Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: Ectopic GPC3 expression was successfully established and validated in isogenic SNU449 cells. ( A ) SNU449 cells were transduced with hGPC3-mGFP and clonally isolated, and GPC3 mRNA expression was quantified by qPCR. A431/GPC3 and HepG2 cells served as positive controls; A431 and GPC3⁻ HepG2 cells served as negative controls. ( B ) GPC3 protein expression was assessed by Western blotting (GAPDH loading control). ( C ) Cell-surface GPC3 was detected in A431/GPC3, SNU449/GPC3, and HepG2 cells, but not in GPC3⁻ SNU449, SNU449/vector, A431, or HepG2 cells. ( D ) Immunofluorescence staining confirmed GPC3 expression in SNU449/GPC3 and A431/GPC3 cells, but not in parental controls. Scale bars: 50 μ m.

Article Snippet: Human tissue specimens were incubated with a mouse monoclonal anti-human GPC3 antibody (clone 1G12; Cell Marque) under the same conditions.

Techniques: Expressing, Transduction, Isolation, Western Blot, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 expression regulates tumor cell proliferation and radiosensitivity. Proliferation of paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A ), Hep3B ( B ), SNU449 and SNU449/V (empty vector control) ( C ), and A431 ( D ) cells was monitored using the IncuCyte® live-cell analysis system under non-irradiated (Non-IR) conditions and following 6 Gy irradiation (IR). Phase-area confluence was normalized to 0 h, and differences in proliferation kinetics were analyzed by two-way ANOVA, with significance assessed at the final time point (****, P <0.0001). Radiosensitivity was evaluated by clonogenic survival assays in the corresponding GPC3⁺ and GPC3⁻ HepG2 ( E ), Hep3B ( F ), SNU449 ( G ), and A431 ( H ) cells exposed to graded doses of γ-irradiation. Colonies were quantified 10–14 days later to generate survival curves. GPC3 loss increased radiosensitivity, yielding dose-modifying factors (DMFs) of 1.25 (HepG2), 1.36 (Hep3B), 1.20 (SNU449), and 1.75 (A431). Data represent mean surviving fraction ± SD from more than three independent experiments.

Article Snippet: Human tissue specimens were incubated with a mouse monoclonal anti-human GPC3 antibody (clone 1G12; Cell Marque) under the same conditions.

Techniques: Expressing, Plasmid Preparation, Control, Cell Analysis, Irradiation

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency increases IR-induced DNA double-strand breaks in tumor cells. DNA damage responses were assessed in paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A , E ), Hep3B ( B , F ), SNU449 ( C , G ), and A431 ( D , I ) cells following 6 Gy irradiation (IR). γ-H2AX immunocytochemistry ( A – D ) was performed at the indicated time points to quantify DNA double-strand break formation and resolution. Representative images at control, 1 h, and 48 h post-IR are shown. γ-H2AX foci (magenta) were quantified from > 50 cells per condition; nuclei were counterstained with DAPI (blue). Scale bars: 25 μ m. Statistical comparisons of foci numbers were performed using a t -test (* p <0.05; *** p <0.001). Comet assays ( E – I ) were performed 24 h after IR. Representative comet images (left) and quantified tail moments (right) are shown. Data are presented as mean ± SD, and significance was determined using a t -test (** p <0.01; *** p <0.001; **** p <0.0001). Scale bars: 50 μ m.

Article Snippet: Human tissue specimens were incubated with a mouse monoclonal anti-human GPC3 antibody (clone 1G12; Cell Marque) under the same conditions.

