high-grade glioma from HPA (left), with quantification of GSDME optical density (OD) across the full cohort (right). (E) Dot plot showing expression of markers used for cell type annotation in the COMET study. (F) COMET images of tumoral (left) and periphery (right) tissue corresponding to the PDCL CME038, showing SOX2, GSDME, and DAPI. (G) Single cell comparison of GSDME RNA expression measured by RNAscope (left) and GSDME protein intensity measured by COMET staining (right) across annotated cell types in the in-house COMET cohort. (H) COMET images of tumor tissue corresponding to the mesenchymal-dominant PDCL CME014, showing SOX2, GSDME, Iba1, TMEM119, and DAPI. (I) Representative COMET images of tumor tissue corresponding to PDCL CME037, showing total caspase-3, cleaved caspase-3, and DAPI (left), with single cell quantification of cleaved caspase-3 abundance across cell types in the full cohort (right). (J) Patient demographic and clinical characteristics for the MILAN study cohort. (K) PCA visualization of all segmented cells from the MILAN study, colored by annotated cell type. (L) Dot plot showing expression of markers used for cell type annotation in the MILAN study. (M) Bar plots showing cell type composition for each image (top) and GSDME intensity per GBM cell in the corresponding image (bottom) in the MILAN study. Box plots show the median, interquartile range, and 10 th -90 th percentile whiskers. Statistical significance is indicated as ****p < 0.0001.

Article Snippet: RNA detection was performed using the RNAscope HiPlex Pro for COMET Reagent Kit (322075, Advanced Cell Diagnostics, Inc.).

Techniques: Expressing, Western Blot, Immunohistochemistry, Single Cell, Comparison, RNA Expression, RNAscope, Staining

Journal: bioRxiv

Article Title: Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

doi: 10.64898/2026.07.22.740157

Figure Lengend Snippet: (A) Nebulosa density plots and box plots show GSDME and GSDMD expression of CME037 maintained in monoculture or co-cultured with human macrophages. (B) Nebulosa density plots and box plots show GSDME and GSDMD expression of CME038 maintained in monoculture or co-cultured with human macrophages. (C) Nebulosa density plots and box plots show GSDME and GSDMD expression of LBT003 maintained in monoculture or co-cultured with human macrophages. (D) qPCR analysis of GSDME and GSDMD expression and immunoblots of GSDME expression in CME014 maintained in monoculture or indirectly co-cultured with THP-1-derived macrophages. (E) Immunoblot analysis of GSDME expression in CME037 and CME038 maintained in monoculture or indirectly co-cultured with THP-1 monocytic cells. (F) Bulk RNA-seq analysis of GSE136751 comparing GSDME and GSDMD expression in GBM cells maintained in monoculture or co-cultured with microglia. (G) Immunoblot analysis of GSDME expression in CME037, CME038, and CME014 maintained in monoculture or indirectly co-cultured with MHC3 microglia. (H) UMAP visualization of scRNA-seq profiles from macrophages maintained in monoculture or co-cultured with PDCLs from the experiment shown in (A) colored by culture condition and annotated using Miller et al. myeloid subtypes. Bar plots show myeloid subtype composition for each PDCL co-culture condition. (I) Bar plots showing cell subtype composition for each image in the MILAN study. (J) Single cell comparison of S100A4 RNA expression measured by RNAscope (left) and S100A4 protein intensity measured by COMET staining (right) across annotated cell types in the in-house COMET cohort. (K) Amphiregulin secretion in THP-1-macrophage monoculture, PDCL monoculture, and co-culture conditions, measured by nELISA. (L) Immunoblots of EGFRvIII expression across GBM PDCLs, including the PDCLs used in the co-culture experiments. (M) PDCL viability following treatment with erlotinib plicamycin, or ruxolitinib measured by CellTiter-Glo 2.0. (N) Immunoblots and quantification of EGFR, phosphorylated EGFR, STAT3, phosphorylated STAT3, and GSDME expression in CME037 maintained in monoculture with or without erlotinib treatment. (O) Immunoblots and quantification of the phosphorylated-to-total STAT3 ratio and GSDME expression following ruxolitinib treatment in the indirect co-culture system. Data are presented as mean ± SEM. Each dot in the bar plots represents an individual replicate. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ****p < 0.0001.

Article Snippet: RNA detection was performed using the RNAscope HiPlex Pro for COMET Reagent Kit (322075, Advanced Cell Diagnostics, Inc.).

Techniques: Expressing, Cell Culture, Western Blot, Derivative Assay, RNA Sequencing, Co-Culture Assay, Single Cell, Comparison, RNA Expression, RNAscope, Staining

Journal: bioRxiv

Article Title: Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

doi: 10.64898/2026.07.22.740157

Figure Lengend Snippet: (A) H&E image of a tumor core region from tumor tissue corresponding to PDCL CME037 (left) and corresponding COMET multiplexed staining for SOX2, GSDME, and DAPI, with a magnified region of interest showing SOX2, Iba1, α-SMA, GSDME, and DAPI. (B) Region of interest from (A) showing CD31, TMEM119, IL1B , CX3CR1 , MSR1 , C1QA , and DAPI (top), and a magnified image showing perivascular Iba1 + cells together with HMOX1 , IL1B , CX3CR1 , MSR1 , C1QA , S100A4 RNA, and S100A4 protein (bottom). (C) Schematic of the GSDME-expressing niche, comprising S100A4+ immunosuppressive macrophages, GSDME high GBM cells, and blood vessels. (D) H&E image of a pseudopalisading region from tumor tissue corresponding to PDCL CME037 (left) and corresponding COMET multiplexed staining for SOX2, EGFR, Iba1, α-SMA, and GSDME in the same tissue region. (E) Region of interest from (B) showing EGFR and OLIG2 staining (top) and CD163, α-SMA, and GSDME staining (bottom). (F) Magnified region of interest from c showing RNAscope staining for IL1B , CX3CR1 , MSR1 , C1QA , and S100A4 .

Article Snippet: RNA detection was performed using the RNAscope HiPlex Pro for COMET Reagent Kit (322075, Advanced Cell Diagnostics, Inc.).

Techniques: Staining, Expressing, RNAscope

Journal: bioRxiv

Article Title: Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

doi: 10.64898/2026.07.22.740157

Figure Lengend Snippet: (A) Correlation between GSDME and NINJ1 expression across pan-cancer cell lines from the CCLE dataset. (B) Odds ratio analysis of GSDME hi NINJ1 hi enrichment across pan-cancer cell lines. (C) Standardized GSDME expression per GBM cell in primary versus recurrent GBM samples from the MILAN cohort. (D) Box plots of CASP3 , GSDME and NINJ1 expression per GBM cell from primary versus recurrent GBM samples. (E) Hyp-PDT signature score in primary versus recurrent GBM. (F) Representative H&E image of the tumor-core region from the tumor corresponding to CME038, together with multiplexed COMET™ staining for SOX2, IBA1, GSDME, NINJ1 and α-SMA and RNAscope detection of MSR1 , C1QA and IL1B . (G) Expression of caspase-3, GSDME and NINJ1 in GBM cells binned by distance deciles from macrophages or endothelial cells as index cells. Box plots show the median, interquartile range, and 10 th -90 th percentile whiskers. Statistical significance is indicated as *p < 0.05, ****p < 0.0001.

Article Snippet: RNA detection was performed using the RNAscope HiPlex Pro for COMET Reagent Kit (322075, Advanced Cell Diagnostics, Inc.).

Techniques: Expressing, Staining, RNAscope