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human u937 monocytic cells (ATCC)

Name: human u937 monocytic cells
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Rating: 99 (7231 reviews)

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ATCC human u937 monocytic cells

Proliferation of pancreatic cancer spheroids and polarization of <t>U937</t> cells in transwell culture. A U937 monocyte cells were treated with PMA to chemically polarize to MØ macrophages. Then MØ cells were chemically polarized with IL-10 to M2 macrophages. Spent media supernatant was collected from M2 macrophages. Cells were stained with arginase to confirm M2-polarization. Figure created in BioRender, Smith, J. (2025); https://BioRender.com/fquwsxl . B Top figure: RNA was collected from the MØ macrophages to confirm SOCS-1 expression by qRT-PCR. PCR was done in replicates of three. Analysis by one-way ANOVA; Significance of **P = 0.004. Bottom figure: Confirmation of M2-polarization of U937 cells after IL-10 was confirmed by arginase staining. C Effects of co-culture or spent media supernatant on growth of AsPC-1 pancreatic cancer spheroids. C-1 Appearance of AsPC-1 spheroids shown at baseline at low magnification (4X) and at a higher magnification (20X). A cartoon of the transwell co-culture is shown below, bottom left. Representative photos show AsPC-1 spheroids after 72 h when grown alone (control-top), grown in culture with spent media supernatant from M2-polarized U937 cells (middle), or when grown in co-culture in transwell plates with U937 cells (bottom). After 72 h the spheroids were aspirated, dissociated and cell numbers counted in each group (replicates of three). C-2 The graphical results show columns representing mean ± SEM for each group with increased AsPC-1 cell number in these spheroids exposed to spent media supernatant or grown in co-culture compared to controls. Analysis was done by Student’s t-test with Bonferroni correction for multiple comparisons to controls. ** P < 0.01 and **** P < 0.0001. C-3 The U937 cells became adherent in the co-culture dish and were stained with arginase to confirm M2-polarization, left. The adherent macrophages were also collected and stained with the M2 macrophage antibody CD206 and with the M0/M1-macrophage antibodies CD80 and CD86 and subjected to flow cytometry, right. The non-adherent cells from the suspension were stained with CD206 and CD86 and subjected to flow cytometry. D Effects of proglumide treatment on MTT viability assay of AsPC-1 cells from spheroids that were untreated (Controls), treated with spent media (SM) or were co-cultured (Co–C) with U937 cells. Columns represent means ± SEM and in replicates of nine. Analysis of each treatment groups was done by student’s two-way t-test. Significance = * P < 0.05; *** P < 0.005. E Growth of human pancreatic cancer BxPC-3 spheroids in transwell inserts alone or in transwell inserts grown with U937 monocytes in co-culture with or without proglumide treatment. Columns represent mean values from N = 6 wells each. Significant differences were analyzed by student’s two-way t-test; * P < 0.05. Representative images of BxPC-3 spheroids are shown to the right

Human U937 Monocytic Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 7231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth"

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-026-02676-8

Proliferation of pancreatic cancer spheroids and polarization of U937 cells in transwell culture. A U937 monocyte cells were treated with PMA to chemically polarize to MØ macrophages. Then MØ cells were chemically polarized with IL-10 to M2 macrophages. Spent media supernatant was collected from M2 macrophages. Cells were stained with arginase to confirm M2-polarization. Figure created in BioRender, Smith, J. (2025); https://BioRender.com/fquwsxl . B Top figure: RNA was collected from the MØ macrophages to confirm SOCS-1 expression by qRT-PCR. PCR was done in replicates of three. Analysis by one-way ANOVA; Significance of **P = 0.004. Bottom figure: Confirmation of M2-polarization of U937 cells after IL-10 was confirmed by arginase staining. C Effects of co-culture or spent media supernatant on growth of AsPC-1 pancreatic cancer spheroids. C-1 Appearance of AsPC-1 spheroids shown at baseline at low magnification (4X) and at a higher magnification (20X). A cartoon of the transwell co-culture is shown below, bottom left. Representative photos show AsPC-1 spheroids after 72 h when grown alone (control-top), grown in culture with spent media supernatant from M2-polarized U937 cells (middle), or when grown in co-culture in transwell plates with U937 cells (bottom). After 72 h the spheroids were aspirated, dissociated and cell numbers counted in each group (replicates of three). C-2 The graphical results show columns representing mean ± SEM for each group with increased AsPC-1 cell number in these spheroids exposed to spent media supernatant or grown in co-culture compared to controls. Analysis was done by Student’s t-test with Bonferroni correction for multiple comparisons to controls. ** P < 0.01 and **** P < 0.0001. C-3 The U937 cells became adherent in the co-culture dish and were stained with arginase to confirm M2-polarization, left. The adherent macrophages were also collected and stained with the M2 macrophage antibody CD206 and with the M0/M1-macrophage antibodies CD80 and CD86 and subjected to flow cytometry, right. The non-adherent cells from the suspension were stained with CD206 and CD86 and subjected to flow cytometry. D Effects of proglumide treatment on MTT viability assay of AsPC-1 cells from spheroids that were untreated (Controls), treated with spent media (SM) or were co-cultured (Co–C) with U937 cells. Columns represent means ± SEM and in replicates of nine. Analysis of each treatment groups was done by student’s two-way t-test. Significance = * P < 0.05; *** P < 0.005. E Growth of human pancreatic cancer BxPC-3 spheroids in transwell inserts alone or in transwell inserts grown with U937 monocytes in co-culture with or without proglumide treatment. Columns represent mean values from N = 6 wells each. Significant differences were analyzed by student’s two-way t-test; * P < 0.05. Representative images of BxPC-3 spheroids are shown to the right