Techniques: Irradiation, Immunocytochemistry, Control

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency enhances radiation response in xenograft models and correlates with clinical outcomes. ( A – D ) HepG2/Luc xenografts derived from GPC3⁺ or GPC3⁻ cells with or without 10 Gy irradiation were monitored by bioluminescence imaging (BLI) and tumor volume measurements. GPC3⁻ tumors showed enhanced growth delay following irradiation. Representative GPC3 and Ki-67 staining is shown (scale bar: 100 μ m). ( E – H ) Similar analyses in A431/Luc xenografts demonstrated greater radiation sensitivity in GPC3⁻ tumors. ( I ) In a clinical cohort, high tumor GPC3 expression was associated with poorer overall survival ( p = 0.008). Statistical comparisons were performed by two-way ANOVA; significance was assessed at the final time point (* p <0.05; *** p <0.001; **** p <0.0001).

Article Snippet: Human tissue specimens were incubated with a mouse monoclonal anti-human GPC3 antibody (clone 1G12; Cell Marque) under the same conditions.

Techniques: Derivative Assay, Irradiation, Imaging, Staining, Expressing

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: Ectopic GPC3 expression was successfully established and validated in isogenic SNU449 cells. ( A ) SNU449 cells were transduced with hGPC3-mGFP and clonally isolated, and GPC3 mRNA expression was quantified by qPCR. A431/GPC3 and HepG2 cells served as positive controls; A431 and GPC3⁻ HepG2 cells served as negative controls. ( B ) GPC3 protein expression was assessed by Western blotting (GAPDH loading control). ( C ) Cell-surface GPC3 was detected in A431/GPC3, SNU449/GPC3, and HepG2 cells, but not in GPC3⁻ SNU449, SNU449/vector, A431, or HepG2 cells. ( D ) Immunofluorescence staining confirmed GPC3 expression in SNU449/GPC3 and A431/GPC3 cells, but not in parental controls. Scale bars: 50 μ m.

Article Snippet: The transferred proteins on the membrane were incubated with specific primary antibodies, including GPC3 (1:1000, 261M-96, Cell Marque, Rocklin, CA, USA), mGFP (1:1000, TA150122, OriGene), luciferase (1:1000, sc-74548, Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #CB1001, Sigma-Aldrich).

Techniques: Expressing, Transduction, Isolation, Western Blot, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 expression regulates tumor cell proliferation and radiosensitivity. Proliferation of paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A ), Hep3B ( B ), SNU449 and SNU449/V (empty vector control) ( C ), and A431 ( D ) cells was monitored using the IncuCyte® live-cell analysis system under non-irradiated (Non-IR) conditions and following 6 Gy irradiation (IR). Phase-area confluence was normalized to 0 h, and differences in proliferation kinetics were analyzed by two-way ANOVA, with significance assessed at the final time point (****, P <0.0001). Radiosensitivity was evaluated by clonogenic survival assays in the corresponding GPC3⁺ and GPC3⁻ HepG2 ( E ), Hep3B ( F ), SNU449 ( G ), and A431 ( H ) cells exposed to graded doses of γ-irradiation. Colonies were quantified 10–14 days later to generate survival curves. GPC3 loss increased radiosensitivity, yielding dose-modifying factors (DMFs) of 1.25 (HepG2), 1.36 (Hep3B), 1.20 (SNU449), and 1.75 (A431). Data represent mean surviving fraction ± SD from more than three independent experiments.

Article Snippet: The transferred proteins on the membrane were incubated with specific primary antibodies, including GPC3 (1:1000, 261M-96, Cell Marque, Rocklin, CA, USA), mGFP (1:1000, TA150122, OriGene), luciferase (1:1000, sc-74548, Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #CB1001, Sigma-Aldrich).

Techniques: Expressing, Plasmid Preparation, Control, Cell Analysis, Irradiation

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency increases IR-induced DNA double-strand breaks in tumor cells. DNA damage responses were assessed in paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A , E ), Hep3B ( B , F ), SNU449 ( C , G ), and A431 ( D , I ) cells following 6 Gy irradiation (IR). γ-H2AX immunocytochemistry ( A – D ) was performed at the indicated time points to quantify DNA double-strand break formation and resolution. Representative images at control, 1 h, and 48 h post-IR are shown. γ-H2AX foci (magenta) were quantified from > 50 cells per condition; nuclei were counterstained with DAPI (blue). Scale bars: 25 μ m. Statistical comparisons of foci numbers were performed using a t -test (* p <0.05; *** p <0.001). Comet assays ( E – I ) were performed 24 h after IR. Representative comet images (left) and quantified tail moments (right) are shown. Data are presented as mean ± SD, and significance was determined using a t -test (** p <0.01; *** p <0.001; **** p <0.0001). Scale bars: 50 μ m.

