neonatal skin fibroblast cell line  (ATCC)

Journal: Genome Medicine

Article Title: Succinate supplementation ameliorates musculoskeletal defects caused by PLOD3 mutations in a BCARD syndrome model

doi: 10.1186/s13073-026-01608-y

Figure Lengend Snippet: Genetic variants in the PLOD3 gene lead to similar phenotypes in zebrafish. A Schematic depiction of the human PLOD3 gene carrying the c.1354 C > T transition in the AC domain (red) that was expected to generate Arg452*, a predicted early stop codon, thereby truncating the peptide. However, sequencing of the cDNA isolated from cultured patient fibroblasts revealed a premature splice site in exon 12, resulting in the deletion of p.Arg452_Val453 and correct splicing to exon 13. B Experimental design of zebrafish (z) and human (h) PLOD3 mRNA overexpression (OE) for rescue experiments. C Rescue experiments with zebrafish (z) and human (h) PLOD3 mRNA OE. Live imaging of larvae at 4 dpf WT, mgt −/− , mgt −/− + zplod3 mRNA, and mgt −/− + hPLOD3 mRNA and two variants (predicted PLOD3 Arg452* and observed Arg452_Val453del). ED = Eye diameter, BL= Body length; lateral views. D , E quantification of the rescue experiments in C. D. Quantification of overexpression (OE) experiments for body length after mRNA microinjection with wild-type zebrafish plod3 and human PLOD3 mRNAs. The n numbers for body length = WT (14) mgt −/− (14), mgt −/− + z- plod3 (8), and mgt −/− + h- PLOD3 (8). E. n = 8 for each group. One-way ANOVA with Tukey’s multiple comparisons test with 95% CL of diff was conducted for analysis, **** p < 0.0001, *** p < 0.001, ** p < 0.01, and ns = non-significant. F Whole-mount staining of WT, mgt −/− and mgt −/− + h-PLOD3 mRNA injected larvae with fluorescently-conjugated WGA lectin (green) to mark N-glycosylated proteins in the pectoral fins. Panels of magnified views of boxed areas show kinked pectoral fins (yellow arrowhead) in mgt −/− fish that were partially rescued with hPLOD3 mRNA. Quantification, WT n = 8, mgt −/− ( n = 9), and mgt −/− + hPLOD3 mRNA ( n = 8). One-way ANOVA with Tukey’s multiple comparisons test with 95% CL of diff was conducted to compare fins between mgt −/− and mgt −/− injected with an mRNA rescue group. **** p < 0.0001. G Rescue experiments of the body length using variants Leu627Pro and Cys690*. The one-way ANOVA with Tukey’s multiple comparisons test with 95% CL of diff was conducted for comparison between mgt −/− and mgt −/− injected with hPLOD3 variants mRNA; **** p < 0.0001. Error bars are the standard error of the mean

Article Snippet: BJ skin fibroblasts (ATCC CRL-2522) controlled all cell culture-related experiments.

Techniques: Sequencing, Isolation, Cell Culture, Over Expression, Imaging, Microinjection, Staining, Injection, Comparison

Journal: Genome Medicine

Article Title: Succinate supplementation ameliorates musculoskeletal defects caused by PLOD3 mutations in a BCARD syndrome model

doi: 10.1186/s13073-026-01608-y

Figure Lengend Snippet: Reduced expression of PLOD3 disrupts procollagen trafficking and induces ER stress in mgt −/− embryos and human BCARD variant fibroblasts. A Western blot (WB) of whole zebrafish protein lysates for Plod3 and type II collagen to assess unprocessed (pNpC), processing intermediates (pN or pC), and processed, mature collagen. α-Tub staining served as a loading control. B WB of protein lysates from human fibroblasts stained with PLOD3 ( n = 3 blots) and COL1 ( n = 4 blots) antibodies from WT (BJ fibroblasts) and PLOD3 (c.1353 C > T) variant dermal fibroblasts. α-Tub staining served as a loading control. C Cryosections of zebrafish craniofacial cartilage stained with Col2 recognizing antibody (red) and WGA (cell membranes, green) treated with proteinase K in WT and mgt −/− at 4 dpf. In WT, Col2 signal localizes to ECM and marks stacked chondrocyte boundaries, whereas in mgt −/− , cartilage elements and cells are misshapen (ECM pink arrow, n = 5 animals per group). DAPI marks nuclei in blue. D Cryosections that were not treated with proteinase K and stained with Col2 (red) and PDI (ER marker, green) antibodies revealed cytoplasmic accumulations and large intracellular deposits of Col2 in mgt −/− cartilage that co-localize with PDI (orange arrows, n = 3 animals per group). DAPI marks nuclei in blue. E IF images of WT and PLOD3 (c.1353 C > T) variant dermal fibroblasts show COL1 (red) staining partially co-localized with ERp57 (ER marker, green). Higher-magnification insets of the boxed areas show partial co-localization. DAPI marks nuclei in blue. F Pearson coefficient analysis of the co-localization of COL1 and ERp57; WT n = 13, variant n = 9. Data were analyzed with a two-tailed Student’s t-test, CI = 95%. Mean and SEM values are indicated with bars; **** p < 0.0001. G Expression analysis by qPCR of ER stress pathways-associated genes atf6 , atf4 , ddit3 (Chop) normalized to β-actin in 4 dpf zebrafish. Data were analyzed with a two-tailed Student’s t-test, CI = 95%. Mean and SEM values are indicated with bars ** p < 0.01, * p < 0.05, and ns = non-significant. H WB analysis in whole zebra-fish lysates at 4 dpf of the ER stress markers elF2α, its phosphorylated form P-elF2α, Chop, and α-Tub (as loading control). I TEM images of 3 dpf zebrafish chondrocytes showing distended rough ER in mgt −/− mutants compared to WT (black arrows). J TEM images of control BJ fibroblasts show tightly packed ER (arrow) and distended rough ER (arrows) in variant PLOD3 (c.1353 C > T) dermal fibroblasts. K WB of protein lysates from human WT and variant PLOD3 (c.1353 C > T) dermal fibroblasts stained with elF2α, P-elF2α, ATF4, CHOP, and α-Tub (as loading control)

