human il 7rα ectodomain  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Structural basis of EGF-repeat O -glucosylation by the protein O -glucosyltransferase POGLUT2

doi: 10.1016/j.jbc.2026.111361

Figure Lengend Snippet: Functional and structural characterization of human POGLUT2 . A , O -glucosylation sites within epidermal growth factor–like (EGF) repeats. POGLUT1 (Rumi in Drosophila ) adds O -Glc to serine within the C 1 –C 2 consensus sequence C 1 -X- S -X-(P/A)-C 2 . POGLUT2/3 adds O -Glc to serine residue within the C 3 –C 4 consensus sequence C 3 -X-N-T-X-G- S -F-X-C 4 . EOGT (EGF domain–specific O -GlcNAc transferase) catalyzes O -GlcNAc addition to serine or threonine within the C 5 –C 6 consensus sequence C 5 -X-X-G-X-( S/T )-G-X-X-C 6 . Six conserved cysteine residues (C1–C6) form three disulfide bonds, indicated by solid lines . B , sequence alignment of representative EGF-repeat substrates of POGLUT2/3 (human Notch3 EGF10, hN3EGF10; human Notch1 EGF11, hN1EGF11; human Notch4 EGF11, hN4EGF11), POGLUT1 (human Notch1 EGF12 and 13, hN1EGF12 and hN1EGF13; human factor IX EGF, hFA9EGF) and EOGT (human Notch1 EGF20, hN1EGF20). C , enzymatic activity of POGLUT2 with different EGF repeats as acceptor substrates (hN3EGF10, hN1EGF11, and hN4EGF11). hFA9EGF (POGLUT1 substrate) and hN1EGF20 (EOGT substrate) serve as negative control. Data represent mean ± SD from three independent assays. D , preparation of homogeneous POGLUT2. SDS-PAGE analysis shows removal of heterogeneous N -glycans by Endo Hf digestion. The resulting deglycosylated POGLUT2 was further purified by gel-filtration chromatography (Superdex 200 Increase 10/300 GL). E , schematic of the domain organization of POGLUT2. Three pairs of disulfide bonds are depicted by yellow lines . F , overall structure of POGLUT2. Cartoon representation highlights A-domain ( green ), B-domain ( pink ), and filamin domain ( dark gray ). The disulfide bonds are depicted as cyan sticks . G , surface representation of POGLUT2 in the top view . H , close-up view of interdomain interactions between the filamin domain and A-domain, as indicated by the box in ( F ). POGLUT2, protein O-glucosyltransferase 2.

Article Snippet: To remove N -glycans, the target protein was incubated with maltose-binding protein (MBP)–tagged Endo Hf (NEB) at a 1:100 (w/w) enzyme-to-protein ratio overnight at 4 °C.

Techniques: Functional Assay, Sequencing, Residue, Activity Assay, Negative Control, SDS Page, Purification, Filtration, Chromatography

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

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 promotes tumor immune evasion by upregulating PD-L1 expression within TME (A–C) scRNA-seq analysis of two paired tumor and paratumor samples from LUAD patients showing cell clustering (A), WFDC2 /HE4-expressing cell populations (B), and quantification of WFDC2 /HE4 expression in epithelial/malignant clusters (C). (D) Online dataset analysis of WFDC2 mRNA expression in tumor versus normal LUAD tissues. (E and F) HE4 overexpression (E) or knockout (F) respectively promoted or suppressed the growth of subcutaneous LLC tumors in mice. (G) Survival of mice intraperitoneally inoculated with HE4-overexpressing or control ID8 cells. (H) HE4 knockout attenuated ID8 peritoneal tumor progression, shown by representative abdominal images and ascites volume. (I) SDS-PAGE with Coomassie blue staining showing the purity of Fc and mouse HE4-Fc (mHE4-Fc) recombinant proteins. (J) Administration of mHE4-Fc promoted MC38 subcutaneous tumor growth. (K) mHE4-Fc administration reversed the growth suppression of HE4-KO LLC subcutaneous tumors. (L) mHE4-Fc failed to reverse tumor growth suppression in HE4-KO LLC tumors implanted in Rag1 -deficient mice. (M) Extracellular HE4 did not promote LLC cell proliferation in vitro , as assessed by CCK8 assay. (N) mHE4-Fc administration suppressed CD8 + T cell activation in the TME of HE4-KO LLC tumors, assessed by IFN-γ + and granzyme B + CD8 + T cells. (O) HE4 upregulated PD-L1 expression on macrophages in the microenvironment of LLC, MC38, and HE4-KO LLC tumors. (P–R) mHE4-Fc administration failed to reverse tumor suppression of HE4-KO LLC subcutaneous tumors in Cd274 -deficient mice, with treatment scheme (P), tumor growth (Q), and tumor weight (R). Statistical analyses were performed using Wilcoxon rank-sum test (D), two-way ANOVA (E, F, J–L, and Q), log rank (Mantel-Cox) test (G), and two-tailed paired (C) or unpaired t tests (H, M, N, O, and R). Data represent two independent experiments (E and M).

Article Snippet: no-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P70667.

