Journal: Science Advances

Article Title: An engineered insulin analog with dual insulin and IGF-1 receptor agonism and distinct signaling

doi: 10.1126/sciadv.aeb7558

Figure Lengend Snippet: Cells expressing IGF-1R ( A and E ), SH-SY5Y ( B and F ), U87MG ( C and G ), and rat primary neuronal culture ( D and H ) were stimulated with 10 nM ligands for 20 min. Phosphorylation of Akt (A to D) and Erk1/2 (E to H) was assessed by Western blot. Data were normalized to GAPDH loading control and are shown relative to IGF-1–induced signal. Identical GAPDH loading controls are shown for the figure pairs (A) and (E), (B) and (F), and (D) and (H) because the samples in each pair were run on the same gel. Representative blots are shown and all blots are shown in the Supplementary Materials. Color coding as in . Asterisks indicate significance versus IGF-1 [(A), (B), and (E)] or versus control [(C), (D), (F), (G), and (H)]. (* P < 0.05; ** P < 0.01; *** P < 0.001; # P < 0.0001; ns, not significant according to ANOVA). X indicates that this sample was not included in the study.

Article Snippet: The U87MG (ATCC, HTB-14) glioblastoma cell line was grown in minimum essential medium supplemented with 10% FBS, 1% streptomycin-penicillin, and 2 mM l -glutamine.

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: International Journal of Nanomedicine

Article Title: Transferrin-Functionalized Conjugated Polymer Nanoparticles for Enhanced Photodynamic Therapy of Glioblastoma

doi: 10.2147/IJN.S592688

Figure Lengend Snippet: Integrated in silico and experimental analysis of transferrin receptor 1 (TfR1 / TFRC) expression in gliomas and representative cell lines. Box-plot summary of TFRC mRNA expression obtained from GEPIA (Gene Expression Profiling Interactive Analysis) based on tumor and normal samples from the TCGA and the GTEx databases (accessed March 2025). ( A ) Comparison of TFRC expression in high-grade gliomas (GBM) vs. low-grade glioma (LGG) (n indicated on each plot). ( B ) TFRC expression stratified by canonical GBM molecular subtype (classical, mesenchymal, neural, proneural). Boxes represent the interquartile range, horizontal lines the median, whiskers extend to 1.5×IQR, and individual data points are overlaid. Brackets with asterisks denote statistically significant pairwise differences (see Methods for statistical test). ( C ) TfR1 expression levels in GBM cell lines and HEK293 (non-tumor control). Data taken and adapted from the Human Protein Atlas. ( D ) Representative flow-cytometry histograms of surface TfR1 staining in U87MG, T98G, MO59K and HEK293 cells. Traces correspond to unstained control (red), secondary-only control (anti-mouse IgG–AF647; Orange) and specific anti-CD71 primary staining followed by anti-mouse-AF647 secondary (blue). The horizontal bracket on each histogram indicates the gate used to define TfR1-positive events. ( E ) gMFI of the CD71 signal (mean ± SD; n = biological replicates indicated in Methods), and ( F ) percentage of TfR1-positive cells. Data were normalized to appropriate controls. Statistical comparisons were performed as described in Methods (* p < 0.05 ).

Article Snippet: Moreover, analysis of mRNA expression data from the Human Protein Atlas indicates that the U87MG cell line (derived from GBM) exhibits elevated TFRC/TfR1 expression, whereas HEK293 cells (of human embryonic kidney origin) display minimal receptor expression ( ).

Techniques: In Silico, Expressing, Gene Expression, Comparison, Control, Flow Cytometry, Staining

Journal: International Journal of Nanomedicine

Article Title: Transferrin-Functionalized Conjugated Polymer Nanoparticles for Enhanced Photodynamic Therapy of Glioblastoma

