Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Sequence alignment of wild-type and STAT1 −/− to STAT1 gene of the human genome (GrCh38.p14). The red bar indicates the binding site of sgRNAs. The red star indicates the 17 bp deletion on the second allele. B Western blot of whole cell lysates of wild-type and STAT1 −/− HEp-2 cells targeting STAT1 (top) and β-Actin (bottom) as loading control. C Bright-field IncuCyte live-cell images of wild-type (left) and STAT1 −/− HEp-2 (right) at 72 hours (h) post-seeding. Images representative of three independent experiments (10 technical replicates). Scale bar indicated next to images. D Quantification of cell eccentricity for wild-type and STAT1 −/− HEp-2 cells at 72 h post-seeding based on live cell images. Eccentricity was assessed at approximately 60% confluence, which was reached at 72 h post-seeding for both cell types. Minimum area, minimum eccentricity, and Hole fill were set to 600 μm 2 , 0.3, and 1000 μm 2 , respectively. Mean ± SD from three independent experiments, with 10 replicates each, are shown. Statistical analysis: Unpaired t-test. E Quantitative analysis of IncuCyte live-cell images for cells per well, with images taken every 3 h for 4.5 days. Mean ± SD of three independent experiments (10 technical replicates) is shown, with non-linear regressions applied for wild-type (green) and STAT1 −/− (red) HEp-2 cells. Statistical analysis: Mann-Whitney test for comparison of ranks. F Volcano plot demonstrating the number of differentially expressed genes between wild-type and STAT1 −/− HEp-2 cells with thresholds p < 0.05 and |Log2 Fold change | ≥ 1. G Bar plot of Gene Set Enrichment Analysis of Gene Ontology. The plot shows significantly enriched biological processes in STAT1 −/− compared to wild-type HEp-2 cells.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Sequencing, Binding Assay, Western Blot, Control, MANN-WHITNEY, Comparison

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Viral replication kinetics of RSV-A-0594 (dashed lines) and rRSV-A-0594-eGFP (solid lines)-infected wild-type (green) and STAT1 −/− (red) HEp-2 cells (MOI 0.05). Mean ± SD of three independent experiments (four technical replicates) is shown. Statistical analysis: Two-way ANOVA with Fisher’s LSD test. B Fluorescence images of rRSV-A-0594-eGFP-infected (MOI 0.05) wild-type (top) and STAT1 −/− (bottom) Hep-2 cells at 48 hours post-infection (hpi). Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images. C Graph showing the comparison of viral transcript numbers from RNA-Seq analysis between wild-type and STAT1 −/− HEp-2 cells from 0 to 72 hpi with thresholds set at p < 0.05 and |Log2 Fold change | ≥ 1. D Presatovir-based fusion inhibition assay on wild-type (green) and STAT1 −/− (red) HEp-2 cells at 72 hpi with rRSV-A-0594-eGFP (MOI 0.05). Fluorescence intensity (eGFP) relative to untreated, infected cells is shown. Solid lines indicate mean values, while dashed lines above and below each curve indicate the corresponding standard deviation (Mean ± SD). Horizontal dashed line and the right graph indicate Half Maximal Inhibitory Concentration (IC 50 ) of wild-type (green) and STAT1 −/− (red) HEp-2 based on non-linear regression fitted to graphs. Mean ± SD of three independent experiments (eight technical replicates) is shown. Statistical analysis for IC 50 comparison: Unpaired t-test with Welsh’s correction.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Infection, Fluorescence, Comparison, RNA Sequencing, Inhibition, Standard Deviation, Concentration Assay

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A UpSet plot demonstrating differentially expressed genes that are shared and unique to infected wild-type and STAT1 −/− HEp-2 after comparison to respective uninfected controls with thresholds p < 0.05 and |Log2 Fold change | ≥ 1. B Cleveland Dot Plot of Gene Set Enrichment Analysis (GSEA) of Gene Ontology comparing processes enriched in both infected wild-type and STAT1 −/− HEp-2 cells based on Normalized Enrichment Scores (NES). C Quantification of total free cholesterol by luminescent enzymatic assay in uninfected wild-type (green) and STAT1 −/− (red) HEp-2 cells. Statistical analysis: Mann-Whitney test. D Quantification of total free cholesterol by luminescent enzymatic assay in wild-type and STAT1 −/− HEp-2 cells, which were uninfected (grey), treated with 5 mM methyl-beta-cyclodextrin (MβCD, orange), infected at MOI 0.05 (light red), infected at MOI (dark red), or treated with 100 ng/ml IFN-α2 and 50 ng/mL IFN-γ at 24 and 48 hpi. Only significant differences between untreated and treated cells within a cell type or within treatments between cell types are shown. Statistical analysis: two-way ANOVA with Fisher’s LSD test. Mean ± SD of four independent experiments (two technical replicates) shown for ( C , D ).