Figure Legend Snippet: Proliferation of pancreatic cancer spheroids and polarization of U937 cells in transwell culture. A U937 monocyte cells were treated with PMA to chemically polarize to MØ macrophages. Then MØ cells were chemically polarized with IL-10 to M2 macrophages. Spent media supernatant was collected from M2 macrophages. Cells were stained with arginase to confirm M2-polarization. Figure created in BioRender, Smith, J. (2025); https://BioRender.com/fquwsxl . B Top figure: RNA was collected from the MØ macrophages to confirm SOCS-1 expression by qRT-PCR. PCR was done in replicates of three. Analysis by one-way ANOVA; Significance of **P = 0.004. Bottom figure: Confirmation of M2-polarization of U937 cells after IL-10 was confirmed by arginase staining. C Effects of co-culture or spent media supernatant on growth of AsPC-1 pancreatic cancer spheroids. C-1 Appearance of AsPC-1 spheroids shown at baseline at low magnification (4X) and at a higher magnification (20X). A cartoon of the transwell co-culture is shown below, bottom left. Representative photos show AsPC-1 spheroids after 72 h when grown alone (control-top), grown in culture with spent media supernatant from M2-polarized U937 cells (middle), or when grown in co-culture in transwell plates with U937 cells (bottom). After 72 h the spheroids were aspirated, dissociated and cell numbers counted in each group (replicates of three). C-2 The graphical results show columns representing mean ± SEM for each group with increased AsPC-1 cell number in these spheroids exposed to spent media supernatant or grown in co-culture compared to controls. Analysis was done by Student’s t-test with Bonferroni correction for multiple comparisons to controls. ** P < 0.01 and **** P < 0.0001. C-3 The U937 cells became adherent in the co-culture dish and were stained with arginase to confirm M2-polarization, left. The adherent macrophages were also collected and stained with the M2 macrophage antibody CD206 and with the M0/M1-macrophage antibodies CD80 and CD86 and subjected to flow cytometry, right. The non-adherent cells from the suspension were stained with CD206 and CD86 and subjected to flow cytometry. D Effects of proglumide treatment on MTT viability assay of AsPC-1 cells from spheroids that were untreated (Controls), treated with spent media (SM) or were co-cultured (Co–C) with U937 cells. Columns represent means ± SEM and in replicates of nine. Analysis of each treatment groups was done by student’s two-way t-test. Significance = * P < 0.05; *** P < 0.005. E Growth of human pancreatic cancer BxPC-3 spheroids in transwell inserts alone or in transwell inserts grown with U937 monocytes in co-culture with or without proglumide treatment. Columns represent mean values from N = 6 wells each. Significant differences were analyzed by student’s two-way t-test; * P < 0.05. Representative images of BxPC-3 spheroids are shown to the right

Techniques Used: Staining, Expressing, Quantitative RT-PCR, Co-Culture Assay, Control, Flow Cytometry, Suspension, MTT Viability Assay, Cell Culture

Differentially expressed genes and proteins by RNA sequencing and RPPA in AsPC-1 spheroids. In the top row volcano plots of differentially expressed genes for each treatment group is shown. A Genes ( N = 724) were significantly downregulated and 182 genes were upregulated in AsPC-1 spheroids treated with proglumide compared to untreated controls. B An equal number of downregulated and upregulated genes were seen in AsPC-1 spheroids treated with supernatant spent media compared to controls. C There were 2045 upregulated genes and only 298 downregulated genes seen in AsPC-1 spheroids treated in co-culture with U937 cells. The most upregulated gene was ELANE (arrow). D Heat map for the top 20 up- (in red) and top 20 down- (in green) regulated genes comparing control spheroids to proglumide-treated spheroids. E Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids to spheroids treated with M2-polarized macrophage supernatant spent media. F Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids AsPC-1 spheroids co-cultured with U937 cells. G Reverse phase protein array (RPPA) of AsPC-1 spheroids shows unsupervised hierarchical clustering capturing the proteins changed when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. The rectangular boxes are highlighting some key proteins and signaling pathways involved in pancreatic cancer cell proliferation that are increased with co-culture with macrophages and decreased with proglumide. H Western blot is shown of the three outlined proteins from panel G. The western blot was performed with AsPC-1 and BxPC-3 spheroids that were grown alone or in co-culture with U937 cells with and without proglumide. I Unsupervised hierarchical clustering capturing the proteins changed in the isolated exosomes when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. J RPPA analysis of pooled exosomes from triplicate replicates of untreated spheroids, co-cultured spheroids, or spheroids treated with proglumide for neutrophil elastase. K Relative mRNA expression of ELANE by qRT-PCR in AsPC-1 spheroids and U937 cells grown alone or in co-culture. * P < 0.05. L Shows relative mRNA expression of ELANE without co-culture in AsPC-1 or U937 cells. * P < 0.05