doi: 10.2147/IJN.S592688

Figure Lengend Snippet: Time-course and competition analysis of cellular uptake of F8BT CPNs with and without holo-Tf. ( A and B ) Representative flow-cytometry histograms showing nanoparticle-associated fluorescence in U87MG and T98G cells at the indicated incubation times (0.5, 1, 2 and 4 h). Unexposed control cells (Ctrl) are shown in red; histograms corresponding to successive time points are overlaid to illustrate the progressive accumulation of nanoparticle signal. ( C and D ) Quantification of uptake kinetics derived from flow-cytometry data, expressed as geometric mean fluorescence intensity (gMFI) as a function of incubation time for non-functionalized F8BT CPNs and holo-Tf-functionalized F8BT-Tf CPNs. Error bars represent SD. ( E ) Competition assay performed 2 h post-incubation showing that the presence of excess free holo-Tf significantly reduces the uptake of F8BT-Tf CPNs relative to non-competed F8BT-Tf CPNs, whereas uptake of non-functionalized F8BT CPNs is minimally affected. ( F ) Representative confocal micrographs showing intracellular localization of CPNs (green), F-actin (rhodamine-phalloidin, red), and nuclei (Hoechst, blue) after 30 min exposure. Orthogonal (xz) projections confirm intracellular, sub-membrane localization of the punctate nanoparticle signal; dashed lines indicate the focal plane used for orthogonal projections. Scale bars = 20 µm. Statistical significance: ns, not significant; * p < 0.05 ; ** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001 .

Article Snippet: Moreover, analysis of mRNA expression data from the Human Protein Atlas indicates that the U87MG cell line (derived from GBM) exhibits elevated TFRC/TfR1 expression, whereas HEK293 cells (of human embryonic kidney origin) display minimal receptor expression ( ).

Techniques: Flow Cytometry, Fluorescence, Incubation, Control, Derivative Assay, Competitive Binding Assay, Membrane

Journal: International Journal of Nanomedicine

Article Title: Transferrin-Functionalized Conjugated Polymer Nanoparticles for Enhanced Photodynamic Therapy of Glioblastoma

doi: 10.2147/IJN.S592688

Figure Lengend Snippet: Photodynamic cytotoxicity of F8BT-PtOEP CPNs with and without holo-Tf in GBM cell lines. ( A ) Cell viability of U87MG and ( B ) T98G cells after 24 h incubation with increasing concentrations (1–10 mg·L −1 ) of F8BT-PtOEP CPNs or F8BT-PtOEP-Tf CPNs, followed by blue light irradiation (40 J·cm −2 ). Cell viability was determined 24 h post-irradiation using the MTT assay. Data are expressed as mean ± SD (n = 6). Curves represent independent nonlinear regression fits generated in GraphPad Prism for each treatment group. Asterisks indicate statistically significant differences between treatments (two-way ANOVA, * ***p < 0.0001 ). ( C ) Representative phase-contrast and fluorescence microscopy images (20×) showing intracellular ROS production immediately after PDT, as measured by DCFDA staining (green fluorescence). In both U87MG and T98G cells, increased green fluorescence intensity correlates with holo-Tf functionalization and increased CPNs internalization, indicating enhanced ROS generation. Control (light only) samples show negligible fluorescence, confirming CPNs and light-dependent activation. Scale bar = 100 µm.

Article Snippet: Moreover, analysis of mRNA expression data from the Human Protein Atlas indicates that the U87MG cell line (derived from GBM) exhibits elevated TFRC/TfR1 expression, whereas HEK293 cells (of human embryonic kidney origin) display minimal receptor expression ( ).

Techniques: Incubation, Irradiation, MTT Assay, Generated, Fluorescence, Microscopy, Staining, Control, Activation Assay

Journal: International Journal of Nanomedicine

Article Title: Transferrin-Functionalized Conjugated Polymer Nanoparticles for Enhanced Photodynamic Therapy of Glioblastoma