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Infection, Comparison, Enzymatic Assay, MANN-WHITNEY

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Representative fluorescence images of rRSV-A-0594-eGFP-infected wild-type ( A ) and STAT1 −/− ( B ) HEp-2 cells (MOI 0.05) at 48 hpi. Cells were left untreated (first row), treated with 50 μM Gemfibrozil (GFZ, second row), treated with 5 μM methyl-β-cyclodextrin (MβCD) and 50 μM GFZ (third row), or with 10 μM 25-hydroxycholesterol (25-HC, bottom panel). Free cholesterol in plasma membranes was visualized by Filipin III staining (0.05 mg/ml), and DNA was visualized by Propidium iodide (PI) staining (5 μg/ml). Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Fluorescence, Infection, Clinical Proteomics, Staining

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: A Fluorescence images from IncuCyte live-cell imaging of rRSV-A-0594-eGFP-infected wild-type (left) and STAT1 −/− (right) HEp-2 cells (MOI 0.05), which were left untreated (left column), treated with 50 μM Gemfibrozil (GFZ, middle column), or with 10 μM 25-hydroxycholesterol (25-HC, right column) at 12, 24, 36, and 48 hpi. Images representative of three independent (two technical replicates) experiments. Scale bar indicated next to images. B Quantification of syncytia size by IncuCyte live-cell imaging for green (fluorescent) area per image relative to green object count per image of rRSV-A-0594-eGFP-infected wild-type (solid lines) and STAT1 −/− (dashed lines) HEp-2 cells (MOI 0.05), either untreated (red), treated with 50 μM GFZ (blue), or 10 μM 25-HC (green). Images of cells were taken every hour for up to 48 hpi. Mean ± SEM of eight independent experiments (two technical replicates) is shown. Statistical analysis was conducted on the overall distribution of syncytia size during the course of infection using the Friedman test with Dunn’s multiple-comparison test.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Fluorescence, Live Cell Imaging, Infection, Comparison

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Confocal microscopy images of RSV-A-0594-infected wild-type ( A ) and STAT1 −/− ( B ) HEp-2 cells (MOI 0.05) at 48 hpi. Infected cells were left untreated (top), treated with 50 μM Gemfibrozil (GFZ, middle), or 10 μM 25-hydroxycholesterol (25-HC, bottom). Cells were stained for RSV postfusion F protein on the cell surface. Intracellular actin was visualized by ActinRed staining and nuclei by NucBlue staining. Images representative of three independent experiments (two technical replicates). Scale bar indicated next to images.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Confocal Microscopy, Infection, Staining

Journal: npj Viruses

Article Title: STAT1 signaling controls cholesterol metabolism in epithelial cells and RSV-induced syncytia formation

doi: 10.1038/s44298-026-00173-w

Figure Lengend Snippet: Western blot of membrane fractions and total cell lysates in uninfected (‘Mock’) and rRSV-A-0594-eGFP-infected (MOI 0.5) wild-type ( A ) and STAT1 −/− HEp-2 ( B ) HEp-2 cells at 48 hpi. Infected cells were left untreated, treated with 50 μM Gemfibrozil (GFZ), treated with 5 μM methyl-β-cyclodextrin (MβCD) and 50 μM GFZ, or with 10 μM 25-hydroxycholesterol (25-HC). Membrane fractions were stained for prefusion F protein, postfusion F protein, and Na + -K + -ATPase (loading control). Total cell lysates were stained for total F protein (prefusion and postfusion conformation) and β-Actin (loading control). Images representative of three independent experiments.

Article Snippet: HEp-2 (ATCC, CCL-23) and STAT1 knockout (STAT1 −/− ) HEp-2 cells were cultured in Minimum Essential medium with Earl’s salts (Capricorn Scientific, MEM-A) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (P/S, Capricorn Scientific), 1% non-essential amino acids (Gibco), and 1% GlutaMAX (Gibco) at 37 °C, 5% CO 2 .

