Journal: Cell Insight

Article Title: Viperin weakens IFN-I-induced immune activity by facilitating STAT1 degradation through E3 ligase UBE4A

doi: 10.1016/j.cellin.2026.100322

Figure Lengend Snippet: Viperin downregulated STAT1 during IFN stimulation requires UBE4A but not UBR5 . (A, B) RT-qPCR analysis of Ifit1 (A) and Isg15 (B) mRNA in Viperin +/+ and Viperin −/− MEF cells treated with IFNβ (500 IU/mL) for 4 and 8 h. (C) Western blot analysis of STAT1 and Viperin protein levels in Ubr5 +/+ and Ubr5 −/− cells treated with IFNα (1 000 IU/mL) for 6, 9 and 12 h. (D) RT-qPCR analysis of Ifit1 , Isg54, Isg15 and Mx1 mRNA in Ubr5 +/+ and Ubr5 −/− cells treated with IFNα (1 000 IU/mL) for 6 and 12 h. (E) Western blot analysis of STAT1 levels in wild type (WT), Ube4a −/− and Ubr5 −/− cells transfected with or without HA-Viperin. (F) Western blot analysis of STAT1 levels in Viperin stable cells transfected with Ctrl, Flag-UBE4A, Flag-UBE3A and Flag-UBE3C. (G) Immunoprecipitation (IP) analysis of the interaction between STAT1 and UBE4A in HT1080 cells transfected with or without Myc-Viperin. (H) IP analysis of the interaction between STAT1 and UBE4A in Viperin +/+ and Viperin −/− MEF cells. (I) Western blot analysis of STAT1 levels in Ube4a +/+ and Ube4a −/− cells treated with IFNα (1 000 IU/mL) for 9, 12 and 15 h. (J) Western blot analysis of PKR and IFIT1 levels in Ube4a +/+ and Ube4a −/− cells treated with IFNα (1 000 IU/mL) for 9 and 12 h. (K, L) Western blot analysis of STAT1 levels in Viperin-sufficient cells (K) or Viperin-deficient cells (L) transfected with Flag-UBE4A in dose manner. (M, N) Viral titers in the supernatant of Ube4a +/+ and Ube4a −/− HT1080 cells treated with IFNα (50 IU/mL, 20 h) and then infected with VSV/HSV (MOI = 1.0, 24 h) were determined by 50% tissue culture infectious dose (TCID50) assay. (O) Western blot analysis of HA (H1N1) protein levels in Ube4a +/+ and Ube4a −/− HT1080 cells treated with IFNα (50 IU/mL, 20 h) and then infected with H1N1 (MOI = 1.0) for 24 h. NS, not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (two-tailed unpaired Student's t -test). Data are shown as means ± SD of at least three biological replicates (A, B, D, M, N), or are representative of three independent experiments (C, E-L, O).

Article Snippet: HEK293T, A549, HT1080, Vero, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Infection, TCID50 Assay, Two Tailed Test

Journal: Cell Insight

Article Title: Viperin weakens IFN-I-induced immune activity by facilitating STAT1 degradation through E3 ligase UBE4A

doi: 10.1016/j.cellin.2026.100322

Figure Lengend Snippet: Viperin-STAT1-UBE4A binding rescues Viperin and promotes STAT1 degradation . (A) Region information of the Viperin deletion mutants. IP analysis of the interaction between Myc-Viperin (WT or deletion mutants) and Flag-UBE4A (left) or STAT1 (right) in HT1080 cells. (B) IP analysis of HA-K6 ubiquitination of Myc-Viperin in HT1080 cells transfected with Myc-Viperin, HA-K6-Ub, and dose of Flag-STAT1. (C) Region information of the STAT1 deletion mutants. IP analysis of the interaction between Flag-HA-Viperin (FH-Viperin) and Myc-STAT1 (WT or deletion mutants) in stable FH-Viperin-expressing HEK293T cells. (D) IP analysis of the interaction between Flag-UBE4A and Myc-STAT1 (WT or deletion mutants) in HT1080 cells. (E) Pattern diagram of interaction between UBE4A, STAT1 and Viperin. Data are representative of three independent experiments.

