Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: Experimental workflow for generating patient-derived GBM tissue slice cultures (PDTC) and neurosphere cultures (PDNS). Fresh tumor tissue was sectioned into 350 μm slices using a tissue chopper. Tissue punches (2–3 mm) were cultured on membrane inserts at an air–liquid interface to establish PDTCs. Remaining tissue was mechanically and enzymatically dissociated and cultured under serum-free conditions to generate PDNS. AAV vectors encoding GFP were added to the culture medium, and transduction was monitored by live confocal microscopy. At endpoint, cultures were analyzed by immunohistochemistry for GFP expression, stem cell markers, and microenvironmental cell populations to assess vector tropism and biological effects

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Derivative Assay, Tissue, Cell Culture, Membrane, Transduction, Confocal, Microscopy, Immunohistochemistry, Expressing, Stem Cell, Plasmid Preparation

Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: AAV2 and AAV6 transduction of patient-derived neurosphere cultures (PDNS) under EGF-supplemented and EGF-deprived conditions. ( A ) Dose-response analysis of AAV-mediated transduction in PDNS. Spheroids derived from at least three independent GBM specimens were transduced with AAV2 or AAV6 vectors at doses ranging from 10 6 to 10 9 vector particles per well. GFP expression was monitored by live confocal microscopy on days 2 and 5 post-transduction. Quantification of GFP fluorescence demonstrated a dose-dependent increase in transduction efficiency for both serotypes. For AAV6, significant increases in GFP signal were observed at 10 8 particles under EGF-supplemented conditions, whereas AAV2 reached significance only at 10 9 particles on day 5. Representative confocal images of AAV6-transduced spheroids cultured in the presence of EGF are shown. To validate live-imaging results and determine cellular tropism, spheroids were fixed on day 5, embedded, and analyzed by immunofluorescence staining for GFP. ( B ) PLAT-normalized AAV vector genome abundance in PDNS 5 days after treatment with 10⁸ vector particles is shown ( n = 4). ( C ) Cell type–specific analysis of AAV transduction in PDNS. Co-immunofluorescence staining for GFP and the lineage-associated markers GFAP, CD44, Nestin, and SOX2 was performed to identify transduced cellular populations. In EGF-deprived media, increased proportions of GFP-positive cells were observed across multiple marker-defined populations. In contrast, under EGF-supplemented conditions, the strongest enrichment of GFP expression was detected within the Nestin-positive population. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001)

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Transduction, Derivative Assay, Analysis, Spheroids, Plasmid Preparation, Expressing, Confocal, Microscopy, Fluorescence, Cell Culture, Imaging, Immunofluorescence, Staining, Marker, Comparison

Journal: Journal of Neuro-Oncology

Article Title: Patient-derived tissue cultures complement neurospheres for preclinical evaluation of AAV-mediated gene delivery in glioblastoma

doi: 10.1007/s11060-026-05807-w

Figure Lengend Snippet: Comparison of AAV-mediated transduction and stem cell marker expression in patient-derived tissue cultures (PDTCs) and neurosphere cultures (PDNS). ( A ) Quantitative comparison of AAV transduction in matched PDTC and PDNS generated from seven independent GBM specimens (shown is the median). GFP fluorescence intensity was used as a readout of AAV-mediated gene delivery following treatment with AAV2 or AAV6 under EGF-supplemented and EGF-deprived conditions. PDTCs exhibited greater inter-patient variability than PDNS across all experimental conditions. In PDTCs, both AAV2 and AAV6 significantly increased GFP expression compared with control cultures. In contrast, only AAV6 induced a significant increase in GFP fluorescence in PDNS, whereas AAV2 did not differ significantly from control conditions. EGF supplementation had minimal effects on transduction efficiency in PDTCs, while omission of EGF reduced GFP expression in PDNS, particularly following AAV6 transduction. ( B ) GFP expression could also be observed in PDTC of non-malignant origin in both serotypes investigated in two independent tissue specimen. ( C ) Expression of stem cell-associated markers following AAV transduction in PDTCs and PDNS. Nestin expression remained stable across culture systems and treatment conditions. In contrast, SOX2 expression exhibited culture- and treatment-dependent variability. A significant increase in SOX2 expression was observed in PDNS transduced with AAV6 under EGF-deprived conditions compared with untreated controls, whereas no significant alterations were detected in PDTCs. These findings indicate that SOX2 expression varied with culture and treatment conditions in PDNS, whereas no significant changes were detected in PDTCs. Statistical analyses were performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison test (* p < 0.05, ** p < 0.01, **** p < 0.0001). Individual data points represent independent patient specimens

Article Snippet: For immunofluorescence staining, samples were incubated overnight with primary antibodies ( stem cell marker : SOX2, Neuromics, GT15098 (1:200); Nestin, Merck, ABD69 (1:1000); OLIG2, Epitomics, AC-0106RUO (1:200); CD44, Cell Signaling Technologies, 156-3C11 (1:400); Survivin, Cell Signaling Technologies, 2808 (1:500) astrocyte marker : GFAP, DAKO, Z0334 (1:500); AAV transduction : GFP, Synaptic Systems, 132005 (1:400); apoptosis and proliferation : Cleaved PARP1, abcam, ab32064 (1:100), Ki67, DCS, KI681C01 (1:200)).