Figure Legend Snippet: Differentially expressed genes and proteins by RNA sequencing and RPPA in AsPC-1 spheroids. In the top row volcano plots of differentially expressed genes for each treatment group is shown. A Genes ( N = 724) were significantly downregulated and 182 genes were upregulated in AsPC-1 spheroids treated with proglumide compared to untreated controls. B An equal number of downregulated and upregulated genes were seen in AsPC-1 spheroids treated with supernatant spent media compared to controls. C There were 2045 upregulated genes and only 298 downregulated genes seen in AsPC-1 spheroids treated in co-culture with U937 cells. The most upregulated gene was ELANE (arrow). D Heat map for the top 20 up- (in red) and top 20 down- (in green) regulated genes comparing control spheroids to proglumide-treated spheroids. E Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids to spheroids treated with M2-polarized macrophage supernatant spent media. F Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids AsPC-1 spheroids co-cultured with U937 cells. G Reverse phase protein array (RPPA) of AsPC-1 spheroids shows unsupervised hierarchical clustering capturing the proteins changed when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. The rectangular boxes are highlighting some key proteins and signaling pathways involved in pancreatic cancer cell proliferation that are increased with co-culture with macrophages and decreased with proglumide. H Western blot is shown of the three outlined proteins from panel G. The western blot was performed with AsPC-1 and BxPC-3 spheroids that were grown alone or in co-culture with U937 cells with and without proglumide. I Unsupervised hierarchical clustering capturing the proteins changed in the isolated exosomes when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. J RPPA analysis of pooled exosomes from triplicate replicates of untreated spheroids, co-cultured spheroids, or spheroids treated with proglumide for neutrophil elastase. K Relative mRNA expression of ELANE by qRT-PCR in AsPC-1 spheroids and U937 cells grown alone or in co-culture. * P < 0.05. L Shows relative mRNA expression of ELANE without co-culture in AsPC-1 or U937 cells. * P < 0.05

Techniques Used: RNA Sequencing, Co-Culture Assay, Control, Cell Culture, Protein Array, Protein-Protein interactions, Western Blot, Isolation, Expressing, Quantitative RT-PCR

Effects of downregulation of ELANE mRNA expression on tumor growth and on the tumor microenvironment. A Relative mRNA expression of ELANE was measured by qRT-PCR after transfection with each of three shRNAs and a scrambled control. shRNA B decreased ELANE expression by 32%. B qRT-PCR analysis comparing ELANE mRNA relative expression in wild-type AsPC-1 cells compared to the first transfection (KD × 1) and the 2nd transfection KD × 2 cells. Assays were done in triplicate and the results were analyzed using a student’s t-test on the normalized mean ΔCT (the difference between the cycle counts of the gene of interest minus the count of an endogenous control) values for each group, with Bonferroni corrections applied to adjust for multiple comparisons; (* P < 0.05 and **** P < 0.0001). C Schematic diagram showing the study design of the co-culture experiment with wild-type AsPC-1 cells or ELANE -KD AsPC-1 cells co-cultured with U937 monocytes ( N = 6 wells per treatment). D 72 h after co-culture, the coverslips from the bottom of the tissue culture wells were stained for M2-polaraized TAMs with arginase. Bar graph representing the mean ± SEM number of the number of arginase positive macrophages adherent on the coverslips per high powered field. None of the U937 monocytes (control) were adherent to the coverslips. Representative images are shown for each group. Assay was done in triplicate. E Final tumor weights comparing wild-type AsPC-1 tumors to that of tumors from AsPC-1 ELANE -KD × 1 and ELANE KDx2 cells; ( N = 10/group). Columns represent the mean ± SEM of the tumor weights in vivo showing the dose response with smaller tumors with the ELANE KD × 2 AsPC-1 cells. Significant by student’s t-test for each group, with Bonferroni corrections applied to adjust for multiple comparisons (** P < 0.01). F Ki67 immunohistochemistry staining for proliferating cells compares wild-type tumors to ELANE -KD tumors (**** P < 0.0001). G A representative photo (top) from a wild-type AsPC-1 tumor showing numerous arginase + M2-polarized macrophages by immunohistochemistry. The bottom image is a representative picture showing the decreased number of arginase + M2-polarized TAMs in the tumors from the ELANE -KD cells. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). H Representative photos of iNOS stained wild-type tumors (top) and ELANE -KD tumors (bottom) for pro-inflammatory M1-polaized TAMs. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). I Flow cytometry results from samples of sections of wild-type or ELANE -KD tumors gated for M2-polarized TAMs (* P < 0.05) or J M1-polarized TAMs (** P < 0,01). K LY6G and arginase positive gating was used to detect N2-polarized neutrophils by flow cytometry in wild-type tumors or ELANE -KD tumors. Analysis using Welch Two Sample t-test showed significant differences between the groups (* P < 0.05). L N1-positive TANs reacted with FasR show an increase in the number of N1 +—neutrophils in the ELANE -KD tumors. (**** P < 0.0001 by Welch Two Sample t-test

Figure Legend Snippet: Effects of downregulation of ELANE mRNA expression on tumor growth and on the tumor microenvironment. A Relative mRNA expression of ELANE was measured by qRT-PCR after transfection with each of three shRNAs and a scrambled control. shRNA B decreased ELANE expression by 32%. B qRT-PCR analysis comparing ELANE mRNA relative expression in wild-type AsPC-1 cells compared to the first transfection (KD × 1) and the 2nd transfection KD × 2 cells. Assays were done in triplicate and the results were analyzed using a student’s t-test on the normalized mean ΔCT (the difference between the cycle counts of the gene of interest minus the count of an endogenous control) values for each group, with Bonferroni corrections applied to adjust for multiple comparisons; (* P < 0.05 and **** P < 0.0001). C Schematic diagram showing the study design of the co-culture experiment with wild-type AsPC-1 cells or ELANE -KD AsPC-1 cells co-cultured with U937 monocytes ( N = 6 wells per treatment). D 72 h after co-culture, the coverslips from the bottom of the tissue culture wells were stained for M2-polaraized TAMs with arginase. Bar graph representing the mean ± SEM number of the number of arginase positive macrophages adherent on the coverslips per high powered field. None of the U937 monocytes (control) were adherent to the coverslips. Representative images are shown for each group. Assay was done in triplicate. E Final tumor weights comparing wild-type AsPC-1 tumors to that of tumors from AsPC-1 ELANE -KD × 1 and ELANE KDx2 cells; ( N = 10/group). Columns represent the mean ± SEM of the tumor weights in vivo showing the dose response with smaller tumors with the ELANE KD × 2 AsPC-1 cells. Significant by student’s t-test for each group, with Bonferroni corrections applied to adjust for multiple comparisons (** P < 0.01). F Ki67 immunohistochemistry staining for proliferating cells compares wild-type tumors to ELANE -KD tumors (**** P < 0.0001). G A representative photo (top) from a wild-type AsPC-1 tumor showing numerous arginase + M2-polarized macrophages by immunohistochemistry. The bottom image is a representative picture showing the decreased number of arginase + M2-polarized TAMs in the tumors from the ELANE -KD cells. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). H Representative photos of iNOS stained wild-type tumors (top) and ELANE -KD tumors (bottom) for pro-inflammatory M1-polaized TAMs. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). I Flow cytometry results from samples of sections of wild-type or ELANE -KD tumors gated for M2-polarized TAMs (* P < 0.05) or J M1-polarized TAMs (** P < 0,01). K LY6G and arginase positive gating was used to detect N2-polarized neutrophils by flow cytometry in wild-type tumors or ELANE -KD tumors. Analysis using Welch Two Sample t-test showed significant differences between the groups (* P < 0.05). L N1-positive TANs reacted with FasR show an increase in the number of N1 +—neutrophils in the ELANE -KD tumors. (**** P < 0.0001 by Welch Two Sample t-test