Article Snippet: HEK293T, A549, HT1080, Vero, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Ubiquitin Proteomics, Transfection, Expressing

Journal: Cell Insight

Article Title: Viperin weakens IFN-I-induced immune activity by facilitating STAT1 degradation through E3 ligase UBE4A

doi: 10.1016/j.cellin.2026.100322

Figure Lengend Snippet: IFN-I promotes ubiquitination and degradation of UBR5 . (A) Western blot analysis of UBR5 protein levels in HT1080 (left) and A549 (right) cells treated with IFNα (1 000 IU/mL) for 3, 6, 9 and 12 h. (B, C) RT-qPCR analysis of Ubr5 and Ifit1 mRNA levels in HT1080 (B) and A549 (C) cells treated with IFNα (1 000 IU/mL) for 3, 6, 9 and 12 h. (D) Western blot analysis of Flag-UBR5 protein levels in HT1080 cells treated with IFNα for 6, 9 and 12 h (left) or dose of IFNα (right). (E) Immunoprecipitation-immunoblotting (IP-IB) analysis of the interaction between Viperin and UBE4A or UBR5 in HEK293T cells transfected with Myc-Viperin and then treated with IFNα (1 000 IU/mL) for 6 and 12 h. (F) Cycloheximide (CHX) chase assay of UBR5 in HT1080 cells treated with CHX (50 μg/mL) for 3 and 6 h. (G) Western blot analysis of Flag-UBR5 protein levels in HT1080 cells transfected Flag-UBR5 for 36 h, and then treated with or without MG132 (10 μM). (H-J) Western blot analysis of UBR5 protein levels in HT1080 cells treated with or without MG132 (10 μM) (H), MA (10 μM) (I) or PR619 (10 μM) (J). (K) IP analysis of ubiquitination of UBR5 in HT1080 cells treated with IFNα (1 000 IU/mL) for 6, 9 and 12 h. (L, M) IP analysis of K48-ubiquitination (48ub) (L) and K63-ubiquitination (63ub) (M) of UBR5 in HT1080 cells treated with or without IFNα (1 000 IU/mL). NS, not significant ( p > 0.05), one-way analysis of variance (ANOVA), two-tailed unpaired Student's t -test. Data are shown as means ± SD of four biological replicates (B, C), or are representative of three independent experiments (A, D-M).

Article Snippet: HEK293T, A549, HT1080, Vero, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Transfection, Two Tailed Test

Journal: Cell Insight

Article Title: Viperin weakens IFN-I-induced immune activity by facilitating STAT1 degradation through E3 ligase UBE4A

doi: 10.1016/j.cellin.2026.100322

Figure Lengend Snippet: IFN-I promotes UBR5 degradation by up-regulating ITCH expression . (A) Potential interacting protein of UBR5 obtained from the PINA database. (B, C) RT-qPCR analysis of Itch mRNA levels in HT1080 (B), A549 and RAW264.7 (C) cells treated with IFN for 3, 6 and 9 h. (D) Western blot analysis of ITCH protein levels in HT1080 and A549 cells treated with IFNα (1 000 IU/mL) for 6, 9 and 12 h. (E, F) IP analysis of the interaction between endogenous UBR5 and ITCH in HT1080 cells (E) or primary mouse heart, liver, spleen, lung and kidney tissues (F). (G) Western blot analysis of UBR5 protein levels in Itch +/+ and Itch −/− cells. (H) Western blot analysis of UBR5 protein levels in HT1080 cells transfected with doses of Flag-ITCH. (I) RT-qPCR analysis of UBR5 mRNA levels in HT1080 cells transfected with doses of Flag-ITCH. (J) Western blot analysis of UBR5 protein levels in HT1080 cells transfected with or without Flag-ITCH, and then treated with MA (10 μM). (K) CHX chase assay of Flag-UBR5 in Itch +/+ and Itch −/− cells transfected with Flag-UBR5 for 36 h, and then treated with CHX (50 μg/mL) for 6 and 12 h. (L) CHX chase assay of UBR5 in HT1080 cells transfected with Flag-ITCH. (M) Western blot analysis of UBR5 protein levels in Itch +/+ and Itch −/− cells treated with IFNα for 6 and 12 h. (N) IP analysis of ubiquitination of UBR5 in Itch +/+ and Itch −/− cells. (O) IP analysis of ubiquitination of UBR5 in HT1080 cells transfected with doses of Flag-ITCH. (P) IP analysis of ubiquitination of UBR5 in Itch +/+ and Itch −/− cells treated with or without IFNα. (Q) RT-qPCR analysis of Ifit1 and Isg1 5 mRNA levels in cells as indicated, treated with IFNα for 6 and 12 h. NS, not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way analysis of variance (ANOVA) (B, C, I), two-tailed unpaired Student's t -test (Q). Data are shown as means ± SD of four biological replicates (B, C, I, Q) or are representative of three independent experiments (D-H, J-P).