Article Snippet: The transferred proteins on the membrane were incubated with specific primary antibodies, including GPC3 (1:1000, 261M-96, Cell Marque, Rocklin, CA, USA), mGFP (1:1000, TA150122, OriGene), luciferase (1:1000, sc-74548, Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #CB1001, Sigma-Aldrich).

Techniques: Irradiation, Immunocytochemistry, Control

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency enhances radiation response in xenograft models and correlates with clinical outcomes. ( A – D ) HepG2/Luc xenografts derived from GPC3⁺ or GPC3⁻ cells with or without 10 Gy irradiation were monitored by bioluminescence imaging (BLI) and tumor volume measurements. GPC3⁻ tumors showed enhanced growth delay following irradiation. Representative GPC3 and Ki-67 staining is shown (scale bar: 100 μ m). ( E – H ) Similar analyses in A431/Luc xenografts demonstrated greater radiation sensitivity in GPC3⁻ tumors. ( I ) In a clinical cohort, high tumor GPC3 expression was associated with poorer overall survival ( p = 0.008). Statistical comparisons were performed by two-way ANOVA; significance was assessed at the final time point (* p <0.05; *** p <0.001; **** p <0.0001).

Article Snippet: The transferred proteins on the membrane were incubated with specific primary antibodies, including GPC3 (1:1000, 261M-96, Cell Marque, Rocklin, CA, USA), mGFP (1:1000, TA150122, OriGene), luciferase (1:1000, sc-74548, Santa Cruz Biotechnology), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, #CB1001, Sigma-Aldrich).

Techniques: Derivative Assay, Irradiation, Imaging, Staining, Expressing

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: Ectopic GPC3 expression was successfully established and validated in isogenic SNU449 cells. ( A ) SNU449 cells were transduced with hGPC3-mGFP and clonally isolated, and GPC3 mRNA expression was quantified by qPCR. A431/GPC3 and HepG2 cells served as positive controls; A431 and GPC3⁻ HepG2 cells served as negative controls. ( B ) GPC3 protein expression was assessed by Western blotting (GAPDH loading control). ( C ) Cell-surface GPC3 was detected in A431/GPC3, SNU449/GPC3, and HepG2 cells, but not in GPC3⁻ SNU449, SNU449/vector, A431, or HepG2 cells. ( D ) Immunofluorescence staining confirmed GPC3 expression in SNU449/GPC3 and A431/GPC3 cells, but not in parental controls. Scale bars: 50 μ m.

Article Snippet: To confirm membranous GPC3 expression, cells were labeled with a mouse anti-GPC3 antibody (clone 1G12; Cell Marque, 1:200 dilution in 2% BSA in PBS) at 4°C for overnight.

Techniques: Expressing, Transduction, Isolation, Western Blot, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 expression regulates tumor cell proliferation and radiosensitivity. Proliferation of paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A ), Hep3B ( B ), SNU449 and SNU449/V (empty vector control) ( C ), and A431 ( D ) cells was monitored using the IncuCyte® live-cell analysis system under non-irradiated (Non-IR) conditions and following 6 Gy irradiation (IR). Phase-area confluence was normalized to 0 h, and differences in proliferation kinetics were analyzed by two-way ANOVA, with significance assessed at the final time point (****, P <0.0001). Radiosensitivity was evaluated by clonogenic survival assays in the corresponding GPC3⁺ and GPC3⁻ HepG2 ( E ), Hep3B ( F ), SNU449 ( G ), and A431 ( H ) cells exposed to graded doses of γ-irradiation. Colonies were quantified 10–14 days later to generate survival curves. GPC3 loss increased radiosensitivity, yielding dose-modifying factors (DMFs) of 1.25 (HepG2), 1.36 (Hep3B), 1.20 (SNU449), and 1.75 (A431). Data represent mean surviving fraction ± SD from more than three independent experiments.