Techniques: Western Blot, Membrane, Infection, Staining, Control

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: Effects of MC1R deletion on the immune system of mice at rest. (a-d) Absolute values of blood leukocytes, lymphocytes, monocytes, and neutrophils in resting WT and MC1R-KO mice. (e-h) Proportions of blood leukocytes, lymphocytes, monocytes, and neutrophils in resting WT and MC1R-KO mice. n = 3. Data are the mean ± Standard deviation. ns: Not significant. MC1R: Melanocortin 1 receptor, MC1R KO: Melanocortin 1 receptor knockout, WT: Wild type.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO mice showing no resistance to DNFB-induced DTH. (a) Increase in the proliferation ability of splenocytes by MC1R deletion. (b) Ear swelling levels of WT and MC1R-KO mice from the same litter subjected to DNFB-induced DTH. (c) Spleen weight. (d) Spleen index of mice in different groups. (e and f) Detection of TNF-a and IL-6 levels in different groups of mice. n = 3. Data are the mean ± Standard deviation. ✶ ✶ P < 0.01, ns: Not significant. MC1R KO: Melanocortin 1 receptor knockout, DNFB: Dinitrofluorobenzene, DTH: Delayed-type hypersensitivity, MC1R: Melanocortin 1 receptor, WT: Wild type, TNF-a: Tumor necrosis factor-a, IL: Interleukin.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO mice being more sensitive to CIA stimulation. (a) Representative images of the feet of WT and MC1R-KO mice treated with CII. (b) Clinical scores of mice treated with CII. (c) Incidence of arthritis in mice treated with CII. (d) Representative histopathological sections of joints in WT and MC1R-KO CIA mice. Untreated mice were used as negative controls. Scale bar: 100 or 200 μm. Objective: ×400 or ×200. The red arrows point to the areas with obvious damage. (e) Statistical data on histopathological scores of mouse tissues. (f) Serum anti-CII antibody levels. n = 3. Data are the mean ± Standard deviation. ✶ ✶ P < 0.01. Ctrl: Control, MC1R KO: Melanocortin 1 receptor knockout, CIA: Collagen-induced arthritis, WT: Wild type, MC1R: Melanocortin 1 receptor.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Standard Deviation, Control, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: Effects of MC1R deletion on T cell subsets in CIA mice. (a-d) Fluorescence-activated cell sorting (FACS) kanalysis of splenic tissue cells from WT and MC1R-KO mice under CIA. Percentages of Th1 (a and c) and Th2 (b and d) cells were measured. (e-h) FACS analysis of splenic tissue cells from WT and MC1R-KO mice under CIA. Percentages of Treg (e and g) and Th17 (f and h) cells were measured. n =3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ns: Not significant. MC1R: Melanocortin 1 receptor, CIA: Collagen-induced arthritis, MC1R KO: Melanocortin 1 receptor knockout, Th: T helper, WT: Wild type, IFN-g: Interferon-g; Treg: Regulatory T, Foxp3: Forkhead box protein P3.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Fluorescence, FACS, Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO promotes the production of inflammatory cytokines in CII-treated mice. (a and b) ELISA measurement of pro-inflammatory cytokines, including IL-17 and IFN-g. (c and d) ELISA measurement of anti-inflammatory cytokines, including IL-10 and IL-4. (e-g) qRT-PCR measurement of cytokines, including IL-6, TNF-a, and IL-1b. (h-m) Western blot analysis to validate changes in immune-related markers by detecting the protein expression levels of p-STAT3, STAT3, T-bet, RORgt, IL-17, and IFN-g. n = 3. Data are the mean ± Standard deviation. ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ns: Not significant. MC1R KO: Melanocortin 1 receptor knockout, ELISA: Enzyme-linked immunosorbent assay, IL: Interleukin, IFN-g: Interferon, TNF-a: Tumor necrosis factor-a, p-STAT3: Phospho-signal transducer and activator of transcription 3, RORgt: Retinoic acid receptor-related orphan receptor g-t, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation, Knock-Out, Reverse Transcription, Polymerase Chain Reaction

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO mouse splenocytes displaying higher CII-specific T cell immune response in vitro . (a-d) Proportions of Th17 cells and Treg cells within the cell population measured by FACS analysis. (e and f) Changes in the levels of pro-inflammatory cytokines IL-17 and IFN-g measured by ELISA. (g and h) Changes in the levels of anti-inflammatory cytokines IL-10 and IL-4 measured by ELISA. n =3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ns: Not significant. MC1R KO: Melanocortin 1 receptor knockout, Th17: T helper 17, ELISA: Enzyme-linked immunosorbent assay, IL: Interleukin, IFN-g: Interferon.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: Stattic inhibits the expression levels of proinflammatory factors in MC1R-KO mice. (a-e) Changes in the levels of pro-inflammatory cytokines IL-17, IFN-g, IL-6, IL-1b, and TNF-a measured by ELISA. (f and g) Changes in the levels of anti-inflammatory cytokines IL-10 and IL-4 measured by ELISA. (h-m) Western blot analysis to validate changes in immune-related markers by detecting the protein expression levels of p-STAT3, STAT3, T-bet, RORgt, IL-17, and IFN-g. n = 3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001. MC1R KO: Melanocortin 1 receptor knockout, IL: Interleukin, IFN-g: Interferon, TNF-a: Tumor necrosis factor-a, ELISA: Enzyme-linked immunosorbent assay, p-STAT3: Phospho-signal transducer and activator of transcription 3, RORgt: Retinoic acid receptor-related orphan receptor g-t.

Article Snippet: The MC1R knockout (MC1R-KO) + stattic group was intraperitoneally injected with 5 mg/kg of stattic (HY-13818, MedChemExpress, Monmouth County, NJ, USA) once every 2 days.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation, Knock-Out