Article Snippet: HEK293T, A549, HT1080, Vero, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Ubiquitin Proteomics, Two Tailed Test

Journal: Cell Insight

Article Title: Viperin weakens IFN-I-induced immune activity by facilitating STAT1 degradation through E3 ligase UBE4A

doi: 10.1016/j.cellin.2026.100322

Figure Lengend Snippet: The multifunctional interfering peptide VS-IP1 enhances IFN-I-induced antiviral immune activity . (A) Western blot analysis of STAT1 protein levels in HT1080 cells treated with VS-IP1 and followed with IFNα for 9, 12 and 15 h. (B) RT-qPCR analysis of Ifit1 and Isg1 5 mRNA levels in HT1080 cells treated with VS-IP1 and IFNα. (C) RT-qPCR analysis of Ifit1 mRNA levels in A549 cells treated with IFNα and dose of VS-IP1. (D) Viral titers in supernatants from HT1080 cells treated with VS-IP1 and IFNα followed by VSV infection (MOI = 1.0), determined by TCID50 assay. (E) RT-qPCR analysis of VSV, H1N1, SeV and HSV viral RNA in HT1080 cells treated with VS-IP1 and IFNα, followed by viral infection. (F) RT-qPCR analysis of Ifit1 , Isg15 and Isg5 4 mRNA levels in Stat1 +/+ and Stat1 −/− HT1080 cells treated with VS-IP1 and IFNα. (G) Viral titers in supernatants from Stat1 +/+ and Stat1 −/− HT1080 cells treated with VS-IP1 followed by VSV infection (MOI = 1.0), determined by TCID50 assay. (H-K) RT-qPCR analysis of VSV (H), H1N1 (I), SeV (J) and HSV (K) viral RNA in Stat1 +/+ and Stat1 −/− cells treated with VS-IP1 and IFNα, followed by viral infection. (L) Viral titers in supernatants from Viperin +/+ and Viperin −/− HT1080 cells treated with VS-IP1 followed by VSV infection (MOI = 1.0), determined by TCID50 assay. (M) RT-qPCR analysis of VSV RNA in Viperin +/+ and Viperin −/− HT1080 cells treated with VS-IP1 and IFNα, followed by VSV infection. NS, not significant ( p > 0.05), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, two-tailed unpaired Student's t -test (B-M). Data are shown as means ± SD of at least three biological replicates (B-M) or are representative of three independent experiments (A).

Article Snippet: HEK293T, A549, HT1080, Vero, and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activity Assay, Western Blot, Quantitative RT-PCR, Infection, TCID50 Assay, Two Tailed Test

Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A) Schematic of the recombinant dCas9 used for chromosome labeling in live cells. Two green fluorophores (sfGFP and mNeonGreen) are fused at the N terminus, and three tandem 2× nuclear localization signal (NLS) modules are positioned at the N terminus, an internal site, and the C terminus to enhance nuclear import. (B) Representative labeling of a mouse artificial chromosome (MAC) in an HT1080 MAC-positive line imaged by confocal microscopy. Nuclei are stained with Hoechst 33342. The inset highlights a representative CRISPR spot (arrow). Scale bars, 50 µm (overview) and 5 µm (inset). (C) Per-cell signal-to-noise ratio (SNR) distribution for MAC-positive and MAC-negative HT1080 cell lines (n = 262 MAC-positive cells and n = 160 MAC-negative cells across three independent experiments). Per-cell SNR was defined as the maximum intranuclear spot SNR within each nucleus. For cells with no detectable spot, the SNR was set to 0. (D) Precision–recall (PR) performance for MAC detection obtained by sweeping the SNR threshold. The curve shows mean performance across three independent experiments; the shaded band indicates ± standard deviation (s.d.); mAP is mean average precision. (E) Two-color colocalization assay using dCas9–green (sfGFP–mNeonGreen) and dCas9– red (2×mScarlet) programmed with distinct guide sets targeting the MAC (sgMajSat_1 for green and sgMajSat_2 for red). Nuclei are stained using NucSpot Live 650. The inset highlights a representative CRISPR spot of MAC focus (arrow). Colocalization was assessed by intersection-over-union (IoU) between the green and red focus masks, defined as the area of overlap divided by the area of union; spots were classified as colocalized at IoU ≥ 0.5. Scale bars, 5 μm.

