Journal: bioRxiv

Article Title: Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of “prime-and-kill” synNotch-CAR-T cells

doi: 10.64898/2026.03.26.713760

Figure Lengend Snippet: ( A–C ) Histograms and empirical cumulative distribution function (ECDF) plots showing distribution of maxUMI CAR values in in vitro E-SYNC cells ( A ), in vitro B-SYNC cells ( B ), and in vivo samples ( C ). In the histograms, the bin width is 1, and the Y-axis is shown on a log 10 scale. In both histograms and ECDF plots, maxUMI CAR values >50 were capped at 50 for visualization. (D) Dot plot showing expression of representative DEGs by sample type (same gene list as in and ), stratified by CAR-expressing versus non-expressing groups. (E) Swarm plot and ECDF plot depicting the distribution of TRM core gene signature scores. Differences between CAR-positive and -negative cells within the brain were assessed using the Kolmogorov–Smirnov test ( P = 4.0 × 10 -5 ). (F) ECDF plots comparing the nCount_RNA and nFeature_RNA values between CAR-expressing and non-expressing cells within the brain-derived CD8 + synNotch-positive population. Differences were assessed using the Kolmogorov–Smirnov test. (G) Schematic overview of the time-course co-culture experiment of B-SYNC cells with the human GBM cell line GBM6 (BCAN + /EphA2 + /IL13Rɑ2 + ). Co-culture was performed using 24-well plates, with each well containing 0.5 × 10 5 GBM cells and 1 × 10 6 CD8 + B-SYNC cells. The data presented are representative of two independent experiments using different donors. Additional data are shown in Fig. S16 . (H) Flow cytometry analysis showing the percentage of cells expressing GFP fused to CAR within the live, singlet, non-tumor (mCherry-negative) population. Corresponding scatter plots are shown in Fig. S16 . (I) RT-qPCR data showing CAR transcript abundance across conditions. Relative expression was calculated using the 2 -ΔΔCq method, with the B-SYNC no-stimulation (no-stim) condition and the RPS18 transcript as references. ( J–K ) Bulk RNA-seq principal component analysis (PCA) plot ( J ) and gene expression heatmap ( K ) illustrating transcriptomic similarities and differences across samples. The heatmap displays scaled expression of 6,885 genes expressed in the B-SYNC no-stim sample at transcripts-per-million (TPM) ≥ 10, sorted by the magnitude of expression changes between no-stim and post-stimulation conditions. ( L ) Representative genes minimally expressed without co-culture but upregulated and sustained after co-culture. The full list of gene expression comparisons is provided in Supplementary Table S13 .

Article Snippet: To prepare the B-SYNC primed sample, 2 × 10 4 cells were cultured for 24 hours on wells coated with Laminin (100 μg/ml, Thermo Fisher Scientific,23017015) and recombinant BCAN (25 μg/ml; R&D Systems, 7188-BC-050 and 4009-BC-050).

Techniques: In Vitro, In Vivo, Expressing, Derivative Assay, Co-Culture Assay, Flow Cytometry, Quantitative RT-PCR, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunohistological staining for BCAN was performed on cerebellar brain sections from Pclo wt/wt and Pclo gt/gt rats. In both genotypes, BCAN is localized in GFAP-positive astrocytes, from which it is secreted. In Pclo wt/wt slices, BCAN is also found in the surrounding tissue, indicating effective secretion. In contrast, in Pclo gt/gt sections, significantly less BCAN was detected outside astrocytes, suggesting impaired secretion of BCAN from astrocytes. B , Quantification of a. BCAN intensity is slightly greater inside astrocytes in Pclo gt/gt cerebellar sections than in Pclo wt/wt cerebellar slices; however, this difference is not significant ( Pclo wt/wt : mean± SEM= 1± 0.05, n=40 astrocytes, 3 independent animals; Pclo gt/gt : mean± SEM= 1.151± 0.06, 2 independent animals; p=0.082, t test). C, Quantification of a. BCAN intensity is significantly lower outside astrocytes in Pclo gt/gt slices than in Pclo wt/wt slices ( Pclo wt/wt : mean± SEM= 1± 0.04, 3 independent animals; Pclo gt/gt : mean± SEM= 0,831± 0.06, 2 independent animals; p=0.018, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining for BCAN on primary Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, BCAN intensity accumulates around the nucleus and extends along a line from the center toward the periphery. In Pclo wt/wt astrocytes, BCAN intensity was distributed more evenly. B , Immunocytochemical staining for TNR on Pclo wt/wt and Pclo gt/gt cortical astrocytes. In Pclo gt/gt astrocytes, TNR intensity accumulates around the nucleus and extends along a line from the cell center toward the cell periphery. In contrast, in Pclo wt/wt astrocytes, the TNR intensity was distributed more evenly. C , Quantification of A. A line was drawn from the nucleus to the periphery and divided into 10 bins. BCAN intensity was subsequently measured within each bin and graphically represented. BCAN intensity is greater close to the nucleus in Pclo gt/gt primary cortical astrocytes than in Pclo wt/wt primary cortical astrocytes. D , Quantification of B. A line was drawn from the nucleus to the periphery and divided into 10 bins, subsequently TNR intensity was measured within each bin. TNR intensity is greater close to the nucleus in Pclo gt/gt cortical astrocytes than in Pclo wt/wt . E , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly less BCAN was detected in Pclo gt/gt cortical supernatants ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.81± 0.045, n=3 independent experiments; p=0.0024, t test). F , Western blot (WB) analysis of the supernatants of Pclo wt/wt and Pclo gt/gt cortical astrocyte cultures. Significantly more TNR was detected in supernatant from Pclo gt/gt cortical astrocytes ( Pclo wt/wt : mean=1, n=3 independent experiments; Pclo gt/gt : mean± SEM= 1.653± 0.283, n=3 independent experiments; p=0.082, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Piccolo Regulates Secretion of the Extracellular Matrix Components Brevican and Tenascin R from Astrocytes to Drive Synapse Formation: Implications for Pontocerebellar Hypoplasia Type 3 (PCH3)

