monocytic cell line u937  (ATCC)

Journal: Frontiers in Immunology

Article Title: A fully human IgG1 antibody targeting MICA α1 domain inhibits interaction with NKG2D and activates immune effector functions against MICA-expressing cells

doi: 10.3389/fimmu.2026.1740184

Figure Lengend Snippet: Antibody-dependent cell phagocytosis mediated by anti-MICA c65 antibody. (A) Dot plots showing phagocytosis by U937-EGFP macrophages. GES-1 cells labeled with the fluorescent probe TFL4 (APC) were co-cultured with U937-EGFP macrophages (FITC) in the presence of anti-MICA c65 antibody, anti-CD20 antibody as an isotype control, or vehicle (PBS) for 2 h Assays at 4 °C served as temperature controls. Dot plots illustrate the percentage of double-positive cells, corresponding to macrophages that phagocytosed GES-1 cells. (B) Bar graph of double-positive cell percentages. Bar graph indicates the mean ± SD of the percentage of APC+ (GES-1) cells on the FITC+ (macrophages) population, compared to 37 °C PBS control, from four independent experiments. *p<0.05, analyzed by Kruskal-Wallis test. (C) Confocal microscopy images of phagocytosed GES-1 cells labeled with CellTracker Orange and co-cultured with U937-EGFP macrophages (green) for 2 h in the presence of anti-MICA c65 antibody or isotype control. Two representative fields per condition are shown. White arrows highlight phagocytosed GES-1 cells. Scale bar: 10 µm.

Article Snippet: U937-EGFP cell line, derived from human promonocytic myeloid leukemia (ATCC ® CRL-1593.2, USA), was treated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) for 48 h to induce macrophage differentiation, following established protocols ( ).

Techniques: Labeling, Cell Culture, Control, Confocal Microscopy

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) Diagram illustrating how CD14 + monocytes were differentiated to MDMs and polarized. CD14 + monocytes were isolated from healthy blood donors, as a model for M2-like Mφs. AML cells were treated with DNR either in monoculture (-Mφ), in the presence of 50% macrophage conditioned media (Mφ-CM) or in direct co-culture with macrophages (Mφ). Representative plots of flow cytometric analysis of Annexin V-FITC and Viability Dye eFluor™ 450 fluorescence are shown for each cell line (Bi, Ci and Di). The treatment conditions are as follows; (Bii) U937: 0.25μM DNR for 24 hours (n=3), (Cii) THP-1: 0.125μM DNR for 72 hours (n=3), (Dii) KG-1a: 3μM DNR for 48 hours (n=4). AML survival (% of non-treated [NT]) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-FITC. Cells were analysed using the MACSQuant® Analyzer 10. Data are Mean ± SEM and were analysed using a one-way ANOVA followed by Tukey’s multiple-comparison test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Isolation, Co-Culture Assay, Fluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) Mφs were loaded with MTDR on Day 9 of culture of M2-like Mφs. CFSE-labelled U937, KG1a and THP-1 were then co-cultured with Mφs at a 1:1 ratio with M2-like Mφs for 24, 48 and 72 hours respectively. Mitochondrial transfer was then assessed in CFSE-labelled FVD - viable AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10. (Bi) Representative histograms of flow cytometric analysis of MTDR fluorescence, U937 = unlabeled U937 cells; U937 + Mφ = MTDR signal from U937 cells in co-culture with M2-like Mφs; U937 + Mφ = MTDR signal from M2-like Mφs in co-culture with U937 cells and Mφ = MTDR signal from M2-like Mφs. (Bii) Data are Mean ± SEM of n=5 and analysed by a Mann-Whitney U Test, **p<0.01. (Ci) Representative histograms of flow cytometric analysis of MTDR fluorescence. (Cii) AML cell lines were cultured on and off MTDR loaded Mφs in the presence or absence of DMSO (vehicle control) or DNR or Ara-C or the combination of DNR & Ara-C, for the indicated times and concentrations: U937: 0.25μM DNR for 24 hours; THP-1: 0.125μM DNR for 72 hours; KG-1a: 3μM DNR for 48 hours, the same times for Ara-C at 2.5μM for all AML cell lines (n=3-4). Data were then analyzed by a one-way ANOVA followed by Dunnett’s multiple-comparison test, *p<0.05, **p<0.01. (D) Primary AML cells were cultured on and off MTDR loaded Mφs in the presence or absence of 0.125μM of DNR for 24h and mitochondrial transfer assessed as above (n=4). (E) U937 cells can interact with macrophages via TNTs. Blue (top left) is indicative of DNA staining (DAPI), green (top right) is the actin staining (ActinGreen488), red (bottom left) corresponds to mitochondria (MTDR), and the merged image is shown bottom right (n=1). Scale bar = 10 μm. White arrows indicate TNTs. (F) U937 cells were cultured alone or cultured either indirectly (tMφ, via transwell inserts) or directly with MTDR loaded Mφs for 24h. (G) U937 cells were cultured on MTDR loaded Mφs in the presence or absence of DNR or 1 μM Cyto B or the combination of DNR & Cyto B, for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=6), and analysed by a one-way ANOVA followed by Tukey’s multiple-comparison test, *p<0.05, ns = non-significant. (Hi) Representative plots of flow cytometric analysis of Annexin V-FITC and Viability Dye eFluor™ 450 fluorescence. (Hii) U937 cells were treated with DNR +/- cyto B either in monoculture (-Mφ) or in direct co-culture with macrophages (+Mφ) for 24h. AML survival (% of NT) was determined by determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Cells were analysed using the MACSQuant® Analyzer 10 (AML cell lines). (H) Data are Mean ± SEM of n=4 and analysed by a two-way ANOVA followed by followed by Tukey’s multiple comparisons test, **p<0.01, ***p<0.0001, ns = non-significant, ns = non-significant.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Cell Culture, Fluorescence, Co-Culture Assay, MANN-WHITNEY, Control, Comparison, Staining

