Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Cortical strength is weaker in mice with type 2 diabetes mellitus (T2DM) treated with metformin compared with those treated with exendin-4. (A) Schematic chart of induction of T2DM in mice (WT, GLP1RKO) and the treatments with metformin or exendin-4. WT: wild type; GLP1RKO: glucagon-like peptide-1 receptor knockout. (B) Micro-CT images representing trabecular formation in control mice, mice with T2DM, mice with T2DM treated with exendin-4/metformin, and GLP-1R knockout mice with T2DM treated with exendin-4 within a region of interest (ROI). n=6 mice per group. Con: control; T2DM: type 2 diabetes mellitus; Met: metformin; Ex-4: Exendin-4; Micro-CT: micro-computed tomography. (C) Quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 mice in each group. (D) Micro-CT images representing cortical formation in control mice, mice with T2DM, mice with T2DM treated with exendin-4/metformin and GLP-1R knockdown mice with T2DM treated with exendin-4 within an ROI, and quantitative analysis of micro-CT images. n=6 mice in each group. (E) Effects of metformin or exendin-4 on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 mice in each group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Knock-Out, Micro-CT, Control, Knockdown, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 reduces Sema4D levels to elevate cortical strength in mice with type 2 diabetes mellitus (T2DM). (A) Schedule of enzyme-linked immunosorbent assay (ELISA) and the corresponding groups. (B) ELISA to determine the levels of ALP, Runx-2, OCN, OPN, and Sema4D in control mice, mice with T2DM, mice with T2DM treated with metformin (Met)/exendin-4 (Ex-4), and GLP-1R knockdown T2DM mice treated with Ex-4. n=6 mice per group. ALP: Alkaline Phosphatase; Runx-2: Runt-related transcription factor 2; OCN: Osteocalcin; OPN: Osteopontin; Sema4D: Semaphorin 4D. (C) Schedule of micro-CT to determine changes in trabecular bone. (D) Micro-CT images representing trabecular formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within a region of interest (ROI). n=6 mice per group. (E) Quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 mice per group. (F, G) Micro-CT images representing cortical bone formation in control mice, mice with T2DM, mice with T2DM treated with Ex-4, mice with T2DM treated with anti-Sema4D, and mice with T2DM treated with a combination of Ex-4 and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 mice per group. (H) Effects of exendin-4 or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Knockdown, Micro-CT, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: The combination of metformin and exendin-4 can neutralize the effect of Sema4D. (A) Schematic chart of the sample preparation of micro-CT and 3-point bending experiments to determine the effect of the combination of metformin and exendin-4. (B) Micro-CT images representing trabecular formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within a region of interest (ROI), and quantitative analysis of micro-CT images. BV: bone volume; TV: tissue volume; Tb.N: trabecular number; Tb.Th: trabecular thickness; Tb.Sp: trabecular spacing; BS/BV: bone superficial area/bone volume. n=6 per group. (C) Micro-CT images representing cortical formation in control mice and in mice treated with metformin, exendin-4, the combination of metformin and exendin-4, and anti-Sema4D within an ROI, and quantitative analysis of micro-CT images. n=6 per group. (D) Effects of metformin, exendin-4, the combination of metformin and exendin-4, or anti-Sema4D on the biomechanical properties of femoral diaphysis evaluated using the 3-point bending test. Femoral maximum load (N), stiffness (N/mm), maximum stress (MPa), and Young’s modulus (GPa). n=6 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Sample Prep, Micro-CT, Control, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 promotes pseudopodia numbers, leading to the stretching and wider spread of BMSCs via CRMP2. (A) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in human bone mesenchymal stem cells (hBMSCs). n=5 per group. White arrows indicate the pseudopodium in BMSCs. (B) Immunofluorescence staining to determine F-actin expression in BMSCs. n=5 per group. (C) Western blotting to determine F-actin expression in different groups. n=3 per group. (D) Proteomics analysis to determine the expression of different phosphorylated proteins. (E) Cell morphology observed using fluorescence microscopy. ImageJ was used to split color channels to observe the pseudopodium in BMSCs. n=5 per group. White arrows indicate the pseudopodium in BMSCs. (F) Immunofluorescence staining showing F-actin in different groups. n=5 per group. (G) Immunofluorescence staining indicating CRMP2 expression in BMSCs. n=5 per group. CRMP2: Collapsin Response Mediator Protein 2. (H) Western blotting to determine ALP, Runx-2, and Osterix expression in BMSCs. n=3 per group. (I) Alizarin red staining to analyze mineral deposits in BMSCs with DPYSL2 KD/OE. n=3 per group. DPYSL2 KD/OE: Dihydropyrimidinase like protein 2 knockdown/overexpression. (J) Western blotting to determine pCRMP2 expression in BMSCs with Sema4D or exendin-4. n=3 per group. (K) Immunofluorescence staining to determine pCRMP2 expression in BMSCs. n=5 per group. **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Staining, Expressing, Western Blot, Knockdown, Over Expression, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Exendin-4 activates bone formation via the GLP-1R/PI3K/GSK-3β/CRMP2 signaling pathway. (A) Western blotting results of pCRMP2 and F-actin expression in BMSCs with GSK-3β knockdown or overexpression. n=3 per group. GSK-3β: Glycogen Synthase Kinase 3 beta. (B) Immunofluorescence staining of F-actin and pCRMP2 expression in BMSCs. n=5 per group. (C) Western blotting to determine pCRMP2 expression after Sema4D treatment with or without GSK-3β knockdown. n=3 per group. (D) Western blotting to determine pCRMP2 expression after exendin-4 treatment with or without GSK-3β knockdown. n=3 per group. (E) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after Akt knockdown or overexpression. n=3 per group. Akt: Protein Kinase B/ Ak strain transforming. (F) Western blotting to determine the expression of pGSK-3β, pCRMP2, and F-actin after PI3K knockdown or overexpression. n=3 per group. PI3K: Phosphoinositide 3-Kinase. (G) Western blotting to determine the expression of pGSK-3β after exendin-4 treatment with or without PI3K knockdown and overexpression. n=3 per group. (H) Western blotting to determine P-PI3K expression after exendin-4 treatment with or without GSK-3β knockdown and overexpression. n=3 per group. (I) Western blotting to determine pGSK-3β expression after Sema4D treatment with or without PI3K knockdown and overexpression. n=3 per group. (J) Western blotting to determine pPI3K expression after Sema4D treatment with or without GSK–3β knockdown or overexpression. n=3 per group. *p < 0.05; **p < 0.01; ***p < 0.001. Data are presented as mean ± standard deviation.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Immunofluorescence, Staining, Standard Deviation

