Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were transfected with control plasmid or plasmids encoding G2E3-FLAG. The cells were then analyzed by SDS-PAGE and immunoblotting with antibodies to the indicate proteins. B Quantification of ratio of LC3B-II, GABARAP and GABARAPL1 to Vinculin. The indicated p-values were calculated using means of three independent experiments ±SEM. n.s., not significant, * p < 0.05, ** p < 0.01. Significance was calculated with Student’s t test. C HEK293TN WT and AsPC-1 WT were transfected with plasmids encoding G2E3-FLAG or control plasmid FLAG. After 48 h, cell lysates and immunoprecipitates were analyzed using SDS-PAGE and immunoblotting with corresponding antibodies. D Immunofluorescent staining of endogenous GABARAP, GABARAPL1, LC3B, and exogenous G2E3 in G2E3 KO AsPC-1cells. G2E3KO AsPC-1 cells were transfected with plasmids encoding G2E3-FLAG. After 48 h, cells were fixed. G2E3-FLAG was stained with anti-FLAG antibody and the co-localization of the endogenous GABARAP, LC3B and GABARAPL1 were visualized under confocal microscope. Scale bar: 10 μm.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Transfection, Control, Plasmid Preparation, SDS Page, Western Blot, Staining, Microscopy

Journal: Cell Death Discovery

Article Title: CRISPR-Cas9 screening reveals G2E3 as a novel ubiquitin-linked factor controlling autophagosome-lysosome fusion and cancer cell progression

doi: 10.1038/s41420-025-02717-0

Figure Lengend Snippet: A WT or G2E3-KO AsPC-1 cells were fixed and stained with Lamp1 (red), LC3 (green), and DAPI (blue). Scale bar 10 μm. B The Pearson’s coefficients of LC3B overlap with Lamp1 in ( A ) were quantified using Fiji software. Mean ± SEM, n = 10. **** p < 0.0001. Significance was calculated with Student’s t test. C Co-localization analysis of endogenous Lamp1 and exogenously expressed G2E3-FLAG. Line plots exemplify degree of co-localization of G2E3 and Lamp1 signals.

Article Snippet: pMD2.G was a gift from Didier Trono (Addgene plasmid # 12259; http://n2t.net/addgene:12259 ; RRID:Addgene_12259), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID: Addgene_12260), FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060 ; RRID:Addgene_110060), Ubiquitination-Related Proteins CRISPR Knockout Library (Addgene #174592; http://n2t.net/addgene:174592 ; RRID:Addgene_174592), pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988 ; RRID:Addgene_62988), G2E3-FLAG plasmids (Sino Biological Cat# HG22847-CF) and FLAG plasmids (Sino Biological Cat# CV012) were provided by Sino Biological.

Techniques: Staining, Software

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: (A) In the amyloidogenic processing of APP, sequential cleavage by β-secretase and γ-secretase produces soluble APPβ (sAPPβ), the APP intracellular domain (AICD), and amyloid-β (Aβ) peptides. Aβ aggregates outside the cell, while AICD moves to the nucleus, where both fragments contribute to AD-related cellular pathology. (B) Schematic of the in vitro AD cell model. SH-SY5Y cells were transfected with either GFP (control) or AICD-GFP (AD model). After 3 hours, control cells were treated with DMSO, whereas AD cells were given Aβ peptide to mimic amyloidogenic stress. The combined presence of AICD and Aβ recapitulates the dual pathogenic signaling axis, and cells were kept at 37 °C for 48 hours before further analysis.

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: In Vitro, Transfection, Control

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: XIST Ectopic Distribution in AD Cell Model. (A) Schematic of the experimental workflow. SH-SY5Y neuroblastoma cells were transfected with AICD-GFP or GFP constructs and incubated for 3 hours at 37°C, then treated with Aβ peptide (AD model) or DMSO (control) for 48 hours. (B) Confocal images showing subcellular localization of XIST RNA (bright red punctum) in AD and control cells. DAPI (blue) stains nuclei. AICD-GFP or GFP (green) indicates successful transfection. Colocalization of XIST with nuclear DAPI is visible in both conditions. In the AD model, XIST shows higher nuclear and enrichment compared to controls. Right panels provide zoomed-in views of XIST-DAPI overlap. (C., C.1– C.4) XIST was significantly upregulated in AD and mainly localized in the nucleus. Both nuclear and cytoplasmic fractions contained significantly higher XIST levels in AD compared to controls. (C.5–C.8) RT-qPCR assessment of relative nuclear versus cytoplasmic expression of small ncRNAs SCARNA9 and tRNA under control and AD conditions confirmed efficient fractionation. (D., D.1., D.2) Quantification of XIST signal intensity in nuclear and cytoplasmic compartments revealed elevated signals in both the nuclear and cytoplasmic compartments in AD. Fold change data are shown as 2 −ΔΔCt ; bars indicate mean ± SEM. *p < 0.05, **p < 0.01, ***p <0.001, ****p <0.0001 (unpaired t-test).

