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

Journal: Aging Cell

Article Title: Inhibition of the cGAS‐STING pathway ameliorates the premature senescence hallmarks of Ataxia‐Telangiectasia brain organoids

doi: 10.1111/acel.13468

Figure Lengend Snippet: A‐T brain organoids secrete enriched levels of pro‐inflammatory factors and fail to transcribe IEGs upon neuronal excitatory input. (a) Culture media from WT and A‐T BOs was collected once a week for 2 months and used to quantify the secreted levels of the indicated SASP proteins. n = 3 independent experiments. * p < 0.05; two‐way ANOVA with concentration and time used as variables. (b‐h) WT and A‐T Brain organoids (BOs) were generated and grown in vitro for 3 months and collected for analysis. (b) Representative images of BO sections stained for p21 (green) and GFAP (red). Scale bar, 100 µm. (c) Quantification of data presented in b. Bar graphs show the percentage of p21 and GFAP positive cells ±95% confidence interval. n = 3 independent biological samples; at least 100,000 cells per sample have been analysed; *** p < 0.001, chi‐squared test. (d) Representative images of BO sections stained for Ki67 (green) and GFAP (red). Scale bar, 100 µm. (e) Quantification of data presented in d. Bar graphs show the percentage of Ki67 and GFAP positive cells ±95% confidence interval. n = 3 independent biological samples; at least 100,000 cells per sample have been analysed; *** p < 0.001, chi‐squared test. (f) Representative images of RNAscope in situ hybridization on BO sections for GFAP (green), IL‐8 (red), IL1B (yellow) and IL‐6 (magenta). Cell nuclei were stained with DAPI (blue). Scale bars, 30 μm. (g) Quantification of data presented in f. Bar graphs show the percentage of cells that are simultaneously labelled by GFAP and the indicated SASP RNAscope HiPlex probes. Error bars represent SD ; n = 4 independent biological samples; Student's t test. (h) 3‐month‐old WT and A‐T BOs were treated with NMDA (100 µM), KCl (55mM) and Bicuculline (50 µM) for 30 min. Immediately after, BOs were collected for RNA extraction. RT‐qPCR analysis of the indicated IEGs was performed, and RPLP0 mRNA was used as normalizer. Bar graphs show fold change of IEG levels in stimulated organoids relative to untreated (depicted by a grid line). Error bars represent SD ; n = 3 independent experiments. * p <0.05, ** p <0.01, Student's t test

Article Snippet: RNA in situ hybridization (ISH) was performed using an RNAscope HiPlex Kit (Advanced Cell Diagnostics) with detection probes against GFAP, IL1B, IL‐8 and IL‐6 mRNAs following directions of the manufacturer.

Techniques: Concentration Assay, Generated, In Vitro, Staining, RNAscope, In Situ Hybridization, RNA Extraction, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Specific fibroblast subpopulations and neuronal structures provide local sources of Vegfc-processing components during zebrafish lymphangiogenesis

doi: 10.1038/s41467-020-16552-7

Figure Lengend Snippet: a – l Whole mount in situ hybridization for adamts14 . a – d At 26 hpf, adamts14 transcripts are detectable in the floorplate (black arrow), in cells along the ventral aspect of the segment boundaries (white arrow) and in cells at the HM (black arrowhead). d Zoom-in of the squared region in c . e – h At 36 hpf, adamts14 expression is visible in the floorplate (black arrow), in cells located at the level of the axial blood vessels (white arrow) and in cells at the HM (black arrowhead). h Zoom-in of the indicated region in g . i – l Expression of adamts14 is still apparent in the floorplate (black arrow) at 48 hpf as well as in cells at the HM (black arrowhead). l Enlargement of the boxed region in k . m , n adamts14 RNA granules can be detected in cells located in the HM region by RNAscope at 48 hpf. n Overlay of the bright field and confocal image for the indicated region in m , showing adamts14 RNA granules in green. o Schematic cross view of the trunk illustrating the adamts14 expression domains (yellow). Scale bars in a , e , i : 100 µm; b , c , f , g , j , k , m : 50 µm. DA dorsal aorta, hpf hours post fertilization, HM horizontal myoseptum, M muscle, N notochord, PCV posterior cardinal vein, PL parachordal lymphangioblast.