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Metformin modulates the expression of plexin B1 and GLP-1R in hBMSCs. (A) Differential expression of miRNAs among hBMSCs, hBMSCs+Sema4D, and hBMSCs+Sema4D+Met. MiR-140-3p and miR-3657 showing a significant increase and reduced expression in hBMSCs treated with Met. (B) Differential expression of miR-140-3p, plxnb1, and GLP-1R in different groups. n=3 per group. (C, D) Western blotting for Plexin B1 and GLP-1R expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (E) MiR-3657 expression in BMSCs, BMSCs+Sema4D, and BMSCs+Sema4D+Met. n=3 per group. (F, G) GLP-1R and Plexin B1 levels in BMSCs (con, miR-140-mimics/inhibitor, miR-3657-mimics/inhibitor). n=3 per group. (H) GLP-1R levels in BMSCs (con, miR-140-mimics, miR-140-mimics + miR-3657 inhibitor, miR-140-3p+miR-3657-mimics). n=3 per group.

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Expressing, Quantitative Proteomics, Western Blot

Journal: Journal of Advanced Research

Article Title: Discovery and identification of semaphorin 4D as a bioindicator of high fracture incidence in type 2 diabetic mice with glucose control

doi: 10.1016/j.jare.2025.03.014

Figure Lengend Snippet: Metformin promotes GLP-1R expression in hBMSCs via the miR-140-3p/STAT3/miR-3657 signaling pathway. (A) Schematic illustration of the sequences for miR-3657 and the wild-type or mutated 3′-UTR of GLP-1R mRNA, and dual Rluc/Fluc luciferase luminescence intensity of GLP-1R wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with miR-3657 mimics or miR–NC mimics. Rluc: Renilla luciferase; Fluc: Firefly luciferase; HEK293: Human Embryonic Kidney 293 Cells. (B, C) Protein expression of GLP-1R in hBMSCs after treatment with miR-3657 mimics (0, 10, 20, 40 μM) and the miR-3657 inhibitor (0, 10, 20, 40 μM). (D, E) Levels of ALP, RUNX-2, and Osterix in hBMSCs treated with exendin-4 and miR-3657 mimics (0, 10, 20, 40 μM)/miR-3657 inhibitor (0, 10, 20, 40 μM). (F) Schematic illustration of the sequences for STAT3 and the wild-type or mutated 3′-UTR of miR–3657, and dual Rluc/Fluc luciferase luminescence intensity of miR-3657 wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with STAT3 or pCDNA3.1. STAT3: signal transducer and activator of transcription 3. (G) qPCR to determine miR-3657 expression in hBMSCs treated with lenti-STAT3 and siR-STAT3 in a dose-dependent manner. n=3. Lenti-: lentivirus; siR-: siRNA; qPCR: quantitative polymerase chain reaction. (H) GLP-1R levels in hBMSCs after treatment with the STAT3 agonist and miR-3657 mimics (0, 10, 20, 40 μM) or treatment with the STAT3 inhibitor and miR-3657 inhibitor (0, 10, 20, 40 μM). (I) STAT3 levels in hBMSCs treated with metformin and the miR-140-3p inhibitor. (J) qPCR to determine miR-3657 expression in hBMSCs treated with metformin and miR-140–30 inhibitor/siR-STAT3. (K) Schematic illustration of the sequences for miR-140-3p and wild-type or mutated 3′-UTR of STAT3 mRNA, and dual Rluc/Fluc luciferase luminescence intensity of STAT3 wild-type or mutated 3′-UTR reporter plasmids in HEK293 cells co-transfected with miR-140-3p mimics or miR-NC mimics. (L, M) Protein expression of STAT3 in hBMSCs after treatment with miR-140-3p mimics (0, 10, 20, 40 μM) and with the miR-140-3p inhibitor (0, 10, 20, 40 μM).

