Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF inhibits NO, IL-6, and TNF-α production in RAW264.7 and MH-S cells. The cells were treated with JGF (50, 100, 150, 300, 600 μg/mL), 2-E (0.1 μM), DXT (10 μM), or LPS (0.1 μg/mL) for 24 h. ( A ) Cell viability was evaluated using crystal violet. ( B ) NO production was measured using the Griess assay. ( C-D ) IL-6 ( C ) and TNF-α ( D ) levels were determined by ELISA. EC 50 was calculated by CompuSyn software. Data was presented as mean ± standard deviation (SD) for groups (n = 3). Significant differences are denoted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Griess Assay, Enzyme-linked Immunosorbent Assay, Software, Standard Deviation

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: Components of JGF inhibit 2-E-induced inflammation. The RAW264.7 and MH-S cells were co-treated with JGF compounds and 2-E for 24 h. ( A ) The 3D-HPLC fingerprint of JGF. Compound structures were sourced from the PubChem database. The detection wavelength ranged from 200 to 400 nm, and the injection volume was 20 μL. ( B ) Cell viability was evaluated using crystal violet. ( C ) NO production was measured using the Griess assay. ( D-E ) IL-6 ( D ) and TNF-α ( E ) levels were determined by ELISA. Data are presented as mean ± SD (n = 3). Statistical significance was determined relative to the 2-E group. Significant differences are denoted as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Injection, Griess Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF downregulates 2-E-induced iNOS and COX-2 in RAW264.7 and MH-S cells. Cells were treated with JGF (0, 50, 200 μg/mL) or 2-E (0.1 μM) for 24 h. ( A ) Protein levels of iNOS and COX-2 in macrophages were measured by Western blot. ( B-C ) Quantification of iNOS and COX-2 in cells without ( B ) and with ( C ) 2-E stimulation, calculated using ImageJ. Actin was used as the internal control. The non-detected data showed as – or ND. Data are presented as mean ± SD (n = 3). Significant differences are denoted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF inhibits 2-E-induced phosphorylation of STAT3 in RAW264.7 and MH-S cells. Cells were treated with JGF (0, 50, 200 μg/mL) or 2-E (0.1 μM) for 3 h. ( A ) Protein levels of phosphorylated JAK2 and STAT3 were measured by Western blot. ( B-C ) Quantification of phosphorylated JAK2 and STAT3 in cells without ( B ) and with ( C ) 2-E stimulation, calculated using ImageJ. Actin was used as the internal control. Data are presented as mean ± SD (n = 3). Significant differences are denoted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF inhibits 2-E-induced phosphorylation of ERK1/2 in RAW264.7 and MH-S cells. Cells were treated with JGF (0, 50, 200 μg/mL) or 2-E (0.1 μM) for 3 h. ( A ) Protein levels of phosphorylated JNK1/2, ERK1/2, p38, and p65 were measured by Western blot. ( B-C ) Quantification of phosphorylated JNK1/2, ERK1/2, p38, and p65 in cells without ( B ) and with ( C ) 2-E stimulation, calculated using ImageJ. Actin was used as the internal control. Data are presented as mean ± SD (n = 3). Significant differences are denoted as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: JGF reduces the 2-E-induced proinflammatory cytokines in vivo . ( A ) The experimental scheme for mouse exposure. ( B-F ) Levels of IL-6 ( B ), TNF-α ( C ), IFN-γ ( D ), IL-1β ( E ), and IL-12 ( F ) in lung tissue and serum were measured by ELISA. Data are presented as mean ± SD (n = 9 for serum, except DXT group n = 6; n = 6 for lung tissue, except DXT group n = 3) ( G ) Representative histological images of lung tissue stained with H&E and IHC images for IL-6, TNF-α, and IL-1β expression. ( H-J ) Quantification of IL-6 ( H ), TNF-α ( I ), and IL-1β ( J ) positive areas using ImageJ (n = 3). Significant differences between the control (CTL) group and other groups are denoted by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Significant differences between the 2-E group and 2-E + JGF group are indicated by #p < 0.05, ##p < 0.01, ###p < 0.001.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Control

Journal: Journal of Traditional and Complementary Medicine

Article Title: Chemical characterization of Jing Guan Fang and its application in alleviating coronavirus envelope protein-induced proinflammatory responses in vitro and in vivo

doi: 10.1016/j.jtcme.2025.12.003

Figure Lengend Snippet: Schematics showing the anti-inflammatory mechanism of JGF in 2-E-induced mice macrophages.

Article Snippet: SARS-CoV-2-envelope (2-E) protein (Cat# NBP2–90986) was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques:

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Generation and characterization of IL-7Rα-targeting antibodies. (a) Schematic workflow of IL-7Rα-specific monoclonal antibodies (mAbs) generation: immunization of IL-7Rα-knockout mice with recombinant human IL-7Rα extracellular domain, hybridoma fusion, and screening/expansion, followed by conversion to human–mouse chimeric IgG. (b) Flow cytometry histograms showing the binding capabilities of in-house clones 577, 2D5, 165, and 24 to IL-7Rα-positive cells compared with a commercial anti-IL-7Rα mAb; secondary-only and unstained controls are included. (c) Competitive binding (epitope binning) assessment between different in-house antibody pairs using flow cytometry. Cells were pre-blocked with an unlabeled antibody and stained with a fluorophore-labelled competitor. The binding ratios were normalized to the non-pre-blocked condition. (d) Surface plasmon resonance analysis of antibody binding to recombinant IL-7Rα.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Bioprocessing, Knock-Out, Recombinant, Flow Cytometry, Binding Assay, Clone Assay, Staining, SPR Assay