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: DGATi and oleate treatment of U2OS cells promotes PML and CCTα association with the INM. (A) U2OS cells treated for 1 h with no addition (NA), oleate (500 µM), DGATi (10 µM) or oleate plus DGATi (10 µM) were immunostained for PML. DNA was visualized with Hoechst 33342 (bar, 10 µm). (B and C) cells were treated with oleate (B) or oleate plus DGATi (C) for up to 24 h, immunostained as described in panel A, and the percentage of cells positive for LAPS and PML patches was quantified. Each time point represents 30–50 cells from a representative experiment. (D) U2OS cells treated as described in panel A for 16 h were immunostained with antibodies against PML and CCTα. LDs were visualized with BODIPY 493/503 (bar, 10 µm). The nucleus is outlined, and regions of interest (ROI) are to the right (bar, 2 µm). (E, F, G and H) quantification of PML patches per cell (E), percentage of cells with PML patches (F), PML NBs per cell (G) and NE enrichment of CCTα (H). Results in E–H are the mean and SD from 9 to 12 fields of cells (6–10 cells/field) per treatment from three independent experiments. Statistical significance determined by two-way ANOVA with multiple comparisons. **** p < 0.0001, *** p < 0.001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Unsaturated fatty acids and DGATi promote PML patch formation and CCTα enrichment of the INM. (A) U2OS cells treated with no addition or 500 µM palmitate (16:0), stearate (18:0), oleate (18:1), linoleate (18:2), or α-linolenate (18:3) in the absence or presence of DGATi for 24 h were immunostained for PML and CCTα, and LDs visualized with BODIPY 493/503 (bar, 10 µm). The percentage of cells with LAPS (B) and PML patches (C), and NE enrichment of CCTα (D) were quantified. Results are the mean and SD 8–16 fields of cells ( n = 1057) from two experiments. Statistical significance determined using two-way ANOVA with multiple comparisons. **** p < 0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML patches are depleted of canonical PML NB-associated proteins. U2OS cells were treated with DGATi and oleate for 16 h and immunostained with antibodies against SUMO (A), SP100 (B) or DAXX (C). The nucleus was visualized with DAPI in the merged image. Small arrows point to PML patches and large arrows point to PML NBs (bar, 10 µm).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML patches are zones of nuclear lamina depletion. (A) Clover-PML U2OS cells treated with DGATi and oleate for treated for 24 h were immunostained for LMNA/C and DNA was visualized with Hoechst 33342 (bar, 5 µm). (B) Clover-PML U2OS cells treated as described above were immunostained for emerin (bar, 10 µm). Arrows indicate PML patches at regions of LMNA/C and emerin depletion.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: PML-II forms patches on the INM during inhibition of TAG synthesis. (A) U2OS PML KO cells transiently expressing GFP-tagged PML-I, PML-II, or PML-IV were treated with no addition (NA), oleate, or oleate and DGATi for 16 h and immunostained with antibodies against LMNA/C and CCTα (bar, 10 µm). The percentage of GFP-expressing cells ( n = 163) with (B) LAPS (identified as ring structures with associated GFP-PML and CCTα) and (C) PML patches were quantified. Results are the mean and SD of three independent experiments. Statistical significance assessed using Student's test. * p < 0.05, ** p < 0.01.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Inhibition, Expressing

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Stabilization of INM-associated CCTα in DGATi and oleate treated cells. (A) Lysates from U2OS cells treated with no addition (NA) or DGATi, with and without oleate for 16 h were immunoblotted for Lipin1, CCTα, and OSBP. (B) CCTα protein expression from panel A was quantified relative to no addition control (mean and SD of three experiments). (C) mRNA expression of PCYT1A in U2OS cells treated as described in panel A (mean and SD of four replicates from two experiments). (D) lysates of U2OS cells treated for 16 h with NA, DGATi (10 µM) in the presence of 0–500 µM oleate or tunicamycin (TM) were immunoblotted for CCTα, PML, eIF2α, pSer51 eIF2α (p-eIF2α) and OSBP (load control). (E) Immunoblots of lysates from cell treated with DGATi and oleate for 0 to 24 h. (F) U2OS cells were pretreated with oleate or oleate plus DGATi for 20 h followed by media without oleate or DGATi but supplemented with MG132 for 4 h. Cell lysates were immunoblotted for CCTα and OSBP. (G) Quantification of CCTα expression from panel H (mean and SD of four experiments) normalized to OSBP load control. Statistical significance was determined using Student's t test. * p <0.05, ** p <0.01, **** p <0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Expressing, Control, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Temporal relationship between CCTα, PML and DAG content of the INM. (A) U2OS cells transiently expressing a nuclear-localized GFP-tagged DAG sensor and treated with oleate or oleate and DGATi for 24 h were immunostained for PML and LMNA/C (bar, 10 µm). A region of interest (ROI) from the merged images is shown (bar, 2 µm). (B, C, and D) Cells expressing the nGFP-DAG sensor were treated with oleate and DGATi for the indicted times and immunostained for PML. The NE enrichment of nGFP-DAG (B), NE enrichment of CCTα (panel C) and percentage of cells with PML patches (panel D) were quantified. (E, F, and G) Cells expressing nGFP-DAG sensor were pretreated with DGATi and oleate for 16 h followed by media with no addition. The NE enrichment of nGFP-DAG (E), NE enrichment of CCTα (F) and percentage of cells with PML patches (G) were quantified by confocal imaging at the indicated times. Results in panels B to G are the mean and SD from five fields of cells from representative experiments. (H) U2OS cells transfected with non-targeting siRNA (siNT) or siRNA targeting LPIN1 (siLPN1) were treated with or without oleate plus DGATi for 24 h. Lysates were immunoblotted with antibodies against Lipin1, CCTα, and actin. (I and J) siNT and siLPN1 transfected cells treated with oleate plus DGATi were immunostained for PML along with BODIPY 493/503 and the percentage cells with PML patches (I) and PML patches per cell (J) was quantified. Results are from two representative experiments (22 fields of cells).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Expressing, Imaging, Transfection