Article Snippet: To confirm membranous GPC3 expression, cells were labeled with a mouse anti-GPC3 antibody (clone 1G12; Cell Marque, 1:200 dilution in 2% BSA in PBS) at 4°C for overnight.

Techniques: Expressing, Plasmid Preparation, Control, Cell Analysis, Irradiation

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency increases IR-induced DNA double-strand breaks in tumor cells. DNA damage responses were assessed in paired GPC3-positive (GPC3⁺) and GPC3-deficient (GPC3⁻) HepG2 ( A , E ), Hep3B ( B , F ), SNU449 ( C , G ), and A431 ( D , I ) cells following 6 Gy irradiation (IR). γ-H2AX immunocytochemistry ( A – D ) was performed at the indicated time points to quantify DNA double-strand break formation and resolution. Representative images at control, 1 h, and 48 h post-IR are shown. γ-H2AX foci (magenta) were quantified from > 50 cells per condition; nuclei were counterstained with DAPI (blue). Scale bars: 25 μ m. Statistical comparisons of foci numbers were performed using a t -test (* p <0.05; *** p <0.001). Comet assays ( E – I ) were performed 24 h after IR. Representative comet images (left) and quantified tail moments (right) are shown. Data are presented as mean ± SD, and significance was determined using a t -test (** p <0.01; *** p <0.001; **** p <0.0001). Scale bars: 50 μ m.

Article Snippet: To confirm membranous GPC3 expression, cells were labeled with a mouse anti-GPC3 antibody (clone 1G12; Cell Marque, 1:200 dilution in 2% BSA in PBS) at 4°C for overnight.

Techniques: Irradiation, Immunocytochemistry, Control

Journal: bioRxiv

Article Title: Ablation of glypican-3 enhances radiosensitivity in liver cancer by prolonging G2/M arrest and activating the ATM/CHK2 pathway

doi: 10.64898/2026.05.11.724294

Figure Lengend Snippet: GPC3 deficiency enhances radiation response in xenograft models and correlates with clinical outcomes. ( A – D ) HepG2/Luc xenografts derived from GPC3⁺ or GPC3⁻ cells with or without 10 Gy irradiation were monitored by bioluminescence imaging (BLI) and tumor volume measurements. GPC3⁻ tumors showed enhanced growth delay following irradiation. Representative GPC3 and Ki-67 staining is shown (scale bar: 100 μ m). ( E – H ) Similar analyses in A431/Luc xenografts demonstrated greater radiation sensitivity in GPC3⁻ tumors. ( I ) In a clinical cohort, high tumor GPC3 expression was associated with poorer overall survival ( p = 0.008). Statistical comparisons were performed by two-way ANOVA; significance was assessed at the final time point (* p <0.05; *** p <0.001; **** p <0.0001).

Article Snippet: To confirm membranous GPC3 expression, cells were labeled with a mouse anti-GPC3 antibody (clone 1G12; Cell Marque, 1:200 dilution in 2% BSA in PBS) at 4°C for overnight.