doi: 10.1101/2025.07.03.662734

Figure Lengend Snippet: A, Immunocytochemical staining BCAN, GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN fluorescence intensity is diffusely distributed in Pclo wt/wt astrocytes, whereas it accumulates along a defined path in Pclo gt/gt astrocytes . B, Immunocytochemical staining of tenascin-R (TNR), GM130, GFAP, and DAPI in Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR fluorescence intensity is concentrated near the nucleus and extends toward the cell periphery in Pclo gt/gt astrocytes, in contrast to a more uniform distribution in Pclo wt/wt astrocytes. C, Schematic illustration of the cargo distribution analysis method. A line from the nucleus to the cell periphery was segmented into 10 equal bins (0–9), and the fluorescence intensity of the cargo was measured in each bin and normalized to the mean intensity per bin in Pclo wt/wt astrocytes. D, Quantification of a. Normalized BCAN intensity decreases progressively from the nucleus to the periphery in Pclo wt/wt astrocytes but slightly increases in Pclo gt/gt astrocytes. E, Quantification of B. Normalized TNR intensity is greater near the nucleus in Pclo gt/gt astrocytes than in Pclo wt/wt cells. F, Western blot (WB) analysis of BCAN levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. BCAN levels are reduced in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 0.641± 0.141, n=3 independent experiments; p=0.064, t test). G, Western blot (WB) analysis of TNR levels in the supernatants of Pclo wt/wt and Pclo gt/gt cerebellar astrocytes. TNR levels are increased in Pclo gt/gt astrocytes ( Pclo wt/wt : mean= 1; Pclo gt/gt : mean± SEM= 1.246± 0.179, n=3 independent experiments; p=0.242, t test). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: Synaptophysin (1:1000; guinea pig; synaptic systems, Göttingen, Germany; Cat# 101 004, RRID:AB_1210382), PSD95 (1:500; mouse; Abcam, Cambridge, UK; Cat# ab2723, RRID:AB_303248), MAP2 (1:1000; chicken; Millipore, Darmstadt, Germany; Cat# AB5543, RRID:AB_571049), Piccolo (1:1000; rabbit; Synaptic Systems, Göttingen, Germany; Cat# 142002, RRID:AB_887759), GFAP (1:1000; chicken; Millipore, Burlington, US, Cat# AB5541, RRID:AB_177521), BCAN (1:200; rabbit; Proteintech, Illinois, US, Cat# 19017-1-AP, RRID:AB_10643526), TNR (1:200; rabbit; Synaptic Systems, Göttingen, germany; Cat# 217 008, RRID:AB_3083013), GM130 (1:300; mouse; BD Biosciences, Franklin Lakes, New Jersey, US, Cat# 610822, RRID:AB_398141), PRA1 (1:200; rabbit; Abcam, Cambridge, UK; Cat# ab213569), VGlut1 (1:1000; guinea pig; Synaptic Systems, Göttingen, germany; Cat# 135 304, RRID:AB_887878).

Techniques: Staining, Fluorescence, Western Blot