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: Transfection, Construct, Incubation, Control, Quantitative RT-PCR, Expressing, Fractionation

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: Impaired EZH2 expression and disrupted EZH2-XIST interaction in AD. (A) Representative immunoblot showing reduced EZH2 protein expression in AD-treated cells compared to controls. β-Actin served as the loading control. (A.1) Quantification of EZH2 protein levels revealed a significant decrease in AD-treated cells (**p < 0.01). (B) qRT-PCR analysis indicated a significant reduction in EZH2 mRNA transcription in AD-treated cells relative to control (**p < 0.01). (C)-(C.1) RIP-qPCR confirmed a significant decrease in XIST RNA bound to EZH2 in AD-treated cells compared to controls (**p < 0.01). (C.2) Enrichment of XIST RNA in EZH2-IP versus IgG-IP in GFP+DMSO-treated control cells validated assay specificity (**p < 0.0001). (C.3) Similarly, AICD+Aβ-treated AD cells showed a significantly higher enrichment of XIST in EZH2-IP compared to IgG-IP (p < 0.0001), further confirming disrupted XIST-EZH2 interaction under AD conditions. Error bars indicate mean ± SEM; significance levels were determined using an unpaired Student’s t test.

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: The global H3K27me3 levels were measured using western blots and was found to be significantly downregulated in AD (AICD+Aβ) compared to control (GFP+DMSO). Error bars represent mean ± SEM; significance was determined using an unpaired Student’s t test.

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: Upregulation of THOC2 and increased XIST–THOC2 interaction in AD. (A) ChIP-qPCR analysis showing increased H3K27me3 enrichment at the THOC2 promoter in AICD+Aβ-treated AD cells compared to GFP+DMSO-treated controls (***p < 0.001). (A.1– A.2) ChIP-qPCR plots showing specific enrichment of H3K27me3 compared to IgG under GFP+DMSO (control) (*p < 0.05) and AICD+Aβ (AD) (**p < 0.01) conditions. (B) Immunoblot of THOC2 protein expression in control and AD-treated cells, with β-Actin as loading control. (B.1) Quantification revealed significant upregulation of THOC2 protein in AD-treated cells (**p < 0.01). (B.2) qRT-PCR analysis of THOC2 mRNA expression indicated a significant decrease in AD relative to control (**p < 0.01). (C) RIP-qPCR demonstrating enhanced association between THOC2 and XIST in AD. Representative gel electrophoresis image showing increased XIST enrichment in THOC2-IP from AD samples compared to controls. (C.1-C.3) qPCR quantification confirmed significant elevation of XIST binding to THOC2 in AICD+Aβ cells (*p < 0.05). The pull-down specificity validated by robust enrichment of XIST RNA in THOC2-IP compared to IgG-IP in both control (***p < 0.001) and AD (****p < 0.0001) groups. Error bars represent mean ± SEM; significance determined using unpaired Student’s t test.

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: ChIP-qPCR, Control, Western Blot, Expressing, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Binding Assay

Journal: bioRxiv

Article Title: XIST Self-regulates its Association with THOC2 and the Nuclear Epigenetic Machinery via miR-186 in Alzheimer’s disease

doi: 10.1101/2025.10.05.680468

Figure Lengend Snippet: Colocalization of THOC2 with XIST in AD using combined ICC and RNA FISH. The images show the spatial relationship between the lncRNA XIST and THOC2 in AD (A) and control (B) samples. Nuclei were stained with DAPI, while AICD or GFP was transfected into the AD and control cells, respectively. XIST was visualized by RNA FISH, and THOC2 was detected through ICC. Notably, in AD cells (A), XIST displays a significant increase in colocalization with THOC2, as seen in merged images, especially in the nuclear area (dotted circles, enlarged panels). Conversely, control cells (B) show much weaker colocalization signals. These findings support a possible AD-specific interaction between XIST and THOC2 within the nuclear compartment. (C) Quantitative analysis of colocalization using Pearson’s correlation coefficient confirms a statistically significant increase in the association between XIST and THOC2 in the AD group compared to controls (**p < 0.01), indicating elevated recruitment of XIST to the TREX-mediated RNA export pathway in AD. Error bars represent mean ± SEM.

Article Snippet: The GFP control plasmid (pGFP-C1, Clontech) and the amyloid precursor protein intracellular domain (AICD) construct cloned into the pGFP-C1 vector (referred to as AICD from here on) were available in the laboratory.

Techniques: Control, Staining, Transfection