Techniques: Derivative Assay, Irradiation, Imaging, Staining, Expressing

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: SPD1 comprises two peptides [SPDa, Ac-K(PpIX)-FF-DHLASLWWGTEL; SPDb, Ac-K(PpIX)-FF-AEEA-C(MAL-PEG 4 -DBCO)], which are composed of four discrete functional domains: (i) DHLASLWWGTEL, a GPC3-targeted motif; (ii) FF, a β sheet motif; (iii) PpIX, a fluorescent dye; (iv) DBCO, a click chemistry group. SPD1 self-assembles into nanoparticles, and SPD1 and Gd-DOTA-N 3 are administered via two sequential injections. SPD1 nanoparticles (first intravenous administration) first accumulate and transform into fibrillar networks on the cell membrane of high GPC3-expressing orthotopic liver tumor through intermolecular π-π stacking between FF. The fibrillar networks, functionalized with DBCO, trapped Gd-DOTA-N 3 (following a second intravenous administration) in the extracellular space via a bioorthogonal reaction and immobilized it in an ordered arrangement, thereby enhancing the T 1 -weighted signal for the precise detection of micro-HCC. Schematic elements were created by eBioart.

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Functional Assay, Membrane, Expressing

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A and B ) UV-vis absorption spectra (A) and fluorescence (FL) emission spectra (B) of PpIX (excitation: 405 nm) following the gradual addition of H 2 O (from 0 to 99.5%) to a DMSO solution of SPD1 nanoparticles. a.u., arbitrary units. ( C ) CAC of SPD1 nanoparticles was determined using pyrene as a fluorescent probe. ( D ) Representative TEM image of self-assembled SPD1 nanoparticles (50 μM) in aqueous solution. ( E ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein [molecular weight (MW) ≈ 61.6 kDa] at varying molar ratios. h, hours. ( F ) TEM images showing the initial SPD1 nanoparticles and nanofibers transformed from SPD1 nanoparticles (50 μM) after incubation with human GPC3 protein at different time points. The molar ratio of human GPC3 protein/SPD1 was ~1:1000. ( G ) CD spectra of SPD1 nanoparticles (50 μM) before and after incubation with human GPC3 protein (1:1000 molar ratio) for various durations, showing the secondary structure transition. mdeg, millidegrees. ( H ) FTIR spectra of SPD1 nanoparticles (50 μM) before and after 24 hours of incubation with GPC3 (1:1000 molar ratio), highlighting a shift in the amide I band consistent with β sheet formation. ( I ) Molecular simulation of the transformation of SPD1 into complex (i.e., SPD1 nanoparticles) in a water box based on the hydrophobic core of PpIX molecules, along with a hydrophilic corona formed by GPC3-targeted ligands. ( J ) Molecular docking simulation for SPD1 and GPC3. Rectangle: the possible binding sites between SPD1 and GPC3. ( K ) MD simulation of fibrillar transformation of SPD1 generated at t = 250 ns after interaction with GPC3. Rectangle: the interaction forces of the hydrogen bond and π-π stacking. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Fluorescence, Transformation Assay, Incubation, Molecular Weight, Circular Dichroism, Binding Assay, Generated

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) SEM-EDX elemental mapping of the Gd-DOTA-N 3 +SPD1+GPC3 probe. False-color maps show the spatial distribution of carbon (C, red), oxygen (O, cyan), nitrogen (N, green), and Gd (magenta), confirming uniform Gd incorporation and colocalization with organic matrix elements. Scale bars, 25 μm. All experiments were independently repeated three times with consistent and reproducible results. ( B ) Schematic illustration of the covalent conjugation attachment of Gd-DOTA-N 3 to SPD1 nanofibers via SPAAC click chemistry, designed to enhance r 1 relaxivity. ( C ) TEM images showing GPC3-modified gold (Au) nanoparticles alone or after incubation with SPD1 or SPD2 nanoparticles (50 μM, 24 hours). Scale bars, 50 nm. ( D ) T 1 -weighted images and pseudocolor T 1 maps comparing Gd-DOTA, Gd-DOTA-N 3 , Gd-DOTA-N 3 +SPD2-GPC3 (noncovalent incubation, molar ratio of 1000:1, 24 hours) for 6 hours, and Gd-DOTA-N 3 +SPD1-GPC3 (bioorthogonal conjugation, molar ratio of 1000:1, 24 hours) for 6 hours. Experiments were independently repeated three times, with consistent results observed across replicates. ( E ) r 1 relaxivity of Gd-DOTA (purple), Gd-DOTA-N 3 (pink), Gd-DOTA-N 3 +SPD2+GPC3 (blue), and Gd-DOTA-N 3 +SPD1+GPC3 (light green). Data are presented as means ± SD ( n = 3 independent experiments). ( F ) Room-temperature VSM showing the enhanced paramagnetic signal from Gd-DOTA-N 3 +SPD1+GPC3 (light green), compared to Gd-DOTA-N 3 +SPD2+GPC3 (blue), Gd-DOTA-N 3 (pink), and Gd-DOTA (purple).

