human monocytic leukemia cell line  (ATCC)

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF promotes lung cancer cell malignancy and M2 macrophage polarization. A549 and H520 cells were transfected with control (Ctrl) or EHF-knockdown (KD-EHF) plasmids. (A-B) Western blot and quantification of EHF protein levels in A549 and H520 cells. (C-D) EdU staining and quantification of EdU + cells in A549 and H520 cells, assessing proliferation. (E-H) Glucose uptake and lactate production assays were conducted using commercial kits to evaluate glycolytic output. (I-J) Flow cytometric analysis of apoptosis. (K-L) Transwell assay was employed to examine cell migration. (M − P) THP-1 cells were differentiated into THP-1-M0 macrophages with 100 ng/mL PMA for 24 h. Culture supernatants from Ctrl- or KD-EHF–transfected A549 and H520 cells were collected and co-incubated with THP-1-M0 macrophages. (M − N) Flow cytometric analysis was performed to assess the proportion of CD206 + M2-polarized macrophages. (O–P) ELISA was conducted to quantify the secretion of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the supernatants of THP-1-M0 macrophages. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Transfection, Control, Knockdown, Western Blot, Staining, Transwell Assay, Migration, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomes derived from lung cancer cells are internalized by THP-1-M0 macrophages and mediate EHF delivery. Exosomes were isolated from A549 and H520 cells. (A) TEM images showed the typical cup-shaped morphology of exosomes isolated from A549 and H520 cell supernatants (scale bar: 100 nm). (B) NTA of exosomes from A549 and H520 cells. (C) Western blot analysis of exosome markers (CD9 and TSG101) and the endoplasmic reticulum marker Calnexin in exosome lysates and parental A549/H520 cell lysates. (D-E) THP-1-M0 macrophages (differentiated from THP-1 cells with 100 ng/mL PMA for 24 h) were co-incubated with DiI-labeled exosomes isolated from A549 and H520 cells. (D) Fluorescence microscopy images of THP-1-M0 macrophages after co-incubation with PKH67-labeled exosomes (scale bar: 20 μm). (E-F) Western blot and quantification of EHF protein levels in THP-1-M0 macrophages after incubation with exosomes from A549 or H520 cells. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Derivative Assay, Isolation, Western Blot, Marker, Incubation, Labeling, Fluorescence, Microscopy

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: Exosomal EHF reprograms THP-1-M0 macrophages to promote lung cancer cell malignancy and angiogenesis via paracrine signaling. (A) Schematic of the paracrine co-culture system: Exosomes from control (Ctrl) or EHF-knockdown (KD-EHF) A549/H520 cells were co-incubated with THP-1-M0 macrophages. The medium from these macrophages was used to treat A549/H520 cells or HUVECs. (B-E) THP-1-M0 macrophages were treated with three conditions: blank control (no exosome), exosomes from control-transfected A549/H520 cells (Exo Ctrl ), or exosomes from EHF-knockdown A549/H520 cells (Exo KD−EHF ). (B–C) Flow cytometry analysis of CD206 + M2 macrophage proportions in THP-1-M0 cells. (D-E) ELISA measurement of M2-associated cytokines (TGF-β1, IL-10, and VEGFA). (F-M) The medium from exosome treated macrophages was then used to incubate A549/H520 cells or HUVECs. (F-G) EdU staining and quantification of EdU + proliferating A549 and H520 cells. (H–I) Flow cytometric analysis of apoptosis in A549 and H520 cells. (J-K) Transwell migration assays and quantification of migrated A549 and H520 cells. (L-M) Tube formation assays and quantification of tube numbers in HUVECs. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Co-Culture Assay, Control, Knockdown, Incubation, Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Migration

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: EHF transcriptionally activates RNF41 and mediates its intercellular transfer via exosomes. (A) JASPAR database prediction of the EHF-binding motif within the RNF41 promoter region, localized to positions −828/-821. (B) ChIP-qPCR analysis in THP-1-M0 macrophages. (C) Dual-luciferase reporter assays in 293T cells. (D) WB analysis of EHF protein levels in THP-1-M0 macrophages with EHF knockdown (KD-EHF) or control (Ctrl). (E-F) Western blot and qRT-PCR analysis of RNF41 protein and mRNA levels in THP-1-M0 macrophages with KD-EHF or Ctrl. (G-H) THP-1-M0 macrophages were treated with culture medium (Blank), control exosomes (Ctrl Exo, from A549/H520 cells), or EHF-knockdown exosomes (KD-EHF Exo, from A549/H520-KD-EHF cells). RNF41 protein levels were detected by Western blot. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Binding Assay, ChIP-qPCR, Luciferase, Knockdown, Control, Western Blot, Quantitative RT-PCR