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Co-Culture Assay, Cell Culture, Staining, In Vivo, Immunohistochemistry, Flow Cytometry

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Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Proliferation of pancreatic cancer spheroids and polarization of U937 cells in transwell culture. A U937 monocyte cells were treated with PMA to chemically polarize to MØ macrophages. Then MØ cells were chemically polarized with IL-10 to M2 macrophages. Spent media supernatant was collected from M2 macrophages. Cells were stained with arginase to confirm M2-polarization. Figure created in BioRender, Smith, J. (2025); https://BioRender.com/fquwsxl . B Top figure: RNA was collected from the MØ macrophages to confirm SOCS-1 expression by qRT-PCR. PCR was done in replicates of three. Analysis by one-way ANOVA; Significance of **P = 0.004. Bottom figure: Confirmation of M2-polarization of U937 cells after IL-10 was confirmed by arginase staining. C Effects of co-culture or spent media supernatant on growth of AsPC-1 pancreatic cancer spheroids. C-1 Appearance of AsPC-1 spheroids shown at baseline at low magnification (4X) and at a higher magnification (20X). A cartoon of the transwell co-culture is shown below, bottom left. Representative photos show AsPC-1 spheroids after 72 h when grown alone (control-top), grown in culture with spent media supernatant from M2-polarized U937 cells (middle), or when grown in co-culture in transwell plates with U937 cells (bottom). After 72 h the spheroids were aspirated, dissociated and cell numbers counted in each group (replicates of three). C-2 The graphical results show columns representing mean ± SEM for each group with increased AsPC-1 cell number in these spheroids exposed to spent media supernatant or grown in co-culture compared to controls. Analysis was done by Student’s t-test with Bonferroni correction for multiple comparisons to controls. ** P < 0.01 and **** P < 0.0001. C-3 The U937 cells became adherent in the co-culture dish and were stained with arginase to confirm M2-polarization, left. The adherent macrophages were also collected and stained with the M2 macrophage antibody CD206 and with the M0/M1-macrophage antibodies CD80 and CD86 and subjected to flow cytometry, right. The non-adherent cells from the suspension were stained with CD206 and CD86 and subjected to flow cytometry. D Effects of proglumide treatment on MTT viability assay of AsPC-1 cells from spheroids that were untreated (Controls), treated with spent media (SM) or were co-cultured (Co–C) with U937 cells. Columns represent means ± SEM and in replicates of nine. Analysis of each treatment groups was done by student’s two-way t-test. Significance = * P < 0.05; *** P < 0.005. E Growth of human pancreatic cancer BxPC-3 spheroids in transwell inserts alone or in transwell inserts grown with U937 monocytes in co-culture with or without proglumide treatment. Columns represent mean values from N = 6 wells each. Significant differences were analyzed by student’s two-way t-test; * P < 0.05. Representative images of BxPC-3 spheroids are shown to the right

Article Snippet: The human pancreatic cancer cell line AsPC-1 (Cat# CRL-1682; RRID: CVCL_0152), human BxPC-3 pancreatic cancer cells (Cat# CRL-1687; RRID: CVCL_0186), and human U937 monocytic cells (Cat# CRL-1593.2; RRID: CVCL_U937) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and obtained from the Georgetown Lombardi Tissue Culture Shared Resource facility.

Techniques: Staining, Expressing, Quantitative RT-PCR, Co-Culture Assay, Control, Flow Cytometry, Suspension, MTT Viability Assay, Cell Culture

Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Differentially expressed genes and proteins by RNA sequencing and RPPA in AsPC-1 spheroids. In the top row volcano plots of differentially expressed genes for each treatment group is shown. A Genes ( N = 724) were significantly downregulated and 182 genes were upregulated in AsPC-1 spheroids treated with proglumide compared to untreated controls. B An equal number of downregulated and upregulated genes were seen in AsPC-1 spheroids treated with supernatant spent media compared to controls. C There were 2045 upregulated genes and only 298 downregulated genes seen in AsPC-1 spheroids treated in co-culture with U937 cells. The most upregulated gene was ELANE (arrow). D Heat map for the top 20 up- (in red) and top 20 down- (in green) regulated genes comparing control spheroids to proglumide-treated spheroids. E Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids to spheroids treated with M2-polarized macrophage supernatant spent media. F Heat map for the top 20 up- and top 20 downregulated genes comparing control spheroids AsPC-1 spheroids co-cultured with U937 cells. G Reverse phase protein array (RPPA) of AsPC-1 spheroids shows unsupervised hierarchical clustering capturing the proteins changed when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. The rectangular boxes are highlighting some key proteins and signaling pathways involved in pancreatic cancer cell proliferation that are increased with co-culture with macrophages and decreased with proglumide. H Western blot is shown of the three outlined proteins from panel G. The western blot was performed with AsPC-1 and BxPC-3 spheroids that were grown alone or in co-culture with U937 cells with and without proglumide. I Unsupervised hierarchical clustering capturing the proteins changed in the isolated exosomes when AsPC-1 spheroids were co-cultured with U937 cells or when treated with proglumide compared to untreated control spheroids. J RPPA analysis of pooled exosomes from triplicate replicates of untreated spheroids, co-cultured spheroids, or spheroids treated with proglumide for neutrophil elastase. K Relative mRNA expression of ELANE by qRT-PCR in AsPC-1 spheroids and U937 cells grown alone or in co-culture. * P < 0.05. L Shows relative mRNA expression of ELANE without co-culture in AsPC-1 or U937 cells. * P < 0.05

