human mm cell line u266  (ATCC)

Journal: Blood Research

Article Title: Hypermethylation-mediated silencing of RASD1 drives multiple myeloma pathogenesis

doi: 10.1007/s44313-026-00125-6

Figure Lengend Snippet: a MSP gel electrophoresis showing methylation (M) and unmethylation (U) status in representative samples. b RASD1 mRNA expression in U266 cells following DAC treatment. c Quantification of apoptosis rates (mean ± SD) in U266 cells (** P < 0.01). d Representative flow cytometry plots of apoptosis (Annexin V-YF647A/PI staining); Total apoptosis: 5.04% in the control group and 12.08% in the DAC-treated group

Article Snippet: The human MM cell line U266 was obtained from the American Type Culture Collection, and was maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum.

Techniques: Nucleic Acid Electrophoresis, Methylation, Expressing, Flow Cytometry, Staining, Control

Journal: Scientific reports

Article Title: Macrophage-derived pro-inflammatory cytokines augment the cytotoxicity of cytokine-induced killer cells by strengthening the NKG2D pathway in multiple myeloma.

doi: 10.1038/s41598-025-99289-x

Figure Lengend Snippet: Fig. 1. Pro-inflammatory cytokines upregulate MICA/B expression in MM. (A) Gating strategy for phenotype analysis. Debris and dead cells were sequentially excluded, and single cells were then gated for calculating the median fluorescence intensity (MFI) of targets. (B) Pro-inflammatory cytokines increased the surface expression of MICA/B in U266 cells, whereas only IL-6 acted on OPM2 and NCI-H929 cells. (C) In U266 cells, 50 ng/mL of single (left) or combined (right) cytokines exhibited the highest effect on MICA/B expression. In NCI-H929 cells, there was no significant difference among the IL-6 concentrations. Data are shown as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, calculated by one-way (B, C) or two-way (C) ANOVA, Bonferroni’s post-hoc test.

Article Snippet: Cell culture and CIK cells generation Human MM cell lines U266, OPM2, and NCI-H929, and human monocyte cell line THP-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO2.

Techniques: Expressing, Fluorescence

Journal: Scientific reports

Article Title: Macrophage-derived pro-inflammatory cytokines augment the cytotoxicity of cytokine-induced killer cells by strengthening the NKG2D pathway in multiple myeloma.

doi: 10.1038/s41598-025-99289-x

Figure Lengend Snippet: Fig. 2. Pro-inflammatory cytokines promote the cytotoxicity of CIK cells against MM. (A) Gating strategy for cytotoxicity analysis. Debris were excluded and FITC + tumor cells were then gated to exclude CIK cells (FITC-) in co-culture system. Live tumor cells were gated and analyzed. (B) Potential cytotoxic effects of cytokines on tumor cells were excluded by FCM and CCK-8 assays. (C) In U266 cells, all cytokines increased the cytotoxicity of CIK cells, whereas in NCI-H929 cells, only IL-6 and TNF-α enhanced the cytotoxic activity of CIK cells, but not IL-1β. (D) TNF-α upregulated NKG2D expression in tumor cells, whereas none of the cytokines affected PD-1 expression in CIK cells. All cytokines augmented PD-L1 expression in U266 cells, while only IL-6 acted on NCI-H929 cells. (E) PD-L1 blockade enhanced the CIK cells’ cytotoxicity against MM cells. Durvalumab, PD-L1 antibody, 10 µg/mL. Data are shown as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, calculated by one-way ANOVA, Bonferroni’s post-hoc test.

Article Snippet: Cell culture and CIK cells generation Human MM cell lines U266, OPM2, and NCI-H929, and human monocyte cell line THP-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO2.

Techniques: Co-Culture Assay, CCK-8 Assay, Activity Assay, Expressing

Journal: Scientific reports

Article Title: Macrophage-derived pro-inflammatory cytokines augment the cytotoxicity of cytokine-induced killer cells by strengthening the NKG2D pathway in multiple myeloma.

