Journal: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a LPA level of KAS-6/1 and U266 cells treated with or without PC. Mean± SD, n = 3 biological replicates, data represent two independent experiments. b The correlation heatmap between Lachnospiraceae abundance or blood PC and PC levels and the rate of GZMB + CD8 T cells in MM patients. c Sb9 mRNA level in KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. d Sb9 protein level in KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. e Representative images of the flow cytometric analysis and quantification of the rate of GZMB + CD8 T cells in CD8 T cells co-cultured with WT or Sb9 KO KAS-6/1 and U266 cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. f Released GZMB level in CD8 T cells co-cultured with WT or Sb9 KO KAS-6/1 and U266 cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. g Representative images of the flow cytometric analysis and quantification of the rate of RFP-labeled WT or Sb9 KO KAS-6/1 and U266 cells co-cultured with CD8 T cells plus PC or LPA treatment. Mean± SD, n = 3 biological replicates, data represent two independent experiments. Sb9 SerpinB9, GZMB granzyme B, KO knockout, PC phosphatidylcholine, LPA lysophosphatidic acid, WT wild type. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post hoc Tukey’s test except for ( b ). Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01; ** * * P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The level of PC derived from KAS-6/1 and U266 cells was identified by PC Colorimetric Assay Kit (E-BC-K796-M, Elabscience, Wuhan, Hubei, China) according to the manufacturer’s instruction.

Techniques: Cell Culture, Labeling, Knock-Out

Journal: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a Sb9 pre-mRNA level in KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. b Levels of junctions of exon-intron in Sb9 pre-mRNA detected by qRT-PCR in KAS-6/1 and U266 cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments c Fragments of exon 2-intron 2-exon 3 or exon 2-exon 3 detected by touchdown PCR in KAS-6/1 and U266 cells. d The GGAGA motif was found in exon 2 and intron 2 of Sb9 pre-mRNA. e Quantification of Sb9 pre-mRNA by qRT-PCR in KAS-6/1 and U266 cells treated with or without PC and LPA following RIP using LIN28A and LIN28B antibodies. Mean± SD, n = 3 biological replicates, data represent two independent experiments. f Sb9 mRNA level in KAS-6/1 and U266 cells transfected with or without LIN28A and LIN28B shRNAs. Mean± SD, n = 3 biological replicates, data represent two independent experiments. g Splicing analysis of Sb9 minigene and indicated deletion mutation of the GGAGA motif in KAS-6/1 and U266 cells. h Splicing of Sb9 pre-mRNA was analyzed by a relative luciferase reporter activity assay in scrambled and LIN28A/B shRNAs-transfected KAS-6/1 and U266 cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. Sb9 SerpinB9, PC phosphatidylcholine, LPA lysophosphatidic acid, E exon, I intron, NC negative control, RIP RNA immunoprecipitation. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey’s test for a , b , e , and f , which was performed utilizing unpaired Student’s t test for h . Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01; ** *P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The level of PC derived from KAS-6/1 and U266 cells was identified by PC Colorimetric Assay Kit (E-BC-K796-M, Elabscience, Wuhan, Hubei, China) according to the manufacturer’s instruction.

Techniques: Quantitative RT-PCR, Touchdown PCR, Transfection, Mutagenesis, Luciferase, Activity Assay, Negative Control, RNA Immunoprecipitation