Techniques: RNA Sequencing, Co-Culture Assay, Control, Cell Culture, Protein Array, Protein-Protein interactions, Western Blot, Isolation, Expressing, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Interruption of cross-communication pathways alters the immune cell signature of pancreatic cancer and decreases tumor growth

doi: 10.1186/s12964-026-02676-8

Figure Lengend Snippet: Effects of downregulation of ELANE mRNA expression on tumor growth and on the tumor microenvironment. A Relative mRNA expression of ELANE was measured by qRT-PCR after transfection with each of three shRNAs and a scrambled control. shRNA B decreased ELANE expression by 32%. B qRT-PCR analysis comparing ELANE mRNA relative expression in wild-type AsPC-1 cells compared to the first transfection (KD × 1) and the 2nd transfection KD × 2 cells. Assays were done in triplicate and the results were analyzed using a student’s t-test on the normalized mean ΔCT (the difference between the cycle counts of the gene of interest minus the count of an endogenous control) values for each group, with Bonferroni corrections applied to adjust for multiple comparisons; (* P < 0.05 and **** P < 0.0001). C Schematic diagram showing the study design of the co-culture experiment with wild-type AsPC-1 cells or ELANE -KD AsPC-1 cells co-cultured with U937 monocytes ( N = 6 wells per treatment). D 72 h after co-culture, the coverslips from the bottom of the tissue culture wells were stained for M2-polaraized TAMs with arginase. Bar graph representing the mean ± SEM number of the number of arginase positive macrophages adherent on the coverslips per high powered field. None of the U937 monocytes (control) were adherent to the coverslips. Representative images are shown for each group. Assay was done in triplicate. E Final tumor weights comparing wild-type AsPC-1 tumors to that of tumors from AsPC-1 ELANE -KD × 1 and ELANE KDx2 cells; ( N = 10/group). Columns represent the mean ± SEM of the tumor weights in vivo showing the dose response with smaller tumors with the ELANE KD × 2 AsPC-1 cells. Significant by student’s t-test for each group, with Bonferroni corrections applied to adjust for multiple comparisons (** P < 0.01). F Ki67 immunohistochemistry staining for proliferating cells compares wild-type tumors to ELANE -KD tumors (**** P < 0.0001). G A representative photo (top) from a wild-type AsPC-1 tumor showing numerous arginase + M2-polarized macrophages by immunohistochemistry. The bottom image is a representative picture showing the decreased number of arginase + M2-polarized TAMs in the tumors from the ELANE -KD cells. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). H Representative photos of iNOS stained wild-type tumors (top) and ELANE -KD tumors (bottom) for pro-inflammatory M1-polaized TAMs. Analysis using Welch Two Sample t-test showed significant differences between the groups (**** P < 0.0001; N = 10). I Flow cytometry results from samples of sections of wild-type or ELANE -KD tumors gated for M2-polarized TAMs (* P < 0.05) or J M1-polarized TAMs (** P < 0,01). K LY6G and arginase positive gating was used to detect N2-polarized neutrophils by flow cytometry in wild-type tumors or ELANE -KD tumors. Analysis using Welch Two Sample t-test showed significant differences between the groups (* P < 0.05). L N1-positive TANs reacted with FasR show an increase in the number of N1 +—neutrophils in the ELANE -KD tumors. (**** P < 0.0001 by Welch Two Sample t-test

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, shRNA, Co-Culture Assay, Cell Culture, Staining, In Vivo, Immunohistochemistry, Flow Cytometry

Exogenous COL4A1, OPN and HA promote development of immunomodulatory CD16 + Mφ. (A, B) After 48 hours of stimulation with exogenous COL4A1 (10 ug/mL), OPN (1 ug/mL) or vehicles, the expression levels of CD16 on U937-induced Mφ ( A ; n=5) and the polarization-related molecules CD86, CD209 and CD206 on CD16 - or CD16 + Mφ ( B ; n=5) were detected by flow cytometry. (C, D) After 48 hours of stimulation with exogenous HA (0, 50, 100 μM), the expression levels of CD16 on U937-induced Mφ ( C ; n=4) and CD86, CD209 and CD206 on CD16 - or CD16 + Mφ ( D ; n=4) were detected by flow cytometry. The data are presented as the mean ± SEM; oneway ANOVA test or two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ****P< 0.0001, NS, no significant difference.