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (Ai) U937 cells were grown with and without M2-like monocyte-derived Mφs for 24 hours and then analyzed independently, using the Seahorse XFp Analyzer with the Mito Stress Test Kit. Sequential injections of Oligomycin (O), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (F), and Rotenone (R) were used to obtain respiration dynamics presented in panel (Aii). Data are mean ± SEM of n=3 and were then analyzed by using a paired student’s t-test, **p<0.01. (Bi) Representative histograms of flow cytometric analysis of CellROX fluorescence. (Bii) CFSE-labelled U937 were co-cultured at a 1:1 ratio with M2-like Mφs and in the presence of absence of DNR for 24 hours. Total ROS levels were then assessed in CFSE-labelled U937 cells using CellROX, which was analysed in the R2 (APC-Cy7) channel, via the MACSQuant® Analyzer 10. Relative CellROX values were calculated as a percentage of values obtained for U937 cells cultured alone in cRPMI (media con). The data are Mean ± SEM of n=3 and analysed by a two-way ANOVA followed by Tukey’s multiple-comparison test, **p<0.01, **p<0.001.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Derivative Assay, Fluorescence, Cell Culture, Comparison

Journal: bioRxiv

Article Title: LIMK Inhibition and Metformin Block Mitochondrial Transfer Overcoming Macrophage Driven Therapy Resistance in Acute Myeloid Leukaemia

doi: 10.64898/2026.02.03.702377

Figure Lengend Snippet: (A) CFSE-labelled U937 cells were directly cultured either in the presence (co-culture) or absence (monoculture) of M2-like Mφs for 24h. CD14 - U937 AML cells were then isolated from CD14 + Mφs, via the use of CD14 magnetic activated cell sorting. Proteomic profiling was then conducted on purified AML whole cell lysates (WCLs), via a TMT11-plex. Data shows the average values of n=4. (B) Association of transcript expression levels of RhoC and (C) Cofilin-1 with overall survival of patients with AML, plots were generated from interrogation of TCGA data via the GEPIA database. (Di) U937 were exposed to 0.25 mM DNR for up to 24h. Levels of ser3 p-cofilin and total cofilin (t-cofilin) were then assessed in purified U937 WCLs by immunoblotting. (Dii) Fold change of normalised ser3 p-cofilin to t-cofilin densitometry values are shown. The data are Mean ± SEM of n=5 and analysed by a one-way ANOVA followed by Dunnett’s multiple comparisons test, *p<0.05. (E) TCGA data were analyzed using GEPIA, and the expression of LIMK2 in AML compared to the normal samples is shown. (Fi) U937 were exposed to 0.25 mM DNR in the presence or absence of DMSO or 10mM of TH-257 for 12h. Levels of ser3 p-cofilin and total cofilin (t-cofilin) were then assessed in purified U937 WCLs by immunoblotting. (Fii) Fold change of normalised ser3 p-cofilin to t-cofilin densitometry values are shown. The data are mean ± SEM of n=3 and analysed by a one-way ANOVA followed by a Tukey’s multiple comparisons test, *p<0.05, **p<0.01. (G) CFSE-labelled U937 cells were cultured in the presence or absence of DNR, DMSO or 10 mM TH-257 either off (-Mφ) or on (+Mφ) MTDR loaded Mφs in the presence or absence of DNR for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=3), and analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, ns = non-significant. (H) AML survival (% of NT) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Data are Mean ± SEM of n=4 and were then analyzed using a two-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, ***p<0.001, ns = non-significant. (I) CFSE-labelled U937 cells were cultured in the presence or absence of DNR, water or 5 mM metformin either off (-Mφ) or on (+Mφ) MTDR loaded Mφs in the presence or absence of DNR for 24 hours. Mitochondrial transfer was then assessed in CFSE-labelled AML cells, with MTDR analysed in the R1 (APC) channel, via the MACSQuant® Analyzer 10 (n=5) with the data analysed by a one-way ANOVA followed by Tukey’s multiple comparisons test, *p<0.05, **p<0.01, ns = non-significant. (J) AML survival (% of NT) was determined by staining with Fixable Viability Dye eFluor™ 450 and Annexin V-APC. Data are Mean ± SEM of n=4 and were then analyzed using a two-way ANOVA followed by Tukey’s multiple-comparison test, **p<0.01, p<0.0001, ns = non-significant.

Article Snippet: Human AML cell lines U937, THP-1, and KG1a (ATCC) were cultured in complete RPMI 1640 (cRPMI, Gibco) with 10% v/v FBS (20% for KG1a), 1% v/v penicillin/streptomycin, and 1% v/v L-glutamine (Invitrogen).

Techniques: Cell Culture, Co-Culture Assay, Isolation, FACS, Purification, Expressing, Generated, Western Blot, Staining, Comparison