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Recombinant, Labeling, Confocal Microscopy, Staining, CRISPR, Standard Deviation

Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A)Schematic of workflow modifications to reduce nonspecific puncta: protease treatment to remove membrane-associated RNP aggregates and transient glutamine deprivation to reduce intranuclear nonspecific accumulation. (B)Representative z-projection image of an OPM volume acquired from MAC-positive HT1080 cells (acquisition time, ~1.5 s per volume). Nuclei and CRISPR spots are shown in magenta and green, respectively. Scale bar, 100 μm. (C)Representative intranuclear CRISPR spots. Maximum-intensity z-projections of the GFP channel are shown; nuclear boundaries are outlined in yellow and arrows indicate discrete spots. Scale bar, 5 µm. (D)Cell-level SNR distributions without and with the workflow optimizations. Vertical lines denote the SNR threshold used for calling and the corresponding limit of detection (LoD) under the defined criteria. Blue, positive-control cells (n = 16,831 without optimization; n = 13,056 with optimization); orange, negative-control cells (n = 20,127 without optimization; n = 16,994 with optimization). (E)Precision–recall curves without and with the workflow optimizations by sweeping the SNR threshold on datasets from 30,723 MAC-positive cells and 37,577 MAC-negative cells across three independent experiments. mAP, mean average precision (without optimization, 0.941; with optimization, 0.971) and standard deviation (shadow).

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Membrane, CRISPR, Positive Control, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: High-throughput CRISPR live-cell imaging of low-frequency chromosomal events quantifies the latent efficiency of chromosome engineering

doi: 10.64898/2026.05.10.724155

Figure Lengend Snippet: (A) Experimental schematic. Following microcell fusion and expansion, recipient HT1080 cells were split into two arms: Hi-CRI imaging after 1 day of culture, or antibiotic selection followed by a clonogenic assay after 8 days. (B) Representative z-projection from an OPM volume in the post-MMCT population. Nuclei (magenta) and CRISPR spots (green) are shown. The inset shows a MAC-positive cell, in which the nuclear boundary is outlined in yellow and the arrow marks an intranuclear CRISPR spot. Scale bars, 75 µm (overview) and 3 µm (inset). (C) Estimated MAC-positive fraction measured by Hi-CRI imaging and by antibiotic-selection-based clonogenic assay across three independent MMCT experiments (Rep 1–3; y-axis, log scale). For Hi-CRI, the fraction is the number of cells called positive divided by the number of analyzed cells; for the clonogenic assay, the fraction is the number of surviving colonies divided by the number of input cells. Dots indicate technical replicates, ranging from 14,097 to 33,032 cells per replicate; error bars indicate ± s.d.

Article Snippet: For the MAC-positive HT1080 cell line (HT1080 MI-MAC27-2), G418 (InvivoGen, #ant-gn-1) was added to the medium at 600 μg/ml.

Techniques: Imaging, Selection, Clonogenic Assay, CRISPR

Journal: bioRxiv

Article Title: Label-free quantitative 3D mapping of collagen architecture by holotomography

doi: 10.64898/2026.05.05.722893

Figure Lengend Snippet: HT enables continuous, label-free monitoring of collagen dynamics through volumetric refractive-index (RI) mapping. a , Time-resolved HT MIPs of type I collagen polymerization showing progressive fibrillar assembly over time. Insets highlight early fibril emergence. Quantification of mean RI change (Δ n ) demonstrates a monotonic increase during gelation, providing a physically calibrated readout of assembly kinetics. b, c , Time-lapse HT imaging of HT1080 cells embedded in collagen under pharmacological perturbation (5-min intervals). b , Type I collagen with ROCK inhibitor (Y-27632). c , Type III collagen with MMP inhibitor (GM6001). For each condition, panels show full-field views at representative time points (0, 1, and 2 h), temporally encoded composite images, and zoomed regions highlighting cell-associated matrix remodeling (dashed circles). Drug treatments alter local collagen organization and remodeling dynamics at the single-fiber level compared to controls.