Techniques: Expressing, Over Expression, Knock-Out, Control, SDS Page, Staining, Recombinant, In Vitro, CCK-8 Assay, Activation Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 competes with IFN-γ for IFN-γR binding and modulates downstream gene expression (A and B) Raw264.7 cells were stimulated with HE4-Fc (20 μg/mL) or IFN-γ (100 ng/mL) for 3 h, followed by RNA-seq; volcano plots of HE4- (A) or IFN-γ-regulated genes (B) are shown. (C) Genes commonly upregulated by HE4 and IFN-γ. (D) AlphaFold-3-predicted interfaces of HE4-IFNGR1/2 and IFN-γ-IFNGR1/2 complexes, with shared receptor-contact residues highlighted. (E) Competitive binding assay: His-tagged IFNGR1/2 was incubated with Flag-HE4 in the presence or absence of IFN-γ, followed by Ni-TED pull-down and immunoblotting. (F and G) SPR sensorgrams showing binding of mHE4-Fc (F) or mIFN-γ-Fc (G) to mIFNGR1-His. (H and I) ELISA quantification of HE4 and/or IFN-γ levels in ascites from ID8-tumor-bearing mice (H) and LLC-tumor-conditioned media (I). (J) High concentrations of HE4 reduced IFN-γ binding to IFNGR1/2 in competitive pull-down assays. (K) PCA of RNA-seq profiles from Raw264.7 cells treated with HE4-Fc, HE4-Fc plus IFN-γ, or IFN-γ for 12 h. (L) Expression (TPM) of representative STAT1- or STAT3-associated genes following the indicated treatments. Statistical analyses were performed using two-tailed paired Student’s t tests (H and I). Data in (E) and SPR sensorgrams (F and G) are representative of three independent experiments.

Article Snippet: no-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P70667.

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Competitive Binding Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 blockade promotes antitumor immunity in the tumor microenvironment (A–C) scRNA-seq analysis of LLC tumors from mice treated with control IgG or anti-HE4 antibody ( n = 3 per group), showing UMAP clustering of 26 cell populations (A), relative abundance of each cluster (B), and aggregated cell types (C). ∗ p < 0.05 (D and E) HE4 neutralization reduced Cd274 (PD-L1) expression in myeloid compartments. UMAP feature plots show Cd274 expression in macrophage/monocyte and epithelial/malignant populations (D), with paired comparison across macrophage clusters (E). (F) UMAP visualization of nine intratumoral T cell subclusters in control IgG– and anti-HE4–treated tumors. (G) In the LLC subcutaneous model, intratumoral IFN-γ + and CD69 + CD8 + T cells were quantified by flow cytometry. (H) In the ID8 intraperitoneal model, IFN-γ + CD8 + T cells were quantified by flow cytometry. (I–K) HE4 neutralization failed to suppress LLC tumor growth in Rag1 −/− mice, shown by treatment scheme and tumor growth/endpoint analyses. (L–O) CD8 + T cell depletion abrogated the antitumor efficacy of HE4 blockade, with treatment scheme, tumor growth/endpoint measurements, and confirmation of depletion efficiency by flow cytometry. (P–S) Macrophage depletion using anti-CSF1R diminished the antitumor efficacy of HE4 neutralization, with tumor growth/endpoint measurements and confirmation of depletion efficiency by flow cytometry. Statistical analyses were performed using unpaired t tests (B, C, H, and K), paired t test (E), one-way ANOVA (G, N, O, R, and S), and two-way ANOVA (J, M, and Q). Data in (G, H, and L–S) are pooled from two independent experiments.

Article Snippet: no-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P70667.

Techniques: Control, Neutralization, Expressing, Comparison, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 neutralization exerts therapeutic activity in human cancer models (A–C) PMA-differentiated THP-1 macrophages were stimulated with Fc or hHE4-Fc, and PD-L1 expression was assessed by flow cytometry (A), immunoblotting (B), and RT-qPCR (C); a commercial hHE4-Fc was included as an independent control. (D) Binding of hHE4 to PMA-differentiated THP-1 cells assessed by flow cytometry. (E and F) PMA-differentiated THP-1 cells were pretreated with ruxolitinib, fludarabine, or Stattic, followed by hHE4-Fc stimulation; PD-L1 was quantified by RT-qPCR (E) and flow cytometry (F). (G and H) Anti-hHE4 monoclonal antibodies inhibited hHE4-induced PD-L1 upregulation in PMA-differentiated THP-1 cells, assessed by RT-qPCR (G) and flow cytometry (H). (I) Anti-hHE4 mAb clone #10 blocked hHE4 binding to PMA-differentiated THP-1 cells. (J) Binding of wild-type or epitope-mutant hHE4-Fc to anti-hHE4 mAb clone #10 was quantified by ELISA. (K) Pharmacokinetic analysis of anti-hHE4 mAb clone #10 in C57BL/6 mice following intravenous administration. (L–O) Fresh human LUAD tumor cell suspensions were treated with anti-hHE4 mAb clone #10, followed by flow cytometric analysis of PD-L1 and ELISA measurement of IFN-γ and granzyme B. (P) Recombinant HE4 suppressed IFN-γ production in human LUAD tumor cell suspensions. (Q–T) HE4 blockade enhanced PBMC-mediated antitumor activity in humanized C-NKG mice bearing OVCAR3 or NCI-H358 tumors, shown by treatment scheme, representative tumors, and tumor weights. Schematics (L and Q) were created using BioRender. Statistical analyses were performed using one-way ANOVA (C, E, and G), paired t tests (M–P), or unpaired t tests (S and T). Data in (A–J) are representative of three independent experiments; data in (Q–T) are pooled from two independent experiments.

Article Snippet: no-tagged mouse IFN-γ protein , MedChemExpress , Cat# HY- P70667.

Techniques: Neutralization, Activity Assay, Expressing, Flow Cytometry, Western Blot, Quantitative RT-PCR, Control, Binding Assay, Bioprocessing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Recombinant