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: The CCTα activators oleoyl alcohol and farnesol induce PML patches. (A) U2OS cells treated with oleate and DGATi, 100 µM farnesol or 100 µM oleyl alcohol (Oleyl-OH) for 4 h were immunostained for PML and CCTα along with BODIPY 493/503 (bar, 10 µm). The percentage of cells with PML patches and (B) CCTα NE enrichment (C) was quantified. Results are the mean and SD of five fields of cells ( n = 303) from a representative experiment. (D) U2OS cells treated with no addition (NA) or increasing concentrations of oleyl alcohol for 16 h were immunoblotted for CCTα and OSBP. (E) U2OS cells treated with NA or increasing concentrations of farnesol for 16 h were immunoblotted for CCTα and OSBP. (F) U2OS cells were treated with 100 µM oleyl alcohol for the indicated times and lysates were immunoblotted for PML, CCTα, and OSBP. (G) U2OS cells treated with NA or 100 µM farnesol for the indicated times and immunoblotted antibodies for PML, CCTα, and OSBP. Results for panels D–G were repeated three times with similar results. Statistical significance was determined using Student's t test. * p <0.05, ** p <0.01, **** p < 0.0001.

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Membrane curvature elastic stress triggers recruitment of PML-II onto the inner nuclear membrane

doi: 10.1091/mbc.E25-09-0443

Figure Lengend Snippet: Knockout of the terminal enzymes in PC synthesis increases PML patch formation. (A) U2OS, CEPT1 KO, and CHPT1 KO cells were immunostained for PML. DNA was stained with Hoechst 33342 (bar, 5 µm). (B) The percentage of cells with PML patches was quantified from images in panel A and is the mean and SD from 12 fields of cells ( n = 911) from three experiments. (C) U2OS, CEPT1 KO, and CHPT1 KO cells transiently expressing nGFP-DAG were immunostained for CCTα and LMNA/C (bar, 10 µm). (D and E) The NE enrichment of the nGFP-DAG (D) and CCTα (E; in GFP-positive nuclei) was quantified from two independent experiments. (F) control SH-SY5Y and SH-SY5Y CEPT1 KO cells were immunostained with antibodies against PML and CCTα (bar, 10 µm). (G and H) the percentage of cells with PML patches (G) and NE enrichment of CCTα (H) were quantified from eight fields of cells ( n = 411) from a representative experiment. (I) Lysates from SH-SY5Y and SH-SY5Y CEPT1 KO cells were immunoblotted for CCTα and OSBP or CEPT1 and actin (asterisk indicates a non-specific band). (J) CCTα expression relative to OSBP was quantified from two experiments. Statistical significance determined by Student's t test; *** p <0.001, **** p <0.0001 (ns, not significant).

Article Snippet: U2OS (ATCC HTB-96), U2OS PML knockout (KO; Attwood et al. , 2019 ), U2OS CEPT1 KO ( Dorighello et al. , 2023 ), U2OS CHPT1 KO ( Dorighello et al. , 2023 ), U2OS Clover-PML knock-in ( Pinder et al. , 2015 ), SH-SY5Y, SH-SY5Y CEPT KO, F8 human skin fibroblasts and Huh7 (JCRB0403, Japanese Collection of Research BioSources Cell Bank) cells were cultured in DMEM containing FBS (10%; medium A) at 37°C in a CO 2 (5%) atmosphere.

Techniques: Knock-Out, Staining, Expressing, Control

Journal: Cell reports

Article Title: Structures of invertebrate PEZO-1 isoforms with a compact architecture and a dispensable pore-distal N-terminal blade

doi: 10.1016/j.celrep.2025.116878

Figure Lengend Snippet: (A) Gene diagrams of pezo-1 G, K, and L isoforms, according to WormBase ( wormweb.org v. WS297), made with Exon-Intron Graphic Maker. Beginning rectangles and triangles denote the 5′ and 3′ UTRs, respectively; rectangles denote exons, and lines denote introns. (B) Schematic representation of monomer topology for PEZO-1 isoforms. Filled lines denote resolved regions, dashed lines denote unresolved regions. (C) Representative whole-cell patch-clamp recordings of mechanically activated currents in N2A Piezo1 −/− cells transfected with pezo-1 G, K, or L isoform. (D) Current densities evoked by maximal mechanical displacement. Bars are mean ± SEM. n.s. not significant by Kolmogorov-Smirnov and Mann-Whitney test ( p = 0.139). (E) Boxplot showing the displacement threshold required to elicit mechanocurrents. Boxplots show the median (bisecting line), as well as the minimum and maximum values (whiskers). n.s. not significant by Kolmogorov-Smirnov and Mann-Whitney test ( p = 0.901). (F) Boxplot of inactivation time constants evoked by maximal displacement. Kolmogorov-Smirnov and Mann-Whitney tests ( p = 0.002). ** p = 0.139 by Kolmogorov-Smirnov and Mann-Whitney test. (G–I) Cryo-EM density maps of PEZO-1 isoforms G (turquoise; PDB: 9ZIS, EMD: 74281), K (aluminum, PDB: 9ZIT, EMD: 74283), and L (red; EMD: 74433), viewed from the top, side, and bottom. See also - ; .

