Journal: Cell reports

Article Title: Metabolic reprogramming during human neuron differentiation indicates glutaminase as a key determinant in Fragile X syndrome

doi: 10.1016/j.celrep.2025.116857

Figure Lengend Snippet: (A) Schematic of metabolites from iPSC-derived forebrain excitatory neurons generated from an FXS and CRISPR-edited isogenic control (iControl) line analyzed by LC-MS. Representative bright-field images and immunofluorescence (IF) images stained for NPC stage (SOX2), neurons (Tuj1), and astrocytes (GFAP) are shown. Scale bar: 70 μM. (B) Western blots and quantification of proteins from neurons matured for 1 or 4 weeks. Data are analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as the mean ± standard deviation (SD) of three replicates. * p < 0.05 and ** p < 0.01. (C) Volcano plot of polar metabolites in FXS and isogenic iControl cells in week 1 and 4 neurons ( N = 1, n = 3). Dotted lines mark log 2 FC = 0 (vertical) and the significance threshold (−log 10 p ≥ 1.3, horizontal). Color-coded metabolites are green for amino acids, red for nucleotide intermediates, and blue for energy metabolism intermediates. Intracellular metabolites are shown on the left and extracellular metabolites on the right (see and ). (D) Pie charts representing functional categories of the differentially expressed metabolites in FXS and CRISPR-edited isogenic control line samples based on intracellular metabolites (C). (E) Table of neurotransmitters and precursors altered in neurons (weeks 1 and 4). The arrows indicate the direction of change (C).

Article Snippet: Rabbit polyclonal Sox2 , Cell Signaling Technology , Cat#2748;RRID:AB_823640.

Techniques: Derivative Assay, Generated, CRISPR, Control, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Western Blot, Standard Deviation, Functional Assay

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: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 2 | NSUN4 inhibition affects cell growth, apoptosis, stemness, invasiveness, and migratory ability in vitro. (A–H) Various experiments were conducted using different treatment methods and cell types (A549 and SK-MES-1 NSCLC cells), with three groups: Sh-ctrl, sh-NSUN4#1, or sh-NSUN4#2. (A) Evaluation of NSUN4 protein expression by immunoblotting in cells transfected as indicated. (B) Assessment of the number of formed colonies with cells transfected as indicated. (C) Determination of cell apoptotic ratio with cells transfected as indicated by flow cytometry. (D and E) Examination of cell migratory rate and invasiveness with transfected cells. Scale bars: 100 μm. (F) Measurement of sphere formation with transfected NSCLC cells. Scale bars: 100 μm. (G and H) Expression of SOX2, CD133, and KLF4 proteins using immunoblot analysis in transfected A549 and SK-MES-1 NSCLC cells. n = 3 in (A–G). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Inhibition, In Vitro, Expressing, Western Blot, Transfection, Flow Cytometry

Journal: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 5 | NSUN4 affects NSCLC cell malignant phenotypes through CDC20. (A–H) A549 and SK-MES-1 NSCLC cells were subjected to intro- duction with vector + sh-ctrl, NSUN4 expression construct + sh-ctrl, vector + sh-CDC20, or NSUN4 + sh-CDC20. (A) CDC20 protein expression by immunoblotting in cells transfected as indicated. (B) The number of formed colonies by colony formation assay with cells transfected as indicated. (C) Cell apoptotic ratio by flow cytometry with cells transfected as indicated. (D and E) Cell migratory rate and invasiveness by transwell assay with transfected cells. Scale bars: 100 μm. (F) Measurement of sphere formation with transfected NSCLC cells. (G and H) Expression of SOX2, CD133, and KLF4 proteins by immunoblot analysis in transfected A549 and SK-MES-1 NSCLC cells. n = 3 in (A–G). *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Plasmid Preparation, Expressing, Construct, Western Blot, Transfection, Colony Assay, Flow Cytometry, Transwell Assay

Journal: Thoracic cancer

Article Title: NSUN4 Facilitates the Activity of Oncogenic Protein CDC20 to Promote NSCLC Development by Mediating m5C Modification of CDC20 mRNA.

doi: 10.1111/1759-7714.70023

Figure Lengend Snippet: FIGURE 6 | NSUN4 depletion hinders the growth of A549 subcutaneous xenografts. (A–E) A549 subcutaneous xenografts were generated by implanting sh-ctrl or sh-NSUN4#2 lentivirus-infected A549 cells. After 30 days, xenografts were harvested. n = 5 for each group. (A) Growth curves of A549 subcutaneous xenografts (n = 3). (B) Representative pictures of A549 subcutaneous xenografts. (C) Tumor average weight was calculated (n = 3). (D) Expression of NSUN4, CDC20, SOX2, CD133, and KLF4 proteins by immunoblot analysis in A549 subcutaneous xenografts (n = 3). (E) Expression of NSUN4, CDC20, SOX2, CD133, and KLF4 proteins by immunohistochemistry in sections of subcutaneous xenografts. **p < 0.01, ***p < 0.001.

Article Snippet: We harvested protein extracts from collected tissue specimens (~50 mg) or cultivated cells (1 × 107) and conducted immunoblot analysis as described previously [18] with rabbit anti- NSUN4 polyclonal (#29786- 1- AP, 1–4000, Proteintech), rabbit antiCDC20 polyclonal (#10252- 1- AP, 1–8000, Proteintech), rabbit anti- SOX2 polyclonal (#11064- 1- AP, 1–600, Proteintech), rabbit anti- CD133 monoclonal (#ab222782, 1–2000, Abcam), rabbit anti- KLF4 polyclonal (#11880- 1- AP, 1–6000, Proteintech), or mouse anti- β- actin monoclonal (#66009- 1- Ig, 1–50 000, Proteintech).

Techniques: Generated, Infection, Expressing, Western Blot, Immunohistochemistry