Journal: Regenerative Therapy

Article Title: Lung cancer cell-derived exosomal EHF drives M2 macrophage polarization via transcriptional activation of RNF41 to promote tumor progression

doi: 10.1016/j.reth.2026.101104

Figure Lengend Snippet: RNF41 mediates exosomal EHF-driven pro-tumorigenic effects in lung cancer. (A) Schematic of the rescue co-culture system: THP-1-M0 macrophages (control or RNF41-overexpressing) were incubated with exosomes from control (Exo Ctrl ) or EHF-knockdown (Exo KD−EHF ) A549/H520 cells. The medium from these co-cultures was used to treat A549/H520 cells or HUVECs. (B–C) Flow cytometry was used to analyze the proportion of CD206 + M2-polarized macrophages in THP-1-M0 cells. (D-E) ELISA was employed to quantify the levels of M2-associated cytokines (TGF-β1, IL-10, and VEGFA) in the CM from macrophages. (F-G) EdU assays were carried out to measure the proliferation of A549 and H520 cells. (H–I) Annexin V/PI flow cytometry was used to assess the apoptosis of A549 and H520 cells treated with CM. (J-K) Transwell assays were performed to evaluate the migration of A549 and H520 cells. (L-M) Tube formation assays were conducted on HUVECs treated with CM. Data are presented as mean ± SD. P < 0.05 denotes statistical significance.

Article Snippet: Human monocyte cell line (THP-1) (Catalog #: YC-D011) were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China) and cultured in RPMI-1640 (Gibco) containing 10% FBS (Gibco).

Techniques: Co-Culture Assay, Control, Incubation, Knockdown, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Migration

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: High‐throughput screening identifies CeeNU as an effective NLRP3 inhibitor. (A and B) The process of selecting the most effective drug CeeNU from the FDA‐approved Drug Library by CellTiter‐Glo. (C) The chemical structure of CeeNU. (D) THP‐1 cells were exposed to graded concentrations of CeeNU for 24 h, and cell viability was subsequently determined by the CCK‐8 assay. (E) LPS‐primed THP‐1 cells were treated with CeeNU for 1 h, stimulated with nigericin for an additional hour, and cell viability was then measured using the CellTiter‐Glo Luminescent Assay. *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: High Throughput Screening Assay, Drug discovery, CCK-8 Assay, Luminescence Assay

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU remarkably represses pyroptosis and IL‐1β release dependent on NLRP3 in human macrophages. (A, B, D, and E) PMA‐differentiated human macrophage THP‐1 cells were either treated with LPS for 3 h plus CeeNU for 1 h, followed by stimulation with nigericin (A and B) or MSU (D and E), the supernatant was collected for ELISA of IL‐1β (A and D) and LDH assay (B and E). (C and F) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant and pro‐IL‐1β, procaspase‐1, NLRP3, GSDMD, GSDMD‐NT, and GAPDH in cell lysates of PMA‐differentiated THP‐1 cells primed with LPS for 4 h in the presence of CeeNU or control, prior to stimulation with nigericin (C) or MSU(F). (G) PMA‐differentiated THP‐1 cells were treated sequentially: first with LPS for 3 h, then with CeeNU for an additional hour. Following this, cells were stimulated with nigericin for 1 h. Subsequently, cells were stained with propidium iodide (PI, 2 µg/mL) to label dying cells and DAPI (1 µg/mL) to label all nuclei for 15 min. Imaging was performed using a 10× objective on an Olympus confocal microscope. Merged images include the corresponding bright‐field view. (H) The number of PI‐positive cells was counted in five randomly chosen microscopic fields. Lytic cell death was defined as the ratio of PI‐positive cells to the total number of DAPI‐positive nuclei and is expressed as a percentage. (I) Scanning electron microscopy of cell surface perforations caused by GSDMD in THP‐1 (Bar = 3 µm). * p < 0.05, ** p < 0.01, *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot, Control, Staining, Imaging, Microscopy, Electron Microscopy