Article Snippet: Mouse: Piezo1 -knockout Neuro2a (N2A Piezo1−/− ) , ATCC , CAT#CCL-131.

Techniques: Patch Clamp, Transfection, MANN-WHITNEY, Cryo-EM Sample Prep

Journal: Cell reports

Article Title: Structures of invertebrate PEZO-1 isoforms with a compact architecture and a dispensable pore-distal N-terminal blade

doi: 10.1016/j.celrep.2025.116878

Figure Lengend Snippet: (A) Lateral view of PEZO-1 isoform G highlighting the cap and central regions. Arrows indicate the spring linker. The cap width is denoted above the respective model. The pore-distal region of the blade is removed for ease of viewing. (B) Lateral view of PIEZO1 (PDB: 7WLT). Arrows indicate the spring linker. (C) Lateral view of PIEZO2 (PDB: 6KG7), whose spring linker is unresolved. (D) Top view of PEZO-1 isoform G showing a larger cap surface area. (E) Top view of PIEZO1. The cap area of PEZO-1G is shown as a dashed outline. (F) Top view of PIEZO2. The cap area of PEZO-1G is shown as a dashed outline. (G) PEZO-1 isoform G multiple hydrogen bonds (R2128–C1278′ , T2127–S1288′, D2123-K1289′, and N2118–E1689′), a salt bridge (K2144–E1689′), and a cation–π interaction (Y2125–K1289′) between the cap and THU7–THU8. (H) Single salt bridge in PIEZO1 (E2257–R1761′). (I) Absence of interactions in PIEZO2. See also .

Article Snippet: Mouse: Piezo1 -knockout Neuro2a (N2A Piezo1−/− ) , ATCC , CAT#CCL-131.

Techniques:

Journal: Cell reports

Article Title: Structures of invertebrate PEZO-1 isoforms with a compact architecture and a dispensable pore-distal N-terminal blade

doi: 10.1016/j.celrep.2025.116878

Figure Lengend Snippet: (A) Ion conduction pathways of PEZO-1 isoforms G and K along the z axis. (B) HOLE profiles of the permeation pathways of PEZO-1G and PEZO-1K compared with available PIEZO1 conformations. Differences in the profile between PEZO-1G and PEZO-1K are indicated by asterisks. (C) Ion conduction pathways of the curved (PDB: 7WLT), intermediate (PDB: 8IXO), and flattened (PDB: 7WLU) conformations of PIEZO1. (D) Ribbon representations showing sites of constriction at the cap gate, transmembrane (TM), and neck regions of PEZO-1G and PIEZO1. Dashed and solid lines represent interactions and distances, respectively. See also .

Article Snippet: Mouse: Piezo1 -knockout Neuro2a (N2A Piezo1−/− ) , ATCC , CAT#CCL-131.

Techniques:

Journal: Cell reports

Article Title: Structures of invertebrate PEZO-1 isoforms with a compact architecture and a dispensable pore-distal N-terminal blade

doi: 10.1016/j.celrep.2025.116878

Figure Lengend Snippet: (A) (Top) Lateral view of PEZO-1G highlighting the beam relative to the pore and its interactions with the proximal and distal blade, close to the N and C terminus, respectively (bottom). (B) Comparison of the beam in PEZO-1G and PEZO-1K with those of the curved, intermediate, and flattened conformations of PIEZO1, as well as PIEZO2. These beams are also depicted as simplified line schematics. An angle illustrating the relative orientation of the beam of PEZO-1G to the beam of PIEZO1 in the curved state is shown. (C) Electrostatic potential surface of the beam-THU7 of PEZO-1G, PEZO1-K, PIEZO1, and PIEZO2. Positively charged regions are shown in blue, and negatively charged regions are shown in red. (D) Lateral view of PEZO-1 isoform G with the cytoplasmic latch-plug denoted in the dashed box. (E) Comparison of the PEZO-1G, PIEZO1 (curved), and PIEZO2 cytoplasmic latch-plugs, highlighting key residues located within this region. See also and .

Article Snippet: Mouse: Piezo1 -knockout Neuro2a (N2A Piezo1−/− ) , ATCC , CAT#CCL-131.