doi: 10.1038/s41598-025-99289-x

Figure Lengend Snippet: Fig. 3. Pro-inflammatory cytokines promote the transcription of MICA/B and PD-L1 genes in U266 cells. (A) Pro-inflammatory cytokines enhanced the transcription of MICA and PD-L1 genes while only TNF-α upregulated MICB transcription. (B) Cytokine treatments did not affect the levels of shed MICA and MICB. (C) The phosphorylation levels of AKT, p38 MAPK, and STAT3 increased upon the treatment of IL-1β, TNF-α, and IL-6, respectively, while ERK1/2, NF-κB, and JNK remained unchanged. (D) Inhibitors of PI3K, JAK, and MAPK completely reversed the upregulation of MICA/B and PD-L1 caused by cytokines. PI3Ki, LY-294,002, 10 µM; MMKi, Gossypetin, 60 µM; JAKi, JAK inhibitor I, 1 µM. (E) STAT3 and AKT were constitutively activated in tumor cells. (F) TNF-α activated p38 MAPK in CIK cells. (G) Inhibition of MKK abolished TNF-α-induced upregulation of NKG2D in CIK cells. Data are shown as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, calculated by one-way ANOVA, Bonferroni’s post-hoc test (A–D, and G) or student’s unpaired t test (E, F).

Article Snippet: Cell culture and CIK cells generation Human MM cell lines U266, OPM2, and NCI-H929, and human monocyte cell line THP-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO2.

Techniques: Phospho-proteomics, Inhibition

Journal: Scientific reports

Article Title: Macrophage-derived pro-inflammatory cytokines augment the cytotoxicity of cytokine-induced killer cells by strengthening the NKG2D pathway in multiple myeloma.

doi: 10.1038/s41598-025-99289-x

Figure Lengend Snippet: Fig. 4. Macrophage-derived pro-inflammatory cytokines upregulate the expression of MICA/B and the cytotoxicity of CIK cells. (A) CM from M1 macrophages upregulated MICA/B and PD-L1 expression in tumor cells, and the cytokine antibodies partially or completely eliminated this effect. (B) CM had no cytotoxic effect on tumor cells. (C) CM promoted the cytotoxicity of CIK cells against MM cells, while the use of cytokine antibodies (anti-IL-1β, anti-IL-6, and anti-TNF-α antibodies for U266 cells, anti-IL-6 antibody for NCI-H929 cells) partially abolished the cytotoxicity of CIK cells. (D) Blockade of PD-L1 enhanced the cytotoxicity of CIK cells. Durvalumab, PD-L1 antibody, 10 µg/mL. Data are shown as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, calculated by one-way ANOVA, Bonferroni’s post-hoc test (A, C, D) or student’s unpaired t test (B).

Article Snippet: Cell culture and CIK cells generation Human MM cell lines U266, OPM2, and NCI-H929, and human monocyte cell line THP-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO2.

Techniques: Derivative Assay, Expressing

Journal: Scientific reports

Article Title: Macrophage-derived pro-inflammatory cytokines augment the cytotoxicity of cytokine-induced killer cells by strengthening the NKG2D pathway in multiple myeloma.

doi: 10.1038/s41598-025-99289-x

Figure Lengend Snippet: Fig. 5. Illustrative diagram of macrophage-derived pro-inflammatory cytokines enhance the cytotoxicity of CIK cells. Macrophage-derived pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) target both tumor cells and CIK cells to strengthen the NKG2D-MICA/B axis and thus promoting the cytotoxicity of CIK cells against MM. In addition, pro-inflammatory cytokines also upregulate PD-L1 expression in tumor cells, and PD-L1 blockade therapy can restore this immunosuppression and enhance the cytotoxicity of CIK cells.

Article Snippet: Cell culture and CIK cells generation Human MM cell lines U266, OPM2, and NCI-H929, and human monocyte cell line THP-1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were maintained in RPMI 1640 medium (Pan-Biotech, Aidenbach, Germany) supplemented with 10% FBS (Gibco, Waltham, MA, USA) and 1% penicillin–streptomycin (Gibco) at 37 °C, 5% CO2.

Techniques: Derivative Assay, Expressing

Journal: Oncology Research

Article Title: ERRγ Promotes Multiple Myeloma Survival by Coordinating NF-κB Signaling and Mitochondrial Apoptosis Regulation

doi: 10.32604/or.2025.063700

Figure Lengend Snippet: Suppression of ERRγ reduces MM cell proliferation. (A) Western blot showing ERRγ protein levels across five MM cell lines (MM1.S, RPMI8226, OPM2, KMS11, and U266). (B) Validation of reduced ERRγ protein levels in RPMI8226 and OPM2 cells 48 h after siRNA transfection. (C) CCK-8 assay demonstrating decreased cell viability in RPMI8226 and OPM2 following ERRγ knockdown. (D) Viability assay of MM1.S and RPMI8226 cells treated with different doses of GSK5182 for 48 h, with IC50 values calculated using GraphPad Prism 8. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Human MM cell lines U266 and KMS11 were purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Biomarker Discovery, Transfection, CCK-8 Assay, Knockdown, Viability Assay