Journal: Frontiers in Immunology

Article Title: Decidual stromal cells drive CD16 + macrophages towards an immunoregulatory phenotype via extracellular matrix-adhesion molecule interaction during early pregnancy

doi: 10.3389/fimmu.2025.1747323

Figure Lengend Snippet: Exogenous COL4A1, OPN and HA promote development of immunomodulatory CD16 + Mφ. (A, B) After 48 hours of stimulation with exogenous COL4A1 (10 ug/mL), OPN (1 ug/mL) or vehicles, the expression levels of CD16 on U937-induced Mφ ( A ; n=5) and the polarization-related molecules CD86, CD209 and CD206 on CD16 - or CD16 + Mφ ( B ; n=5) were detected by flow cytometry. (C, D) After 48 hours of stimulation with exogenous HA (0, 50, 100 μM), the expression levels of CD16 on U937-induced Mφ ( C ; n=4) and CD86, CD209 and CD206 on CD16 - or CD16 + Mφ ( D ; n=4) were detected by flow cytometry. The data are presented as the mean ± SEM; oneway ANOVA test or two-tailed Student’s t-test; *P < 0.05, **P < 0.01, ****P< 0.0001, NS, no significant difference.

Article Snippet: Human monocyte cell line U937 from American Type Culture Collection (ATCC, CRL-3253) was cultured with RPMI-1640 medium (HyClone, SH30027.01) containing 10% FBS.

Techniques: Expressing, Flow Cytometry, Two Tailed Test

CD16 + Mφ in the co-culture system are suppressed with the inhibition of COL4A1, OPN and HA in DSCs. (A) DSCs were transfected by plasmid of siRNA targeting COL4A1 (si COL4A1 ; n=3), SPP1 (si SPP1 ; n=3) or control plasmids (n=3) for 72 hours and the efficacy was verified by RT-qPCR. (B-D) After co-cultured with COL4A1 -silenced DSCs, SPP1 -silenced DSCs or control DSCs for 48 hours, CD16 expression of U937-induced Mφ ( B, C ; n=4) and the polarization markers (CD86, CD209 and CD206) of CD16 - or CD16 + macrophages ( D ; n=4) were explored by flow cytometry. (E, F) After co-cultured with 4-MU (a hyaluronic Acid synthesis inhibitor, 500 μM) or vehicle treated DSCs for 48 hours, CD16 expression of U937-induced Mφ ( E ; n=6) and the polarization markers of CD16 - or CD16 + macrophages ( F ; n=6) were explored by flow cytometry. The data are presented as the mean ± SEM; oneway ANOVA test or two-tailed Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, NS significant difference.

Journal: Frontiers in Immunology

Article Title: Decidual stromal cells drive CD16 + macrophages towards an immunoregulatory phenotype via extracellular matrix-adhesion molecule interaction during early pregnancy

doi: 10.3389/fimmu.2025.1747323

Figure Lengend Snippet: CD16 + Mφ in the co-culture system are suppressed with the inhibition of COL4A1, OPN and HA in DSCs. (A) DSCs were transfected by plasmid of siRNA targeting COL4A1 (si COL4A1 ; n=3), SPP1 (si SPP1 ; n=3) or control plasmids (n=3) for 72 hours and the efficacy was verified by RT-qPCR. (B-D) After co-cultured with COL4A1 -silenced DSCs, SPP1 -silenced DSCs or control DSCs for 48 hours, CD16 expression of U937-induced Mφ ( B, C ; n=4) and the polarization markers (CD86, CD209 and CD206) of CD16 - or CD16 + macrophages ( D ; n=4) were explored by flow cytometry. (E, F) After co-cultured with 4-MU (a hyaluronic Acid synthesis inhibitor, 500 μM) or vehicle treated DSCs for 48 hours, CD16 expression of U937-induced Mφ ( E ; n=6) and the polarization markers of CD16 - or CD16 + macrophages ( F ; n=6) were explored by flow cytometry. The data are presented as the mean ± SEM; oneway ANOVA test or two-tailed Student’s t -test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, NS significant difference.

Article Snippet: Human monocyte cell line U937 from American Type Culture Collection (ATCC, CRL-3253) was cultured with RPMI-1640 medium (HyClone, SH30027.01) containing 10% FBS.

Techniques: Co-Culture Assay, Inhibition, Transfection, Plasmid Preparation, Control, Quantitative RT-PCR, Cell Culture, Expressing, Flow Cytometry, Two Tailed Test

Curcuminoids increase global 5hmC in leukemia cells. Graded concentrations of curcumin (Cur) or DMC were used to treat U937 ( a , b ) and HL60 leukemia cells ( c , d ) for 48 h, followed by genomic DNA extraction and quantitation of global 5hmC as described in the methods. The data represent the mean of 3 replicates ± SD. * indicates a significant difference from the control at p < 0.05.

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Curcuminoids increase global 5hmC in leukemia cells. Graded concentrations of curcumin (Cur) or DMC were used to treat U937 ( a , b ) and HL60 leukemia cells ( c , d ) for 48 h, followed by genomic DNA extraction and quantitation of global 5hmC as described in the methods. The data represent the mean of 3 replicates ± SD. * indicates a significant difference from the control at p < 0.05.

Article Snippet: Human promyeloid leukemia (HL60) and human monocytic leukemia (U937) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium (Sigma-Aldrich, Milwaukee, WI, USA) containing 10% fetal bovine serum (FBS) and 2.5 mM L-glutamine.

Techniques: DNA Extraction, Quantitation Assay, Control

Curcuminoids increase TET enzymatic activity in leukemia cells. Graded concentrations of curcumin (Cur) or DMC were used to treat U937 ( a , b ) and HL60 leukemia cells ( c , d ) for 48 h, followed by nuclear protein extraction and quantitation of TET activity, as described in the methods. The data represent the mean of 3 replicates ± SD. * indicates significant difference from the control at p < 0.05.

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Curcuminoids increase TET enzymatic activity in leukemia cells. Graded concentrations of curcumin (Cur) or DMC were used to treat U937 ( a , b ) and HL60 leukemia cells ( c , d ) for 48 h, followed by nuclear protein extraction and quantitation of TET activity, as described in the methods. The data represent the mean of 3 replicates ± SD. * indicates significant difference from the control at p < 0.05.