Article Snippet: BJ skin fibroblasts (ATCC CRL-2522) controlled all cell culture-related experiments.

Techniques: Expressing, Variant Assay, Western Blot, Staining, Control, Marker, Two Tailed Test

Journal: Genome Medicine

Article Title: Succinate supplementation ameliorates musculoskeletal defects caused by PLOD3 mutations in a BCARD syndrome model

doi: 10.1186/s13073-026-01608-y

Figure Lengend Snippet: PLOD3 deficiency leads to increased autophagy. A Schematic diagram of autophagosome formation. B Cryosections of the craniofacial cartilage chondrocytes stained with Col2 antibodies (red) in Tg (eGFP-GABARAP) genetic background (green), with protease K treatment from WT and mgt −/− zebrafish at 4 dpf. The arrowhead marks an eGFP + chondrocyte in WT, and the yellow arrows in mgt −/− ; nuclei stained with DAPI (blue). C Quantification of eGFP-GABARAP + chondrocytes as a percentage of the total number of chondrocytes; WT, n = 288 cells ( N = 6 WT larvae); mutant, n = 250 cells ( N = 8 mgt −/− larvae). Data were analyzed with a two-tailed Student’s t-test, CI = 95%. Mean and SEM values are indicated with bars. **** p < 0.0001. D WB of whole zebrafish protein lysates in WT and mgt −/− at 4dpf labeled for Atg5, p62, Lc3l, Lc3ll, and Cathepsin B. α-Tub served as a loading control. E WB of protein lysates from human fibroblasts, WT and variant (c.1353 C > T), stained for ATG5, p62, LC3, LC3ll, and LAMP1, α-Tub served as loading control. F IF images of co-immunostaining for LC3B (green) and Lysosomes (Red, Lysotracker) in control fibroblasts (WT) and PLOD3 (c.1353 C > T) variant cells, nuclei stained with DAPI (blue). Inset of the boxed area highlights co-localization of the two markers. G Pearson coefficient analysis of the co-localization of LC3B and Lysotracker, WT n = 20 and variant n = 8. Data were analyzed with a two-tailed Student’s t-test, CI = 95%. Mean and SEM values are indicated with bars; **** p < 0.0001. H TEM image of 3 dpf WT zebrafish shows a healthy cytoplasmic compartment in a chondrocyte immersed in dense ECM, while mgt −/− chondrocyte contains large autophagosomes (arrowheads) in the cytoplasm. I TEM image of human WT fibroblast shows normal ER, Golgi, and other secretory compartments, while the human PLOD3 variant (c.1353 C > T) fibroblast shows degradative compartments: autophagosome (arrowhead 1), lysosome (arrowhead 2), autolysosome (arrowhead 3), and multilamellar bodies (arrowhead 4)

Article Snippet: BJ skin fibroblasts (ATCC CRL-2522) controlled all cell culture-related experiments.

Techniques: Staining, Mutagenesis, Two Tailed Test, Labeling, Control, Variant Assay, Immunostaining

Journal: Bioresources and Bioprocessing

Article Title: Box-Behnken optimized copper oxide nanoparticles from Thymus vulgaris potentiate efficacy against multidrug-resistant bacterial pathogens and exhibit anticancer activity

doi: 10.1186/s40643-026-01008-5

Figure Lengend Snippet: Comprehensive Evaluation of TE-CuONPs Anticancer Activity and Apoptotic Mechanism in MCF-7 Breast Cancer Cells. Concentration-dependent cytotoxicity and mechanistic analysis. a , b MTT dose–response curves against MCF-7 breast cancer cells and normal human skin fibroblasts (HSF). c , d Representative phase-contrast micrographs (200 × magnification) of MCF-7 cells after 24 h treatment at respective IC₅₀ concentrations, showing c minimal morphological changes with TE treatment versus d characteristic apoptotic features with TE-CuONPs treatment, including cell shrinkage (red arrows), membrane blebbing (yellow arrows), chromatin condensation, and cellular detachment. e Flow cytometric scatter plots (FSC-H vs. SSC-H) demonstrating consistent cell populations across treatments (10,000 events gated per sample). ( f ) Schematic proposal for anticancer mechanisms. g Annexin V-FITC/propidium iodide (PI) dual-staining flow cytometric analysis

Article Snippet: MCF-7 breast adenocarcinoma cells (ATCC HTB-22) and normal human skin fibroblasts (HSF, CRL-2522) were obtained from Nawah Scientific (Cairo, Egypt) and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2.

Techniques: Activity Assay, Concentration Assay, Membrane, Staining