Journal: The Journal of Biological Chemistry

Article Title: Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs

doi: 10.1016/j.jbc.2025.110328

Figure Lengend Snippet: Construction of human Focicle vector based on PiggyBac transposon system . A , design of PiggyBac transposon-based human Focicle vector. Tricistronic inserts of mRuby-hCdt1, Ypet-h53BP1 foci-forming region (FFR), and mTagBFP2-hGmnn are connected to P2A and T2A peptides and expressed under the CAG promoter. B , scheme of 53BP1, hCdt1, and hGmnn expression in cell cycle. C , generation of Focicle vector-integrated human RPE1-hTERT cells. Expression of 53BP1FFR ( green ), hCdt1 ( red ), and hGmnn ( blue ) was observed using a fluorescence microscope. hCdt1 positive cells indicate cells in G1 phase ( top ). hGmnn-positive cells indicate cells in the G2/M phase ( bottom ). Scale bars represent 10 μm. D , the distribution of DNA content in the hCdt1 - /hGmnn - ( red ), hCdt1 + ( orange ), and hGmnn + ( blue ) populations. E , Focicle RPE1-hTERT cells were exposed to 2 Gy gamma-ray, and 1 h after irradiation, 53BP1 foci were observed using fluorescence microscopy. F , Focicle RPE1-hTERT cells were treated with camptothecin (CPT) for 1 h, and 53BP1 foci were observed by fluorescence microscopy. G , cell cycle population after ionizing radiation (IR) exposure in Focicle RPE1-hTERT. Cells were untreated or treated with 2 Gy gamma-ray irradiation and analyzed at the indicated times after exposure. Cell cycle population was assessed by flow cytometry. hCdt1 positive cells were counted as G1 cells, and hGmnn-positive cells as G2 cells. Statistical analyses were performed using a one-way ANOVA and Tukey’s post hoc test. ns indicates no significant difference. ∗∗∗∗ p < 0.0001. All experiments were performed at least three times.

Article Snippet: The cells were blocked with 1% BSA/PBS-T at 4 °C for 1 h and stained with primary antibodies: SOX2 rabbit polyclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), Nestin mouse monoclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), MAP2 chicken polyclonal antibody (1:500, #ab5392, Abcam), Tublin β3 (Tuj1) mouse monoclonal antibody (#MMS-435P, Covance), 53BP1 rabbit polyclonal antibody (1:1000, #A300–272A; Bethyl), S139P γH2AX mouse monoclonal antibody (1:1000, #05–636; Merck Millipore), RAD51 mouse polyclonal antibody (1:500, #H00005888-B01P; Abnova), phosphor S2056 DNA-PKcs rabbit polyclonal antibody (1:500, #ab18192, Abcam) for 4 h–24 h at 4 °C.

Techniques: Plasmid Preparation, Expressing, Fluorescence, Microscopy, Irradiation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs

doi: 10.1016/j.jbc.2025.110328

Figure Lengend Snippet: Focicle vector-integrated human iPS cells . A , generation of Focicle vector-integrated human induced pluripotent stem cells (hiPSCs). Cell morphology of Focicle hiPSCs was observed by phase contrast microscopy in two independent clones (clones one and 2) and parental cells (C2). The scale bar represents 500 μm. B , to assess growth rate, cell numbers were counted after seeding on the indicated days in two independent clones (clone one and clone 2) and parental cells (C2). ( C and D ) Generation of Focicle vector-integrated hiPSCs. The expression of 53BP1FFR ( green ), hCdt1 ( red ), hGmnn ( blue ), and differential interference contrast image (DIC) were observed by confocal microscopy ( C ) and fluorescence microscopy ( D ) in two independent clones (clone one and clone 2). Scale bars represent 100 μm ( C ) and 50 μm ( D ). ( E and F ) Focicle hiPSCs were either untreated or irradiated with 2 Gy. One hour after irradiation, 53BP1 foci formation were counted and graphed in F. Scale bars represent 10 μm. ( G ) Distribution of DNA content in the hCdt1 - /hGmnn - ( red ), hCdt1 + ( orange ), and hGmnn + ( blue ) population in an hiPSC clone.

Article Snippet: The cells were blocked with 1% BSA/PBS-T at 4 °C for 1 h and stained with primary antibodies: SOX2 rabbit polyclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), Nestin mouse monoclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), MAP2 chicken polyclonal antibody (1:500, #ab5392, Abcam), Tublin β3 (Tuj1) mouse monoclonal antibody (#MMS-435P, Covance), 53BP1 rabbit polyclonal antibody (1:1000, #A300–272A; Bethyl), S139P γH2AX mouse monoclonal antibody (1:1000, #05–636; Merck Millipore), RAD51 mouse polyclonal antibody (1:500, #H00005888-B01P; Abnova), phosphor S2056 DNA-PKcs rabbit polyclonal antibody (1:500, #ab18192, Abcam) for 4 h–24 h at 4 °C.