Techniques: Activity Assay, Protein Extraction, Quantitation Assay, Control

Curcuminoids induce TET isoform transcription in leukemia cells. U937 leukemia cells were treated with graded concentrations of either curcumin for 24 and 48 h ( a ) and ( b ), respectively) or DMC (( c ) and ( d ), respectively), followed by RNA extraction and single-step RT-PCR, as described in the methods. The data represent the mean ± SD for 3 replicates. * indicates a significant difference at p < 0.05, ** indicates a significant difference at p < 0.01, *** indicates a significant difference at p < 0.001, and **** indicates a significant difference at p < 0.0001. Note that TET3 induction is not visible because of the scale.

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Curcuminoids induce TET isoform transcription in leukemia cells. U937 leukemia cells were treated with graded concentrations of either curcumin for 24 and 48 h ( a ) and ( b ), respectively) or DMC (( c ) and ( d ), respectively), followed by RNA extraction and single-step RT-PCR, as described in the methods. The data represent the mean ± SD for 3 replicates. * indicates a significant difference at p < 0.05, ** indicates a significant difference at p < 0.01, *** indicates a significant difference at p < 0.001, and **** indicates a significant difference at p < 0.0001. Note that TET3 induction is not visible because of the scale.

Techniques: RNA Extraction, Reverse Transcription Polymerase Chain Reaction

Volcano plots for curcumin- and DMC-induced changes in 5hmC at the single-CpG level. U937 cells were treated with curcumin (5 µM, ( a )), DMC (1 µM, ( b )), and DMC (2 µM, ( c )) for 48 h, followed by oxidative bisulfite sequencing, as described in the methods. In the volcano plot, each black circle represents a CpG site with a nonsignificant change ( p < 0.05) in 5hmC, while the red circles represent a CpG site with a significant change at p < 0.05. Positive values on the x-axis indicate an increase in 5hmC relative to the control untreated samples, and negative values indicate a decrease in 5hmC relative to the control (active demethylation). The numbers at the top indicate the number of CpG sites showing an increase in DNA 5hmC (positive values on the x-axis) or a decrease in DNA 5hmC (negative values on the x-axis).

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Volcano plots for curcumin- and DMC-induced changes in 5hmC at the single-CpG level. U937 cells were treated with curcumin (5 µM, ( a )), DMC (1 µM, ( b )), and DMC (2 µM, ( c )) for 48 h, followed by oxidative bisulfite sequencing, as described in the methods. In the volcano plot, each black circle represents a CpG site with a nonsignificant change ( p < 0.05) in 5hmC, while the red circles represent a CpG site with a significant change at p < 0.05. Positive values on the x-axis indicate an increase in 5hmC relative to the control untreated samples, and negative values indicate a decrease in 5hmC relative to the control (active demethylation). The numbers at the top indicate the number of CpG sites showing an increase in DNA 5hmC (positive values on the x-axis) or a decrease in DNA 5hmC (negative values on the x-axis).

Techniques: Oxidative Bisulfite Sequencing, Control

Genomic distribution of the increase in 5hmC induced by curcumin and DMC in leukemia cells. U937 leukemia cells treated by curcumin (Cur) (5 μM) or DMC (1 μM) for 48 h, followed by single CpG analysis of 5hmC distribution using RRBS, as described in the methods. The upper panel shows the distribution of the 5hmC increase in promoters, exons, introns, and intergenic regions after treatment with Cur or DMC. The lower panel shows the distribution of 5hmC increase in CpG islands (CpGi), shores, and other regions after treatment with Cur or DMC.

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Genomic distribution of the increase in 5hmC induced by curcumin and DMC in leukemia cells. U937 leukemia cells treated by curcumin (Cur) (5 μM) or DMC (1 μM) for 48 h, followed by single CpG analysis of 5hmC distribution using RRBS, as described in the methods. The upper panel shows the distribution of the 5hmC increase in promoters, exons, introns, and intergenic regions after treatment with Cur or DMC. The lower panel shows the distribution of 5hmC increase in CpG islands (CpGi), shores, and other regions after treatment with Cur or DMC.

Techniques:

Volcano plots for curcumin- and DMC-induced changes in 5hmC at CpG islands and promoter regions. U937 cells were treated with curcumin (5 μM) and DMC (1 μM) for 48 h, followed by oxidative bisulfite sequencing, as described in the methods. For CpG island analysis, ( a , b ) represent curcumin and DMC treatment, respectively. For gene promoter analysis, ( c , d ) represent curcumin and DMC treatment, respectively. In the volcano plot, each black circle represents a CpG site with a nonsignificant change ( p < 0.05) in 5hmC, while the red circles represent a CpG site with a significant change at p < 0.05. Positive values on the x-axis indicate an increase in 5hmC relative to the control untreated samples, and negative values indicate a decrease in 5hmC relative to the control (active demethylation). The numbers at the top indicate the number of CpG sites showing an increase in DNA 5hmC (positive values on the s-axis) or a decrease in DNA 5hmC (negative values on the x-axis).