Journal: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a Level of exosomal Sb9 derived from KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. b Representative images of the flow cytometric analysis for CD8 T cells cultured with PKH67-labeled exosomes (green) derived from WT or Sb9 KO KAS-6/1 and U266 cells treated with or without PC and LPA. c Sb9 protein levels in CD8 T cells cultured with PKH67-labeled exosomes (green) derived from WT or Sb9 KO KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. d GZMB mRNA levels in CD8 T cells cultured with exosomes derived from WT or Sb9 KO KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. e Released GZMB level in CD8 T cells cultured with exosomes derived from WT or Sb9 KO KAS-6/1 and U266 cells treated with or without PC and LPA. Mean± SD, n = 3 biological replicates, data represent two independent experiments. f Representative images of the flow cytometric analysis and quantification of the rate of RFP-labeled KAS-6/1 and U266 cells co-cultured with WT or Sb9-overexpressed CD8 T cells. Mean± SD, n = 3 biological replicates, data represent two independent experiments. Sb9 SerpinB9, PC phosphatidylcholine, LPA lysophosphatidic acid, GZMB granzyme B, KO knockout, OE overexpression, WT wild type. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey’s test except for ( b ). Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01; ** *P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The level of PC derived from KAS-6/1 and U266 cells was identified by PC Colorimetric Assay Kit (E-BC-K796-M, Elabscience, Wuhan, Hubei, China) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Cell Culture, Labeling, Knock-Out, Over Expression

Journal: Nature Communications

Article Title: Microbiota-reprogrammed phosphatidylcholine inactivates cytotoxic CD8 T cells through UFMylation via exosomal SerpinB9 in multiple myeloma

doi: 10.1038/s41467-025-57966-5

Figure Lengend Snippet: a Representative image of tumors developing from bioluminescent WT or Sb9 KO KAS-6/1 and U266 cells in NOD-SCID mice co-injected with PC and WT or Sb9-overexpressed human CD8 T cells at days 9 and 25 postinjection of MM cells. b Quantification of the volume of xenograft tumors derived from WT or Sb9 KO KAS-6/1 and U266 cells in the presence and absence of PC and WT or Sb9-overexpressed human CD8 T cells in NOD-SCID mice. Mean± SD, n = 5 biologically independent mice. c Images of xenograft tumors derived from WT or Sb9 KO KAS-6/1 and U266 cells in the presence and absence of PC and WT or Sb9-overexpressed human CD8 T cells in NOD-SCID mice. Histograms indicate the quantification of the weight of tumors. Mean± SD, n = 5 biologically independent mice. Sb9 SerpinB9, MM multiple myeloma, WT wild type, PC phosphatidylcholine, KO knockout, OE overexpression. All statistical tests were two-sided. Statistical significance was assessed using one-way ANOVA followed by post-hoc Tukey’s test for ( b ) and ( c ). Adjustments were made for multiple comparisons. * P < 0.05; ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: The level of PC derived from KAS-6/1 and U266 cells was identified by PC Colorimetric Assay Kit (E-BC-K796-M, Elabscience, Wuhan, Hubei, China) according to the manufacturer’s instruction.

Techniques: Injection, Derivative Assay, Knock-Out, Over Expression

Journal: Cancers

Article Title: Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines

doi: 10.3390/cancers15082199

Figure Lengend Snippet: Drug-library screening in multiple myeloma cell lines identifies EHMT2 inhibition as synthetic lethal to the proteasome inhibitor carfilzomib. Experimental design of the drug-library screening. Primary screening was performed on U266 PIR cell line. The top 40 candidates were selected using excess over Bliss (EOB) value (cut-off ≥ 0.4) and subjected to a secondary screening that was performed on U266 PIR , U266, KMM-1, AMO-1, and KMS28-BM cell lines. The EHMT2 inhibitor UNC0642 ranked among the top ten candidates, being synergistic with CFZ in five out of five cell lines at two or more different concentrations (Created by BioRender.com accessed on 26 February 2023).

Article Snippet: Human multiple myeloma (MM) cell lines KMM-1, U266, OPM-2, KMS28-BM, U266 PIR , and KMM1 PIR were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB), National Institutes of Biomedical Innovation, Health and Nutrition, Ibakari, Osaka, Japan; the Leibniz Institute-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany; or generated in our lab and authenticated by DNA fingerprinting using the GenePrint system (Promega, Madison, WI, USA).