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU is a specific inhibitor of the NLRP3 inflammasome. (A and C) Production of cleaved‐IL‐1β from LPS‐primed BMDMs treated with CeeNU before transfected with poly(dA:dT) (A), flagellin (C). (B and D) LDH release from LPS‐primed BMDMs treated with various doses of CeeNU before transfected with poly(dA:dT) (B), flagellin (D). (E–H) Western blotting analysis of cleaved IL‐1β p17 and caspase‐1 p20 levels in cell supernatant (SN) and pro‐IL‐1β, procaspase‐1, NLRP3, and GAPDH in cell lysates of BMDMs (E and F) and THP‐1 (G and H), LPS‐primed BMDMs and THP‐1 cells treated with CeeNU for 1 h before transfected with poly(dA:dT) for 16 h (E and G), flagellin for 16 h (F and H). (I and K) Production of cleaved‐IL‐1β from LPS‐primed THP‐1 cells treated with CeeNU before transfected with poly(dA:dT) (I), flagellin (K). (J and L) LDH release from LPS‐primed THP‐1 cells treated with various doses of CeeNU before transfected with poly(dA:dT) (J), flagellin (L). (M) BMDMs were treated with or without LPS, and plus different concentrations of CeeNU for 4 h, cell lysates were collected for Western blotting. (N) Western blotting analysis of p20 in SNs, pro‐casp1, and NLRP3 in input from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (O) ELISA of IL‐1β in SNs from LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. (P) LDH release from SN of LPS‐primed wild type, nlrp3 knockout, and gsdmd knockout BMDMs treated with 50 µM CeeNU, and then stimulated with ATP for 30 min. *** p < 0.001. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Transfection, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: MedComm

Article Title: High‐Throughput Screening Reveals That CeeNU Acts as a New NLRP3 Inflammasome Inhibitor

doi: 10.1002/mco2.70695

Figure Lengend Snippet: CeeNU specifically inhibits NLRP3 inflammasome assembly. (A–D) The treated THP‐1 (A and C) or BMDMs (B and D) cells were incubated with ASC primary antibody overnight, then re‐stained with secondary antibody for 1.5 h, and then stained with DAPI for 10 min before immunofluorescence images were taken by Olympus confocal microscope. Green represents ASC, DAPI (blue) shows nuclei. Five fields were randomly selected for each group of samples. The circular strong fluorescent spot indicated in the figure by the arrow was ASC speck. After that, the average fluorescence intensity was analyzed by ImageJ software, and the data were processed by GraphPad. (E) Western blotting analysis of ASC oligomerization level after cross‐linking with DSS in BMDMs. (F) Western blotting analysis of NLRP3 self‐oligomerization level of BMDMs pretreated with different doses of CeeNU after LPS stimulation for 3 h, then plus with nigericin. (G and H) BMDMs were primed with LPS, treated with 50 µM CeeNU, and then stimulated with 5 mM nigericin for 1 h. The interaction between endogenous NEK7 and NLRP3 was assessed by co‐immunoprecipitation (co‐IP) using an anti‐NEK7 (or anti‐NLRP3) antibody, followed by immunoblotting with antibodies against the reciprocal protein. (I) HEK293T cells were co‐transfected with plasmids expressing GFP‐NLRP3 and FLAG‐NEK7. Following transfection, cells were treated with 50 µM CeeNU or vehicle control as indicated. Cell lysates were subjected to IP using an anti‐GFP or FLAG antibody, followed by immunoblotting with antibodies against FLAG and GFP. ** p < 0.01. Data are mean ± SEM.

Article Snippet: Human THP‐1 cell line (THP‐1 ATCC TIB‐202) was from ATCC. iBMDMs and THP‐1 cells were cultured in DMEM‐high glucose and RPMI 1640 medium, respectively.

Techniques: Incubation, Staining, Immunofluorescence, Microscopy, Fluorescence, Software, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Expressing, Control

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: Human: THP-1 , ATCC , Cat# TIB202.

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