Article Snippet: The RNAscope transcript detection was performed on embryos that were fixed in 4% paraformaldehyde at RT for 20 min using the RNAscope Fluorescent Multiplex Reagent Kit (ACD) and following standard procedures : Fixed embryos were washed three times for 5 min in 1 ml PBT (0.1% Tween), followed by a series of dehydration steps: 25, 50, 75, and 100% methanol for 5 min each.

Techniques: In Situ Hybridization, Expressing, RNAscope

Journal: bioRxiv

Article Title: Extensive, transient, and long-lasting gene regulation in a song-controlling brain area during testosterone-induced song development in adult female canaries

doi: 10.1101/2022.06.13.495861

Figure Lengend Snippet: A , schematic of the songbird brain with song control areas (white) including the HVC. B and C , representative photomicrographs of in situ hybridizations of androgen receptor ( AR ) mRNA in B and 5α-reductase ( SRD5A2 ) mRNA in C from controls (CON) and testosterone-treated (T8h resp. T14d) birds. AR mRNA expression is increased in the HVC of T14d (B, arrowheads) and SRD5A2 mRNA is increased in the HVC of T8h animals (C, arrowheads). D , testosterone increased the expression of genes in the HVC compared with controls (e.g., AR , SRD5A2 , UTS2B ), and the expression of many genes differed between time points after testosterone treatment started (e.g., ALDH1A2 , HSD17B12 ). The in situ hybridization data of the five depicted genes confirmed the statistical differences (Supplementary Table 2 and Supplementary Table 3) observed in the microarray-derived expression measurements (see ). Data represent the proportion of the labeled HVC area (RNAscope staining of gene mRNA, see B and C) relative to the total HVC area of that section. Each gray dot represents the value of one HVC section. Black dots indicate the predicted estimates of the linear mixed-effects models, and vertical orange bars indicate the 95% confidence intervals (CrI) of the predicted estimates. N = 3 birds per group. ALDH1A2 : aldehyde dehydrogenase 1 family member A2; AR : androgen receptor; HSD17B12 : 17β-hydroxysteroid dehydrogenase; SRD5A2 : 5α-reductases 2; UTS2B : urotensin 2B. E , a heat map showing the expression levels of selected groups of genes during testosterone treatment as compared to the control HVCs (log 2 fold change): genes of gonadal steroid receptor genes (blue), genes with known functions in steroidogenesis (gray), genes involved in testosterone-induced angiogenesis (red), genes involved in neuronal recruitment (green), and the gene encoding transcription factor SP8 (yellow).

Article Snippet: In situ hybridization for mRNA expression of ALDH1A1 , AR , HSD17B12 , SRD5A2 , and UTS2B was performed on sections (20 or 14 μm thick) using the RNAScope ®2.0 HD Detection Kit (Advanced Cell Diagnostics, Newark, California) ( ) according to the protocols indicated by the manufacturer.

Techniques: Control, In Situ, Expressing, In Situ Hybridization, Microarray, Derivative Assay, Labeling, RNAscope, Staining

Journal: bioRxiv

Article Title: Extensive, transient, and long-lasting gene regulation in a song-controlling brain area during testosterone-induced song development in adult female canaries

doi: 10.1101/2022.06.13.495861

Figure Lengend Snippet: Photomicrographs of RNAScope® fluorescence multiplex in situ hybridizations of the HVC from T14d animals. Colocalization of SP8 mRNA and ESR2 mRNA in both SLC17A6 expressing (glutamatergic) neurons (A 1 to A 5 ) and SLC17A6 negative neurons (B 1 to B 3 ). Ninety-six percent of SP8 positive cells were also ESR2 positive; among SP8 and ESR2 double-labeled cells, 60% were in SLC17A6 expressing neurons. Scale bars in both top and bottom panels correspond to 50 µm. SLC17A6 : Solute Carrier Family 17 Member 6, formerly known as vGluT2.

Article Snippet: In situ hybridization for mRNA expression of ALDH1A1 , AR , HSD17B12 , SRD5A2 , and UTS2B was performed on sections (20 or 14 μm thick) using the RNAScope ®2.0 HD Detection Kit (Advanced Cell Diagnostics, Newark, California) ( ) according to the protocols indicated by the manufacturer.

Techniques: RNAscope, Fluorescence, Multiplex Assay, In Situ, Expressing, Labeling