Article Snippet: To figure out the different osteogenesis effect of Exendin-4 and metformin, mice were randomly divided into 5 groups (n = 6): control group, T2DM group, T2DM + metformin group (300 mg/kg/day, MCE), T2DM + Exendin-4 group (20 mg/kg, MCE,13241–33-3) and knockout GLP-1R T2DM mice + Exendin-4 group.

Techniques: Expressing, Luciferase, Transfection, Real-time Polymerase Chain Reaction

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: 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

Journal: Journal of Hematology & Oncology

Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

doi: 10.1186/s13045-025-01774-3

Figure Lengend Snippet: Targeting CD47 with SRF231 induces phagocytosis and cell death in lymphoid malignant cells. A. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in Jurkat cells cocultured with hMDM ( n = 2). B. Phagocytosis induction evaluated in the presence of 2-hour treatment with SRF231 or hIgG4 isotype in 11 CLL patient samples cocultured with hMDMs from 3 different donors (MAC1—MAC3). Reported P values were calculated by paired Student’s t test. C. Diagram showing gating strategy – Phagocytosis is identified as the CD14 + population with uptake of CFSE + lymphocytes. SRF231-mediated cell death is identified as the non-phagocytosed CD14 − cells with CFSE and Violet Live/Dead uptake. Violet Live/Dead (Zombie Violet™) is an amine-reactive fluorescent dye that stains cells that have compromised membranes as compared to AnnV stain that binds phosphatidylserine. Representative flow cytometry analyses showing the effect of hIgG4 isotype (10 µg/ml, top panel) and SRF231 (10 µg/ml, bottom panel) on phagocytosis and cell death of non-phagocytosed cells, cocultured with hMDMs. D. Population of non-phagocytosed Jurkat cells following treatment with either hIgG4 isotype or SRF231 were analyzed by flow cytometry. Tumor cell death denoted by % of death of non-phagocytosed cell population ( n = 2). E . Cells from 24 CLL patient samples were subjected to Protein G-bound SRF231 (10 µg/ml) and cell viability were measured by AnnV/Hoechst cell death assay. Decrease in percent live (AnnV-/Hoechst+) cells in SRF231 vs. hIgG4 isotype treatment was reported. Reported P values were calculated by paired Student’s t test. F. Healthy B cells (HB, n = 8), healthy T cells (HT, n = 8), healthy monocytes (HM, n = 3) and PBMC cells from CLL patients (CLL, n = 24) were subjected to Protein G-bound SRF231 or hIgG4 isotype treatment (10 µg/ml). Cell death induction was measured using AnnV/Hoechst assay at 0, 2, 4, 6 and 10 h, and data presented were % live cells of SRF231 normalized to their respective hIgG4 isotype. Reported P values were calculated by Sidak’s multiple comparison test. G-H. Western Blot showing CD47 and β-actin protein expressions in primary HB and CLL cells, Ri-1, Raji and Jurkat (Wild type and CD47 Knockout) cells. I. Cell death inductions of Ri-1 ( n = 3, 6 h; n = 4, 24 h), Jurkat ( n = 4) and Raji ( n = 4) cells were measured following Protein G-bound SRF231 incubation with AnnV/Hoechst assay at indicated timepoints. Reported P values were calculated by paired Student’s t test