Techniques: Comparison

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) ClinVar classifications of RAD51D missense variants each year. P/LP = Pathogenic/Likely Pathogenic, B/LB = Benign/Likely Benign, VUS/CON = unknown significance/conflicting reports. ( b-c ) RAD51D forms an obligate heterodimer with XRCC2 to form the ( b ) BCDX2 (PDB: 8GBJ) or ( c ) XRCC3 (X3CDX2) complexes (PDB: 9SVX) through its interaction with RAD51C. ( d ) Schematic of pooled RAD51D variant function assay. RAD51D mutant library cell population is treated with 250 nM olaparib over several passages, selecting for cells with normal RAD51D function. RAD51D variants are quantified within the starting (P0) and final (P2) cell populations by sequencing and scored by their relative enrichment in P2 versus P0.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Functional Assay, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Histograms of RAD51D variant abundance, log10(counts/million counts), within pre-selection libraries for each mutagenesis tile. Cutoff line is at 100 cpm (1/10,000).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Selection, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Distributions of function scores by variant class; nonsense variants in codons 1–304 are perfectly separated from synonymous variants. Vertical lines denote cutoffs for function classifications (LOF, loss-of-function; INT, intermediate; NEU, neutral). ( b ) Per-residue mean missense function scores, grouped by residue surface area exposure status and secondary structure. Unique LOF exposed residues are labeled. ( c ) Variant-to-function heatmap across RAD51D, shaded by function score (white, WT-like; red, null-like) for each mutant amino acid (rows) at each codon position (columns). Variants that do not pass multiple testing correction (lfsr>0.01) are shaded from white to gray, WT residues are boxed, and dark gray denotes missing data. Tracks above heatmaps, from top to bottom: conservation score, protein secondary structure, key domains. Asp (D) or Glu (E) are shown in boldface to highlight stronger effects of these substitutions at some sites. ( d ) Function scores separate pathogenic from benign variants among single-residue variants reported in ClinVar, and provide evidence for missense VUS and those with conflicting reports (all variants plotted SpliceAI score <0.2). Point color denotes variant type, and filled/open points denote statistical significance (i.e. filled: lfsr ≤0.01). ( e ) Precision-recall curve showing classification performance between a set of 211 SNVs with confident P/LP and B/LB variant classification, included in ( a ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Residue, Labeling, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Surface representation of RAD51D within the BCDX2 complex (PDB: 8GBJ). Surface residues are colored according to either red (50% pathogenicity) or yellow (20% pathogenicity). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ( b ) Specific loss-of-function variants (red, 50% pathogenicity) listed above or (yellow, 20% pathogenicity) listed below the RAD51D schematic are shown with the indicated interaction interfaces indicated. ( c-g ) Binding sites for RAD51C ( c ), ATP site 1 ( d ), XRCC2 ( e ), ssDNA ( f ), and ATP site 2 ( g ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Intolerance of negatively charged (i.e. Asp (D) and Glu (E)) residues throughout RAD51D, as measured by the difference between the mean function scores of D/E and non-D/E mutations. Residues with a difference ±0.5 are labeled. ( b ) Mean function scores of residues making contact with other proteins, ATP, or DNA via H-bonds or salt bridges (noted with circles or starbursts, respectively) in the BCDX2 complex (as predicted in PDB: 8GBJ). ( c ) AlphaMissense pathogenicity scores compared to MAVE function scores. AlphaMissense scores were binned as benign, pathogenic, and ambiguous using the published cutoff values. Counts of each plotted category are reported in the table.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Labeling

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet:

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques:

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Schematic of sister chromatid exchange assay to measure homologous recombination. In this assay, a cassette with a non-functional copy of GFP is integrated into RAD51D CRISPR/Cas9 U2OS cells. This GFP has a unique I-SceI restriction cut site. A DSB can be induced by expression of a plasmid expressing the I-SceI restriction enzyme. GFP expression is restored by use of a homologous template provided on the cassette following homologous recombination. ( b-c ) A plasmid with indicated synonymous or truncation variant was transiently transfected RAD51D CRISPR/Cas9 U2OS cells with a plasmid coding for the I-SceI restriction enzyme. The percentage of GFP+ cells was measured after three days, indicating a recombination event using a GFP fragment on the cassette. The HR proficiency threshold was determined based on comparison with the range of synonymous variants (green bars) to a wild-type RAD51D expressing plasmid (HR >0.75). The threshold for loss of HR function was calculated using the range of truncation variants compared to a wild-type RAD51D expressing plasmid, as <0.6 (indicated in red for the variants). Note that a subset of those variants analyzed here are replotted in as representative variants. The experiment was performed three to seven times with standard deviations plotted. An empty vector was used as a negative control.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Homologous Recombination, Functional Assay, CRISPR, Expressing, Plasmid Preparation, Variant Assay, Transfection, Comparison, Negative Control