doi: 10.2147/IJN.S592688

Figure Lengend Snippet: In vivo therapeutic evaluation, biodistribution, and experimental workflow of F8BT-PtOEP CPNs with and without holo-Tf in an orthotopic U87MG-tdiRFP GBM model. ( A ) Schematic overview of the experimental design. U87MG-tdiRFP cells were stereotactically implanted on day 0. Nanoparticle formulations (1 mg·kg −1 , i.v.) were administered on day 10, and PDT was applied 24 h later (day 11). Illumination was delivered through a minimal cranial trepanation using a fiber-optic LED source (irradiance 20 mW·cm −2 at the fiber tip, 10 min; total fluence 12 J·cm −2 ) and was delivered through a minimal cranial trepanation created at the tumor implantation site to allow direct transmission of light into the brain parenchyma while minimizing attenuation by the skull. Animals were subsequently monitored for survival. ( B ) Biodistribution of Pt-containing CPNs 24 h after systemic administration in tumor-bearing mice (n = 4 per group). Platinum levels were quantified by ICP-MS. Both formulations showed predominant hepatic accumulation, consistent with reticuloendothelial clearance of polymeric nanoparticles. Detectable Pt levels in brain tissue were observed only in the F8BT-PtOEP-Tf CPNs group, whereas Pt concentrations in mice receiving F8BT-PtOEP CPNs remained below the analytical limit of detection (LOD = 1 ppb). ( C ) Representative ex vivo NIR fluorescence images of excised brains collected at humane endpoint. Tumor burden derived from the tdiRFP reporter is shown in pseudocolor (red) over a grayscale anatomical background. Images were acquired under identical imaging conditions and correspond to the following experimental groups (displayed uniformly across panels): Control, Light only, F8BT-PtOEP CPNs, F8BT-PtOEP CPNs + Light, F8BT-PtOEP-Tf CPNs, and F8BT-PtOEP-Tf CPNs + Light. Fluorescence intensity scale is shown below. Scale bar = 5 mm. ( D ) Quantification of ex vivo tumor fluorescence area (mean ± SD, n = 4 per group), demonstrating the greatest reduction in tumor signal in animals treated with F8BT-PtOEP-Tf CPNs followed by light irradiation. ( E ) Kaplan–Meier survival analysis for all treatment groups (n = 6 per group). The F8BT-PtOEP-Tf CPNs + Light group showed the most pronounced survival benefit, followed by the F8BT-PtOEP CPNs + Light group, whereas nanoparticle-only and light-only controls produced no significant improvement. Statistical significance was evaluated using the log-rank (Mantel–Cox) test. ** p < 0.01 , **** p < 0.0001 . Illustrations created with BioRender.

Article Snippet: Moreover, analysis of mRNA expression data from the Human Protein Atlas indicates that the U87MG cell line (derived from GBM) exhibits elevated TFRC/TfR1 expression, whereas HEK293 cells (of human embryonic kidney origin) display minimal receptor expression ( ).

Techniques: In Vivo, Tumor Implantation, Transmission Assay, Ex Vivo, Fluorescence, Derivative Assay, Imaging, Control, Irradiation, Produced

Journal: International Journal of Nanomedicine

Article Title: Transferrin-Functionalized Conjugated Polymer Nanoparticles for Enhanced Photodynamic Therapy of Glioblastoma

doi: 10.2147/IJN.S592688

Figure Lengend Snippet: Histopathological evaluation of orthotopic U87MG-tdiRFP GBM following CPNs treatment and PDT. Representative hematoxylin–eosin–stained brain sections from each experimental group (n = 4) are shown, highlighting key morphological features associated with treatment response. Control tumors exhibit compact, highly cellular GBM architecture with moderate–marked pleomorphism and abundant blood vessels (black arrowheads: mitotic figures). Light-only controls display diffuse infiltration into adjacent parenchyma and neoformed aberrant vasculature (black arrow: perivascular neoplastic cells infiltrating into the brain parenchyma. The absence of a boundary with normal tissue is noted). In the non-irradiated F8BT-PtOEP-Tf CPNs -treated group, regions of partially preserved parenchyma are interspersed with infiltrating tumor cells (white arrows). GBM treated with non-functionalized F8BT-PtOEP CPNs + PDT shows extensive coagulative necrosis and hemorrhage, irregular hyperemic vessels, and palisading tumor-cell arrangements. The F8BT-PtOEP-Tf CPNs + PDT group demonstrates the most severe tissue disruption, with widespread necrosis, hemorrhage, and marked vascular damage, together with reduced mitotic activity and abundant cell death (yellow arrowhead: pyknosis). Scale bars = 100 (10x magnification) and 50 μm (40x magnification). The non-irradiated F8BT-PtOEP CPNs group is not shown for clarity, as its histological features were comparable to those observed in the other nanoparticle-treated group under dark conditions.

Article Snippet: Moreover, analysis of mRNA expression data from the Human Protein Atlas indicates that the U87MG cell line (derived from GBM) exhibits elevated TFRC/TfR1 expression, whereas HEK293 cells (of human embryonic kidney origin) display minimal receptor expression ( ).

Techniques: Staining, Control, Irradiation, Disruption, Activity Assay