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Conjugation Assay, Modification, Incubation

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative Western blot analysis of GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells ( n = 3 independent experiments). ( B ) Quantitation of relative GPC3 protein level from (A). Data are presented as means ± SD ( n = 3). Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test. ( C ) Flow cytometry analysis of surface GPC3 expression in HepG2, WRL-68, and Hepa1-6 cells. ( D ) Representative IHC images of GPC3 expression (brown) in tumor tissues from orthotopic Hepa1-6 tumor-bearing mice. Scale bar, 50 μm. ( E ) CLSM images of HepG2 and Hepa1-6 cells treated with SPD1 or SPD2 nanoparticles (50 μM; red fluorescence) for 6 hours. Scale bars, 20 μm. ( F ) Time-dependent CLSM imaging of HepG2 cells treated with SPD1 nanoparticles (50 μM) showing membrane-localized fibrillar transformation. Scale bars, 20 μm. ( G ) CLSM analysis of HepG2 cells sequentially incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours; red) and FITC-labeled anti-GPC3 antibody (green; 1:200; Abcam, #ab207080). Colocalization (yellow) indicates specific binding of SPD1 to membrane-bound GPC3. Fluorescence intensity and colocalization were quantified using MATLAB. Data are presented as means ± SD ( n = 3); n.s., not significant (one-way ANOVA with Tukey’s post hoc test). Scale bars, 20 μm. ( H ) SEM images of untreated HepG2 and WRL-68 cells or incubated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours). Magnified insets highlight membrane-associated fibrillar structures. ( I ) TEM images of untreated HepG2 cells (top) and those treated with SPD1 nanoparticles (50 μM, 24 hours; bottom). Red arrows indicate membrane-associated nanofibers. Scale bars, 500 nm. ( J ) SEM images showing the persistence of SPD1-derived fibrillar networks on HepG2 cells at 6, 24, and 72 hours posttreatment (50 μM). Scale bars, 2 μm. All experiments were independently repeated three times with consistent and reproducible results.

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Western Blot, Expressing, Quantitation Assay, Flow Cytometry, Fluorescence, Imaging, Membrane, Transformation Assay, Incubation, Labeling, Binding Assay, Derivative Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Representative CLSM images of HepG2 cells incubated with SPD1 nanoparticles (red, 50 μM) for 6 hours, followed by treatment with FITC-N 3 (10 to 50 μM, green) for an additional 6 hours. Merged yellow fluorescence indicates successful copper-free click conjugation between DBCO and N 3 on the cell membrane. Cells treated with SPD2+FITC-N 3 (50 μM) served as the nontargeted controls, showing minimal colocalization. Scale bars, 20 μm. ( B ) Time-dependent kinetics of bioorthogonal conjugation quantified by BCA protein assay and ICP-MS. HepG2 cells were pretreated with SPD1 or SPD2 nanoparticles (50 μM, 6 hours), followed by incubation with Gd-DOTA-N 3 (50 μM) for 0.5, 1, 6, or 12 hours. Cells treated with Gd-DOTA-N 3 alone served as baseline controls. ( C ) T 1 -weighted MR images of HepG2 cells treated with Gd-DOTA (50 μM), Gd-DOTA-N 3 (50 μM), or sequentially with SPD1 or SPD2 (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( D ) Quantitative r 1 relaxivity under the corresponding treatment conditions in (C). ( E ) r 1 relaxivity of HepG2 cells preblocked with anti-GPC3 antibody (5 μg/ml, 12 hours; Abcam, #ab207080) before SPD1 treatment (50 μM, 6 hours) followed by Gd-DOTA-N 3 (50 μM, 6 hours). ( F to H ) Cell viability of WRL-68 (F), HepG2 (G), and Hepa1-6 (H) cells after sequential treatment with SPD1 or SPD2 for 6 hours followed by Gd-DOTA-N 3 (50 μM, 6 hours). Cell viability was quantified using the CCK-8 assay. Data are presented as means ± SD { n = 3 for [(A) to (E)]; n = 6 for [(F) to (H)]}. Statistical significance was performed using one-way ANOVA followed by Tukey’s post hoc test. P < 0.05 was considered statistically significant; n.s., not significant. All experiments were independently repeated three times with consistent results.