Techniques: Comparison, Transduction, Stem Cell, Marker, Expressing, Derivative Assay, Tissue, Generated, Fluorescence, Control

Journal: The Plant Journal

Article Title: The pathogenic effector PHYL1 JWB interacts with ZjCURT1A to mediate jujube witches' broom‐induced photosynthesis impairment of jujube trees

doi: 10.1111/tpj.71104

Figure Lengend Snippet: PHYL1 JWB physically interacts with ZjCURT1A. (a) The yeast two‐hybrid assay showed that PHYL1 JWB interacted with ZjCURT1A but not with the negative controls. (b) In vitro pull‐down assays of PHYL1 JWB with ZjCURT1A. Equal amounts of affinity‐purified PHYL1 JWB ‐MBP were incubated with ZjCURT1A‐GST and GST. Pull‐down proteins were subjected to immunoblotting with anti‐MBP antibodies. PHYL1 JWB ‐MBP was specifically pulled down by ZjCURT1A‐GST but not the GST control. (c) Luciferase complementation imaging (LCI) assay in Nicotiana benthamiana . Signals were only detected with the co‐transformation of PHYL1 JWB and ZjCURT1A . Empty vectors were used as negative controls. (d) Co‐immunoprecipitation (Co‐IP) assay showed an interaction between PHYL1 JWB and ZjCURT1A in vivo . Following incubation of total protein extracts with anti‐GFP agarose, immunoblotting with an anti‐MYC antibody revealed that ZjCURT1A co‐precipitated with PHYL1 JWB ‐GFP, but not with the GFP control. * indicated PHYL1 JWB ‐GFP. (e) Bimolecular fluorescence complementation (BiFC) assay in N. benthamiana . Plasmids of the indicated gene sets were transiently co‐expressed in tobacco leaves. A reconstituted YFP signal was observed only in cells co‐infiltrated with PHYL1 JWB ‐cYFP and ZjCURT1A‐nYFP , whereas control combinations with empty vectors showed no fluorescence. pBI121‐35S‐Chlo‐mCherry was used as a chloroplast marker. Bars, 50 μm.

Article Snippet: ZjCURT1A‐MYC was detected with an anti‐MYC antibody (CWBIO; Cat# CW0299M), while GFP and GFP‐tagged fusion proteins were probed with an anti‐GFP antibody (Cat# AE012; ABclonal).

Techniques: Y2H Assay, In Vitro, Affinity Purification, Incubation, GST Pulldown Assay, Western Blot, Control, Luciferase, Imaging, Transformation Assay, Co-Immunoprecipitation Assay, In Vivo, Bimolecular Fluorescence Complementation Assay, Fluorescence, Marker

Journal: bioRxiv

Article Title: IRES-mediated translation of Δ160p53 regulates p53 functions and fine-tunes cancer homeostasis

doi: 10.64898/2026.08.21.744132

Figure Lengend Snippet: (A) Western blot analysis of cell extracts from H1299 transfected with pcDNA and Δ133p53, and treated with 5-fluorouracil (5-FU; 20 µM) and thapsigargin (0.1 µM) for 16 h, probed with BiP antibody, p53 polyclonal antibody, and β-actin antibody. (B) Partial sequence of p53 transcript variants 5,6 and 7, which are known to produce Δ133p53, and the putative IRES sequence was marked. (C) Schematic representation of bicistronic plasmids; H1299 cells were transfected with the plasmids along with pRL-TK. At 48 h post-transfection, relative luciferase activity was calculated in the presence of different IRES sequences and the control sequence (null). (D) Schematic representation of the peGFPΔEMCVI3Δ160p53 construct; western blot analysis of cell extracts from H1299 cells transfected with pcDNA (negative control), Δ160p53 (positive control), and peGFPΔEMCVI3Δ160p53, probed with p53 polyclonal antibody, β-actin antibody, and green fluorescence protein (GFP) antibody. (E) Splicing assay: Semiquantitative polymerase chain reaction (PCR) analysis using two sets of primers (P1/P2 and P3/P4), RNA isolated from pRI3F-transfected (Lanes 2–7) and untransfected (Lane 1) H1299 cells. Lanes 2 and 5 show the reverse transcriptase negative control, and lanes 4 and 7 show the PCR product amplified from the pRI3F bicistronic plasmid (Positive control). (F) Cryptic promoter activity assay: pGL3-basic (Promoter-less luciferase vector as negative control), PG13 (p53 binding sites containing luciferase plasmid as positive control), and IRES-pGL3 plasmids were transfected into H1299 cells along with pRL-TK, and relative luciferase activity was measured. (G) Real-time PCR of Fluc mRNA in H1299 cells transfected with pGL3-basic, PG13, and IRES-pGL3 plasmids. Error bars indicate standard deviation (SD). All experiments were performed in three biological replicates (n = 3). The criterion for significance was determined using a two-tailed Student’s t-test (**P ≤ 0.01 or ***P ≤ 0.001).

Article Snippet: Rabbit anti-GFP-Tag antibody (Abclonal, Cat. No. AE011) and β-actin antibodies (Sigma) were horseradish peroxidase-conjugated, and no secondary antibodies were required for detection.

Techniques: Western Blot, Transfection, Sequencing, Luciferase, Activity Assay, Control, Construct, Negative Control, Positive Control, Fluorescence, Splicing Assay, Polymerase Chain Reaction, Isolation, Reverse Transcription, Amplification, Plasmid Preparation, Binding Assay, Real-time Polymerase Chain Reaction, Standard Deviation, Two Tailed Test