Journal: International Journal of Molecular Sciences

Article Title: Curcuminoids Activate TET Enzymes and Increase DNA Hydroxymethylation and Active Demethylation in Leukemia Cells

doi: 10.3390/ijms27010310

Figure Lengend Snippet: Volcano plots for curcumin- and DMC-induced changes in 5hmC at CpG islands and promoter regions. U937 cells were treated with curcumin (5 μM) and DMC (1 μM) for 48 h, followed by oxidative bisulfite sequencing, as described in the methods. For CpG island analysis, ( a , b ) represent curcumin and DMC treatment, respectively. For gene promoter analysis, ( c , d ) represent curcumin and DMC treatment, respectively. In the volcano plot, each black circle represents a CpG site with a nonsignificant change ( p < 0.05) in 5hmC, while the red circles represent a CpG site with a significant change at p < 0.05. Positive values on the x-axis indicate an increase in 5hmC relative to the control untreated samples, and negative values indicate a decrease in 5hmC relative to the control (active demethylation). The numbers at the top indicate the number of CpG sites showing an increase in DNA 5hmC (positive values on the s-axis) or a decrease in DNA 5hmC (negative values on the x-axis).

Techniques: Oxidative Bisulfite Sequencing, Control

( A ) First-generation Siglec-Fc. ( B ) V1 and V2 constructs of Siglec-Fc with a genetically encoded ST. ( C ) Schematic of site-specific conjugation of SC to DSPE-PEG-maleimide. ( D ) SDS-PAGE of SC and SC-PEG-DSPE. ( E ) Schematic of making SC-liposomes. ( F ) Binding of V1 and V2 WT Siglec-7– and R124A Siglec-7–liposomes to U937 cells. Data are represented as flow cytometry histograms and median fluorescent intensity (MFI) of Siglec-7 binding. ( G ) Binding of V2 WT Siglec-7 and R124A Siglec-7 in different densities on liposomes to U937 cells. Data are represented as flow cytometry histograms and MFI of Siglec-7 binding. Binding of ( H ) WT and R124A V2 Siglec-7, ( I ) WT and R116A V2 Siglec-1, and ( J ) WT and R120A V2 Siglec-2 precomplexed with Strep-Tactin (Strep.) and conjugated to liposomes (lipo.) to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way analysis of variance (ANOVA). **** P < 0.0001.

Journal: Science Advances

Article Title: An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

doi: 10.1126/sciadv.adz8096

Figure Lengend Snippet: ( A ) First-generation Siglec-Fc. ( B ) V1 and V2 constructs of Siglec-Fc with a genetically encoded ST. ( C ) Schematic of site-specific conjugation of SC to DSPE-PEG-maleimide. ( D ) SDS-PAGE of SC and SC-PEG-DSPE. ( E ) Schematic of making SC-liposomes. ( F ) Binding of V1 and V2 WT Siglec-7– and R124A Siglec-7–liposomes to U937 cells. Data are represented as flow cytometry histograms and median fluorescent intensity (MFI) of Siglec-7 binding. ( G ) Binding of V2 WT Siglec-7 and R124A Siglec-7 in different densities on liposomes to U937 cells. Data are represented as flow cytometry histograms and MFI of Siglec-7 binding. Binding of ( H ) WT and R124A V2 Siglec-7, ( I ) WT and R116A V2 Siglec-1, and ( J ) WT and R120A V2 Siglec-2 precomplexed with Strep-Tactin (Strep.) and conjugated to liposomes (lipo.) to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way analysis of variance (ANOVA). **** P < 0.0001.

Article Snippet: Human monocytic U937 cells (American Type Culture Collection) were grown in RPMI 1640 (Thermo Fisher Scientific), supplemented with 10% (v/v) heat-inactivated fetal bovine albumin, penicillin (100 U/ml), and streptomycin (100 μg/ml).

Techniques: Construct, Conjugation Assay, SDS Page, Liposomes, Binding Assay, Flow Cytometry

Binding of WT and arginine-mutated of ( A ) Siglec-7–, ( B ) Siglec-1–, ( C ) Siglec-2–, ( D ) Siglec-3–, ( E ) Siglec-9–, and ( F ) Siglec-10–liposomes expressed in CMAS +/+ and CMAS −/− CHO cells to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way ANOVA. **** P < 0.0001.

Journal: Science Advances

Article Title: An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

doi: 10.1126/sciadv.adz8096

Figure Lengend Snippet: Binding of WT and arginine-mutated of ( A ) Siglec-7–, ( B ) Siglec-1–, ( C ) Siglec-2–, ( D ) Siglec-3–, ( E ) Siglec-9–, and ( F ) Siglec-10–liposomes expressed in CMAS +/+ and CMAS −/− CHO cells to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way ANOVA. **** P < 0.0001.

Techniques: Binding Assay, Liposomes, Flow Cytometry

( A ) Schematic for multiplexing liposomes with different fluorophores. ( B ) Binding of single color and multiplexed of AF488–Siglec-1–liposome to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. ( C ) Binding of multiplexed and single color of AF488–Siglec-1–, AF555–Siglec-7–, and AF647–Siglec-2–liposomes in different combinations to U937 cells. Data are presented as a heatmap. Binding of ( D ) Siglec-2/Siglec-7 and ( E ) Siglec-1/Siglec-2 on the same liposomes to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technique replicates was calculated using an unpaired one-way ANOVA. ns, not significant; P > 0.05, **** P < 0.0001.

Journal: Science Advances

Article Title: An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

doi: 10.1126/sciadv.adz8096

Figure Lengend Snippet: ( A ) Schematic for multiplexing liposomes with different fluorophores. ( B ) Binding of single color and multiplexed of AF488–Siglec-1–liposome to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. ( C ) Binding of multiplexed and single color of AF488–Siglec-1–, AF555–Siglec-7–, and AF647–Siglec-2–liposomes in different combinations to U937 cells. Data are presented as a heatmap. Binding of ( D ) Siglec-2/Siglec-7 and ( E ) Siglec-1/Siglec-2 on the same liposomes to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technique replicates was calculated using an unpaired one-way ANOVA. ns, not significant; P > 0.05, **** P < 0.0001.

Techniques: Multiplexing, Liposomes, Binding Assay, Flow Cytometry

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