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Generation and cytotoxicity assessment of IL-7Rα-targeting ADCs. (a) Internalization kinetics of four IL-7Rα-targeting monoclonal antibodies in IL-7Rα-positive REH cells. Surface-bound antibody levels of the four antibodies at 0, 15, 60, and 240 minutes were determined by flow cytometry and normalized to the signal at minute 0. (b) Schematic representation of the conjugation process for generating IL-7Rα-targeting ADCs. Antibodies were partially reduced with 20 mM 2-mercaptoethylamine (2-MEA) for 0.5 hours at 37°C, followed by conjugation with 10 mM mc–vc–PAB–MMAE for 16 hours at 4°C, yielding an average drug-to-antibody ratio of 3–4. (c) Quantification of IL-7Rα expression (molecules per cell) in three different leukemia cell lines, CCRF-CEM (low), NALM6 (medium), and REH (high), using flow cytometry. (d) Cytotoxicity of free MMAE, isotype control IgG–MMAE, and four IL-7Rα-targeting ADCs in CCRF-CEM, NALM6, and REH cells. Cell viability was assessed using the WST-8 assay 72 hours after each treatment. Data are presented as mean ± SEM; n = 6 technical replicates from a single experiment.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Bioprocessing, Flow Cytometry, Conjugation Assay, Expressing, Control

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: In vivo efficacy and biodistribution of IL-7Rα-targeting agents. (a) Schematic representation of the subcutaneous tumor model and treatment schedule with four IL-7Rα-targeting ADCs. (b) Tumor volumes over time for each treatment group. (c) Relative body weight changes during treatment. PBS, phosphate-buffered saline. Lines show mean ± SEM, n = 6–9 per group. (d) Serial in vivo fluorescence imaging of fluorophore-labelled parent anti-IL-7Rα mAbs and an isotype antibody control in a separate tracer-dose cohort (representative animals). (e) Quantification of tumor region-of-interest (ROI) fluorescence; each animal was normalized to its own 5-min post-injection value. NC, negative control. Data are presented as mean ± SEM; n = 3–5 per group. (f) Relative performance of the four anti-IL-7Rα mAbs (577, 2D5, 165, and 24) was compared across five parameters. Binding activity, SPR-derived apparent binding affinity, internalization, and pIC 50 (-log10 IC 50 [M]) and in vivo efficacy were evaluated using the respective ADCs. Ratings were assigned based on the experimental data shown in , using a semi-quantitative scale from “+” (lowest) to “++++” (highest). The scale reflects the relative ranking within each parameter and does not represent absolute quantitative values.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: In Vivo, Saline, Fluorescence, Imaging, Control, Injection, Negative Control, Binding Assay, Activity Assay, Derivative Assay

Journal: mAbs

Article Title: Targeting IL-7Rα with PNU-159682 antibody–drug conjugates in acute lymphoblastic leukemia: translational implications

doi: 10.1080/19420862.2026.2663639

Figure Lengend Snippet: Enhanced anti-tumor activity of IL-7Rα-targeting ADCs with novel payload PNU-159682. (a) Schematic representation of the conjugation process for generating PNU-159682-linked ADCs. Antibodies were partially reduced with 20 mM 2-mercaptoethylamine (2-MEA) for 0.5 hours at 37 °C, followed by conjugation with 10 mM Mal–PEG4–VC–PAB–DMEA–PNU-159682 for 16 hours at 4 °C, yielding a drug-to-antibody ratio of 3–4. (b) In vitro cytotoxicity of 577-PNU, 577-MMAE, isotype control IgG–PNU, and free PNU-159682 in NALM6 cells. Cell viability was measured using the WST-8 assay 72 hours after treatment. Data are shown as mean ± SEM. (c) Comparison of IC 50 values between 577-MMAE and 577-PNU in NALM6 cells, calculated from nine independent experiments performed on separate days; IC 50 values analyzed after log10 transformation; paired t-test (two-tailed), p < 0.0001; geometric mean ratio (MMAE/PNU) = 85.3 (95% CI 57.7–126.0). (d) In vivo anti-tumor efficacy of each treatment in NALM6 xenografts (subcutaneous model). Mice were treated with a single dose of 577-MMAE (10 mg/kg; n = 4), 577-PNU (0.5 mg/kg; n = 5), isotype control IgG–PNU (0.5 mg/kg; n = 4), free PNU-159682 (17 µg/kg, the dose of PNU equal to 0.5 mg/kg 577-PNU; n = 3), or phosphate-buffered saline (PBS) vehicle ( n = 5). Tumor volumes were measured twice weekly. (e) Complete response (CR) rate on day 28 following treatment with 577-MMAE (10 mg/kg; n = 4) or 577-PNU (0.5 mg/kg; n = 5). Two-sided Fisher’s exact test comparing groups: p = 0.0476. (f) Relative body weight change (%) during treatment. Data are presented as mean ± SEM; n = 3–5 per group. * p < 0.05; **** p < 0.0001.

Article Snippet: His-tagged recombinant human IL-7Rα ectodomain (R&D Systems, Minneapolis, MN, USA, Cat. No. 10758-IR) was captured on the active flow cell to approximately 50 response units (RU).

Techniques: Activity Assay, Conjugation Assay, In Vitro, Control, Comparison, Transformation Assay, Two Tailed Test, In Vivo, Saline