Article Snippet: Ri-1 (Sigma-Aldrich, cat# 96090512), Raji (ATCC, cat# CCL-86), Jurkat (Clone E6-1, ATCC, cat# TIB-152) and Jurkat CD47 CRISPR/Cas9-knockout (KO) (Applied StemCell), OCI-Ly3 (DSMZ, cat# ACC761), OCI-Ly1, OCI-Ly1-R, TMD8 (Wu Lab, DFCI), MOLM14-S (Chng Lab, CSI, NUS), MOLM14-R, HL60 (Pervaiz Lab, NUS) cell lines were maintained in R10.

Techniques: Staining, Flow Cytometry, Comparison, Western Blot, Knock-Out, Incubation

Journal: Journal of Hematology & Oncology

Article Title: CD47 blockade-driven necroptosis complements BCL-2 inhibition-driven apoptosis in lymphoid malignancies

doi: 10.1186/s13045-025-01774-3

Figure Lengend Snippet: SRF231 induces cell death through necroptotic pathway. A . Schematic illustration of necroptosis signalling activation involving the phosphorylation of key proteins such as RIPK1/RIPK3 and MLKL as well as their respective inhibitors. Created in BioRender. Chamberlain, S. (2026) https://BioRender.com/toqddow . B-C. Western Blot showing the increased phosphorylation of RIPK1 (S166) and MLKL (S358) after Protein G-bound SRF231 treatment of Ri-1 cells (10 µg/ml, 24 h) or primary CLL cells (10 µg/ml, 6 h). D . Images of 6-hour hIgG4 isotype and SRF231 treated primary CLL cells displaying the intensity of p-RIPK1 and p-MLKL (all green), DAPI (blue) and merge using confocal microscopy (60x). E. Western blot showing the p-MLKL of wild-type Jurkat and CD47-KO Jurkat cells following treatment with Protein G-bound hIgG or SRF231 (10 µg/ml, 24 h). F. Cell viability of Jurkat cells treated with Protein G-bound hIgG or SRF231 (10 µg/ml, 24 h) following 48-hour siMLKL was measured with AnnV/Hoechst assay ( n = 4). Reported P values were calculated by Sidak’s multiple comparison test. G. Effect of 1-hour pre-treatment with RIPK3 inhibitor (GSK’872, 50 nM) or RIPK1 inhibitor (Necrostatin-1, 1 µM) on 6-hour Protein G-bound SRF231-induced cell death in 14 patient samples, was measured by AnnV/Hoechst assay. Reported P values were calculated by paired two-tailed Student’s t test. H. Western blot showing the effect of 1-hour pre-treatment with RIPK3 inhibitor (GSK’872, 50 nM) or RIPK1 inhibitor (NEC-1, 1 µM) followed by 6-hour treatment with Protein G-bound SRF231 (10 µg/ml) on p-MLKL in primary CLL cells. Total ERK and PGAM5 were used as loading controls. I. p-RIP1K and p-MLKL immunohistochemistry staining of hIgG control- or SRF231-treated formalin-fixed paraffin embedded tissues from subcutaneously implanted Raji tumors in CB17.SCID mice. J. Quantification of 6 representative region of interests (ROI) per sample of p-RIP1K and p-MLKL IHC staining in Fig. 2I. ( n = 3 per treatment). Reported P values were calculated by unpaired two-tailed Welch’s t test. OD: Optical Density

Article Snippet: Ri-1 (Sigma-Aldrich, cat# 96090512), Raji (ATCC, cat# CCL-86), Jurkat (Clone E6-1, ATCC, cat# TIB-152) and Jurkat CD47 CRISPR/Cas9-knockout (KO) (Applied StemCell), OCI-Ly3 (DSMZ, cat# ACC761), OCI-Ly1, OCI-Ly1-R, TMD8 (Wu Lab, DFCI), MOLM14-S (Chng Lab, CSI, NUS), MOLM14-R, HL60 (Pervaiz Lab, NUS) cell lines were maintained in R10.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Confocal Microscopy, Comparison, Two Tailed Test, Immunohistochemistry, Staining, Control, Formalin-fixed Paraffin-Embedded