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Incubation, Fluorescence, Conjugation Assay, Membrane, Bicinchoninic Acid Protein Assay, CCK-8 Assay

Journal: Science Advances

Article Title: In vivo membrane engineering traps Gd-based MRI contrast agents for detecting microhepatocellular carcinoma

doi: 10.1126/sciadv.aec9913

Figure Lengend Snippet: ( A ) Pharmacokinetic profiles of SPD1 [10 mg/kg, intravenously (iv)] and SPD2 (10 mg/kg, iv) in healthy mice. Plasma concentrations were determined via PpIX fluorescence, and pharmacokinetic parameters were calculated using PKSolver 2.0 software. Data are expressed as means ± SD ( n = 3). ( B ) Blood Gd 3+ levels measured by ICP-MS in orthotopic Hepa1-6 tumor-bearing mice after Gd-DOTA-N 3 administration (0.1 mmol/kg, iv), with or without 6 hours pretreatment with SPD1 or SPD2 (10 mg/kg, iv). ( C ) Ex vivo fluorescence (FL) images of the tumors (T), heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (K), and intestine (I), at indicated time points following SPD1 injection (10 mg/kg, iv) in orthotopic Hepa1-6 tumor-bearing mice. Red dashed circles indicate tumors. Representative images from three mice per time point are shown. ( D ) Quantification of the fluorescence intensity in tumor and major organs of (C). ( E ) Representative micrographs demonstrate H&E staining, GPC3 IHC, and TUNEL staining in orthotopic Hepa1-6 tumor specimens from C57BL/6 mice treated with the following regimens: SPD1 pretreatment followed by Gd-DOTA-N 3 6 hours later, SPD2 pretreatment followed by Gd-DOTA-N 3 6 hours later, Gd-DOTA-N 3 alone, and Gd-DOTA alone. Representative images are shown from three biologically independent experiments. Scale bars, 100 μm. ( F ) Schematic illustration of the molecular imaging mechanism of the SPD1+Gd-DOTA-N 3 probe, highlighting self-assembly into nanofibers and signal amplification via r 1 enhancement. d, days. ( G ) In vivo T 1 -weighted images of subcutaneous Hepa1-6 tumor-bearing mice at multiple time points under four different experimental protocols. Red circles mark tumor regions. Representative images from three mice per group are shown. ( H ) In vivo T 1 -weighted images of orthotopic Hepa1-6 tumor-bearing mice under four different treatment protocols. Red circles mark tumor regions. Representative images from three mice per group are shown.

Article Snippet: Cells were then incubated overnight at 4°C with FITC-conjugated anti-GPC3 antibody (Novus, NBP2-47760V; 1:200 in 10% BSA/PBS).

Techniques: Clinical Proteomics, Fluorescence, Software, Ex Vivo, Injection, Staining, TUNEL Assay, Imaging, Amplification, In Vivo