Article Snippet: HT1080 cells (ATCC, CCL-121) were prepared as a single-cell suspension and mixed with neutralized collagen at 8,000 cells per 60 μL (1.3 × 10 cells/mL) before casting.

Techniques: Refractive Index, Imaging

Journal: Clinical and Translational Medicine

Article Title: Oncogenic driver and therapeutic target: Prolactin signalling axis in retroperitoneal sarcoma

doi: 10.1002/ctm2.70669

Figure Lengend Snippet: Functional characterisation of PRL‐mediated proliferation and chemoresistance in sarcoma models. (A and B) Secretory PRL quantification by ELISA confirming knockdown efficiency in the culture medium, n = 3. (C–F) Growth suppression following PRL depletion: CCK‐8 time‐course assay ( n = 5) and colony formation capacity ( n = 3) in PRL‐knockdown models. (G–J) Recombinant PRL (50 ng/mL)‐induced proliferative enhancement: (G and H) CCK‐8 ( n = 5) and (I and J) colony formation ( n = 3) in HT1080 and SW872 lines. (K–P) PRLR‐dependent proliferation modulation: (K–N) CCK‐8 dose‐response ( n = 5) and (O‐P) colony formation ( n = 3) analysis post‐PRLR perturbation. (Q and R) After treating SW872 and HT1080 cells with PRLR antibody rolinsatamab talirine (20 µg/mL), the effect on cell proliferation was detected by the CCK8 method, with n = 5. (S and T) Xenograft tumourigenesis assay demonstrating impaired SW872 growth with PRL knockdown ( n = 9). (U) A single SW872 clone exhibiting the lowest PRL expression among the pooled PRL‐knockout cells was isolated by limiting dilution cloning, expanded in culture and validated for PRL protein levels via ELISA. (V) Cell proliferation was assessed using the CCK‐8 assay following stable PRL knockout ( n = 5 biological replicates). (W) Bromocriptine‐mediated antiproliferative effects were evaluated in parallel in wild‐type and PRL‐knockout SW872 cell lines using the CCK‐8 assay ( n = 5). (X) In vivo efficacy was determined in a subcutaneous xenograft mouse model, wherein tumour growth derived from wild‐type or PRL‐knockout SW872 cells was monitored following bromocriptine treatment, n = 7. (Y and Z) Chemosensitisation effects: PRL pretreatment (50 ng/mL) enhances cytotoxicity of RG7112/abemaciclib/doxorubicin/gemcitabine, n = 5, RG7112 (10 µM), abemaciclib (5 µM), doxorubicin (2 µM), gemcitabine (10 µM). (a) Western blot analysis was performed to detect MDM2 expression in human adipocytes, liposarcoma cell lines (SW872, 93T449, 94T778), fibrosarcoma cell line HT1080 and clinically isolated liposarcoma cell lines established in our laboratory. (b) Western blot analysis was performed to detect MDM2 in 12 clinical retroperitoneal liposarcoma tissues and its corresponding paracancerous tissues, 6 clinical retroperitoneal fibrosarcoma tissues and corresponding paracancerous tissues. (c) Therapeutic synergy evaluation: bromocriptine combined with RG7112 in WEHI164 fibrosarcoma murine model, RG7112: 100 mg/kg per day, bromocriptine: 10 mg/kg, twice daily, ( n = 6). Data expressed as mean ± SD unless specified; * p < .05, ** p < .01, *** p < .001 by two‐tailed Student's t ‐test; ns: not significant.

Article Snippet: The SW872 (HTB‐92) and HT1080 (CCL‐121) cell lines were purchased from ATCC.

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Knockdown, CCK-8 Assay, Recombinant, Expressing, Knock-Out, Isolation, Cloning, In Vivo, Derivative Assay, Western Blot, Two Tailed Test