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) The HR proficiency of 70 RAD51D variants was tested using the sister chromatid recombination assay calibrated against synonymous and truncation variants. Loss of HR function was calculated based on the range of truncation variants (indicated in red) as <0.6 (missense LOF in light). HR proficient variants (gray), were determined based on comparison with the range of synonymous variants (green) if they exhibited HR >0.75. Variants with intermediate HR proficiency, in the range of 0.6–0.75, were color-coded in yellow. See for all synonymous and truncation variants tested, and note that a subset of those variants analyzed are replotted here as representative variants. The experiment was performed 4–9 times and plotted as mean values ± s.d. ( b-e ) Olaparib and cisplatin sensitivity of breast/ovarian cancer identified RAD51D variants with reduced HR. Representative images of U2OS cell lines stably expressing WT or the indicated RAD51D variant that were treated with increasing concentrations of Olaparib ( b ) or cisplatin ( d ). ( c & e ) Clonogenic survival assays were quantified by percent colony area and normalized to the area of untreated or vehicle control. Means of 4–12 trials are plotted ± s.d., and drug concentrations with colony area <0.001 are omitted. ( f ) RAD51D variants with reduced HR are expressed. Western blot analysis of U2OS cell lines stably expressing WT or the indicated RAD51D variants. RAD51D protein expression was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-Tubulin antibody. Note that L4H exhibits reduced protein expression. Experiment performed in triplicate. ( g ) Structures of the RAD51 paralog pentamer, XRCC3 complex, (PDB: 9SVX) and the BCDX2 complex (PDB: 8GBJ). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), XRCC3 (yellow), RAD51 (orange), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ssDNA is shown in orange. ( h-i ) Variants causing complete (red, h ) or intermediate (yellow, i ) deficiency in homologous recombination are highlighted on RAD51D. The interacting surfaces of RAD51C and XRCC2 are outlined with black dashed lines. The contact interfaces are identical between the XRCC3 and BCDX2 complex. ( j-k ) Variants that disrupt RAD51C-RAD51D interactions are shown as red sticks, mapped onto the BCDX2 complex structure.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Recombination Assay, Comparison, Stable Transfection, Expressing, Variant Assay, Control, Western Blot, Homologous Recombination

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a-b ) Interaction proficiency of RAD51D VUS was measured via ( a ) yeast 2-hybrid (Y2H) of pGAD-RAD51D or its variant, expressed in a GAL4 DNA activating domain expressing plasmid with pGBD-XRCC2 expressed in the GAL4 DNA binding domain expressing plasmid and ( b ) pGAD-RAD51D or its variant, expressed in the pGAD (GAL4 DNA activating domain) plasmid with pGBD-RAD51C (GAL4 DNA binding domain) plasmid with pADH1-RAD51B via yeast 3-hybrid (Y3H). RAD51B serves to stabilize RAD51C protein levels. Empty vectors are used as a negative control. Quantification of yeast growth from three experiments is plotted as mean ± s.d relative to the WT control. Representative images for variants and controls are shown. Variants with <50% interaction are classified as deficient and plotted in red. ( c ) RAD51D variants with reduced interaction with XRCC2 or RAD51C are expressed. Western blot analysis of protein extract from yeast cells in (a) expressing WT RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed by measuring the amount of Kar2 using an anti-Kar2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. See for Western blots of all variants. ( d-f ) MAVE function scores vs individual variant assay results of (d) HR activity, (e) XRCC2 binding, and (f) RAD51C binding. Missense variant point shape denotes functional status (LOF/INT/NEU) based upon MAVE result.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Control, Western Blot, Activity Assay, Functional Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Western blot analysis of protein extract from U2OS cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-tubilin antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. ( b ) HR analysis of RAD51D variants that are stably expressed in the RAD51D KO cell line used for the clonogenic survival assays shown in .

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Western blot analysis of protein extract from yeast cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-KAR2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Pull-down analysis of WT BC (RAD51B-His/RAD51C) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( b ) ssDNA binding of BCDX2 paralog complexes reconstituted by mixing WT BC and DX2 sub-complexes with indicated RAD51D variants. ( c ) Pull-down analysis of WT CX3 (RAD51C-His/XRCC3-STREP) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( d ) ATPase analysis of 0.5 μM WT BC and DX2 sub-complexes with indicated RAD51D variants in the presence of ssDNA after 60 min incubation. Pi indicates released inorganic phosphate after hydrolysis. ( e ) ssDNA binding of X3CDX2 paralog complexes reconstituted by mixing WT CX3 and DX2 sub-complexes with indicated RAD51D variants. For ( b, d-e ), results from three independent experiments were plotted as mean values ± s.d.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Incubation

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Summary of cellular findings. Schematic of RAD51D (1-328 aa) is shown with the Walker A and B motifs (green), and DNA binding loops (blue) indicated. The variants analyzed are shown above with the results from the cellular studies (MAVE variant-function map; ), RAD51D Y2H interaction with XRCC2 and its Y3H interaction with RAD51C , SCR recombination (HR), as well as olaparib and cisplatin clonogenic survival assays are summarized based on functional score. Loss-of-function is shown in red, an intermediate function is shown in yellow, and wild-type function is indicated in green. ( b ) Summary of biochemical findings. Variants included in biochemical analysis for BCDX2 or XRCC3 (X3CDX2) complex formation, DNA binding, and ATPase restrain from are shown. ( c ) Model for BCDX2 and X3CDX2 function in DSB repair. Upon DSB formation, the 3’ ssDNA end is resected and coated by RPA. The BCDX2 and X3CDX2 complexes facilitate the displacement of RPA and the loading of RAD51 onto ssDNA. This is achieved by the regulation of BC ATPase activity by DX2, which facilitates its binding to ssDNA. CX3 in complex with DX2 then targets RAD51 to ssDNA. The combined functions of the BCDX2 and X3CDX2 complexes are necessary to facilitate RAD51 filament assembly and subsequent RAD51-mediated homology search and strand exchange activities.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Variant Assay, Functional Assay, Activity Assay