Techniques: Plasmid Preparation, Microscopy, Clone Assay, Expressing, Confocal Microscopy, Fluorescence, Irradiation

Journal: The Journal of Biological Chemistry

Article Title: Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs

doi: 10.1016/j.jbc.2025.110328

Figure Lengend Snippet: Induction of human neural progenitor cells (hNPCs) from Focicle hiPSCs . A , Scheme for neural differentiation of hiPSCs. Day 7–Day 12 cells were used as hNPCs, and after Day cells were used as neurons. B , hNPCs were derived from Focicle hiPSCs. To confirm neural differentiation, cells were stained for the NPCs markers SOX2 ( green ) and nestin ( red ). 4′,6-diamidino-2-phenylindole (DAPI: blue ) was used for cell nucleus staining. The scale bar represents 10 μm. C , to confirm whether 53BP1FFR works in hNPCs, cells were exposed to 2 Gy gamma rays, and 53BP1 foci were observed 1 h after irradiation. Scale bars represent 10 μm. ( D ) The distribution of DNA content in the hCdt1 - /hGmnn - ( red ), hCdt1 + ( orange ), and hGmnn + ( blue ) populations in a hNPC clone. E , hNeurons were derived from Focicle hiPSCs. To confirm neural differentiation, cells were stained for the mature neuron markers MAP2 ( green ) and Tuj1 ( red ). DAPI ( blue ) was used for cell nucleus staining. Scale bars represent 10 μm.

Article Snippet: The cells were blocked with 1% BSA/PBS-T at 4 °C for 1 h and stained with primary antibodies: SOX2 rabbit polyclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), Nestin mouse monoclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), MAP2 chicken polyclonal antibody (1:500, #ab5392, Abcam), Tublin β3 (Tuj1) mouse monoclonal antibody (#MMS-435P, Covance), 53BP1 rabbit polyclonal antibody (1:1000, #A300–272A; Bethyl), S139P γH2AX mouse monoclonal antibody (1:1000, #05–636; Merck Millipore), RAD51 mouse polyclonal antibody (1:500, #H00005888-B01P; Abnova), phosphor S2056 DNA-PKcs rabbit polyclonal antibody (1:500, #ab18192, Abcam) for 4 h–24 h at 4 °C.

Techniques: Derivative Assay, Staining, Irradiation

Journal: The Journal of Biological Chemistry

Article Title: Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs

doi: 10.1016/j.jbc.2025.110328

Figure Lengend Snippet: Homologous recombination repair activity in hiPSCs, hNPCs, and hNeuro ns. A , hiPSCs, hNPCs, and hNeurons were exposed to 2 Gy gamma rays and fixed at the indicated times. Non-IR cells were used as an untreated control. Cells were stained with 53BP1 and the homologous recombination marker RAD51 antibody. The cell nucleus was stained with DAPI. Scale bars represent 10 μm. B , quantification of 53BP1 and RAD51. The DNA damage foci per cell were quantified using CellProfiler. Statistical analyses were performed using a one-way ANOVA and Tukey’s post hoc test. ns indicates no significant difference. ∗ p < 0.1, ∗∗ p < 0.001, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. All experiments were performed at least three times.

Article Snippet: The cells were blocked with 1% BSA/PBS-T at 4 °C for 1 h and stained with primary antibodies: SOX2 rabbit polyclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), Nestin mouse monoclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), MAP2 chicken polyclonal antibody (1:500, #ab5392, Abcam), Tublin β3 (Tuj1) mouse monoclonal antibody (#MMS-435P, Covance), 53BP1 rabbit polyclonal antibody (1:1000, #A300–272A; Bethyl), S139P γH2AX mouse monoclonal antibody (1:1000, #05–636; Merck Millipore), RAD51 mouse polyclonal antibody (1:500, #H00005888-B01P; Abnova), phosphor S2056 DNA-PKcs rabbit polyclonal antibody (1:500, #ab18192, Abcam) for 4 h–24 h at 4 °C.

Techniques: Homologous Recombination, Activity Assay, Control, Staining, Marker

Journal: The Journal of Biological Chemistry

Article Title: Live-cell imaging of DNA damage and cell cycle progression uncovers distinct responses during neural differentiation of hiPSCs

doi: 10.1016/j.jbc.2025.110328

Figure Lengend Snippet: Live cell imaging after laser microirradiation in Focicle hiPSCs, hNPCs, and hNeurons . A , hiPSCs, hNPCs, and hNeurons were exposed to 405 nm laser ablation, and YFP-53BP1 foci tracks were observed at the indicated time points (minutes). Scale bars represent 5 μm. B , YFP-53BP1 signal intensity in Focicle hiPSCs, hNPCs, and hNeurons (two independent clones) was measured and graphed. Error bars indicate standard error. At least 30 cells were analyzed at each time point (min).

Article Snippet: The cells were blocked with 1% BSA/PBS-T at 4 °C for 1 h and stained with primary antibodies: SOX2 rabbit polyclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), Nestin mouse monoclonal antibody (1:500, Human Neural Stem Cell Immunocytochemistry Kit, A24354, Invitrogen, Thermo Fisher Scientific), MAP2 chicken polyclonal antibody (1:500, #ab5392, Abcam), Tublin β3 (Tuj1) mouse monoclonal antibody (#MMS-435P, Covance), 53BP1 rabbit polyclonal antibody (1:1000, #A300–272A; Bethyl), S139P γH2AX mouse monoclonal antibody (1:1000, #05–636; Merck Millipore), RAD51 mouse polyclonal antibody (1:500, #H00005888-B01P; Abnova), phosphor S2056 DNA-PKcs rabbit polyclonal antibody (1:500, #ab18192, Abcam) for 4 h–24 h at 4 °C.

Techniques: Live Cell Imaging, Clone Assay