Techniques: Drug discovery, Inhibition

Journal: Cancers

Article Title: Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines

doi: 10.3390/cancers15082199

Figure Lengend Snippet: EHMT2 inhibition synergically increases proteasome inhibitor-mediated cell death in PI-resistant and PI-sensitive multiple myeloma cell lines. ( a ) Heatmap representing cell viability after single and combinatorial treatment in U266 PIR -, KMM1 PIR -, AMO1 PIR -, RPMI PIRBTZ -, and RPMI PIRCFZ -resistant cell lines and U266-, KMM-1-, KMS28-BM-, AMO-1-, RPMI-8226-, and OPM-2-sensitive cell lines. Analysis was performed 72 h post-treatment with FACS or the CellTiterGlo assay ( n = 3). ( b – d ) SynergyFinder analysis of UNC0642/CFZ dose–response matrixes. Percentage of inhibition was retrieved with the CellTiter Glo assay 72 h post-treatment and normalized to the luminescent signal measured at day 0. Synergy score was calculated with Bliss.

Article Snippet: Human multiple myeloma (MM) cell lines KMM-1, U266, OPM-2, KMS28-BM, U266 PIR , and KMM1 PIR were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB), National Institutes of Biomedical Innovation, Health and Nutrition, Ibakari, Osaka, Japan; the Leibniz Institute-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany; or generated in our lab and authenticated by DNA fingerprinting using the GenePrint system (Promega, Madison, WI, USA).

Techniques: Inhibition, Glo Assay

Journal: Oncotarget

Article Title: Glutaminase inhibition in multiple myeloma induces apoptosis via MYC degradation

doi: 10.18632/oncotarget.20691

Figure Lengend Snippet: (a) Western Blot analysis of MYC and selected candidates of the glutamine metabolism in untreated myeloma cell lines. All MYC-expressing cell lines (INA-6, RPMI-8226, OPM-2, MM.1S) show marked protein expression of glutaminase (GAC) and the substrate transporters (ASCT2, LAT1). U266 cells lack MYC protein expression along with the weakest levels of glutaminase isoform GAC and low of ASCT2 expression. (b) Growth-inhibitory effect targeting glutaminase. Enzyme inhibition was performed in a 2-fold serial dilution and measured after 5 days of incubation by MTT-assay using the small molecules 968 (n=3), its inactive control compound 335 (n=2) and BPTES (n=3). INA-6 cells are most susceptible to 968-treatment, whereas U266 cells show the weakest response to 10 μM 968 in relation to DMSO-control. BPTES reduced cell proliferation only moderately with a maximum of 40% independent of the treated cell line. (c) Comparison of INA-6 and U266 cell proliferation during 10 μM 968 treatment for 4 days analyzed by MTT. U266 proliferation was reduced by 50 % compared to DMSO-control on day four, while 968-treated INA-6 cells show no proliferation at all. (d) Treatment of INA-6 and U266 cells with 5μM 968 for 3 days reduced the percentage of viable Annexin V-FITC/PI negative cells in INA-6 by 50% whereas U266 cells showed no signs of apoptosis (n=3). Western Blot analysis on day 1 and 2 after 968 treatment confirms induction of apoptosis in INA-6 cells by PARP-1 cleavage (cl. PARP-1 indicates cleaved PARP-1 fragment). (e) Primary MM and peripheral blood mononuclear cells (PBMCs) were treated with 10μM 968 or DMSO as control for 72h and the percentage of viable cells (Annexin V-FITC negative, PI negative) compared to the DMSO treated sample were quantified using Annexin V-FITC and PI staining.

Article Snippet: Quantitative transcriptome analysis using random-primed cDNA library from endogenous cell lines INA-6, U266 and the generated MYC-positive U266-CDS-MYC cells (GATC Biotech).

Techniques: Western Blot, Expressing, Enzyme Inhibition Assay, Serial Dilution, Incubation, MTT Assay, Control, Comparison, Staining

Journal: Oncotarget

Article Title: Glutaminase inhibition in multiple myeloma induces apoptosis via MYC degradation

doi: 10.18632/oncotarget.20691

Figure Lengend Snippet: (a) Western Blot shows protein expression in U266 cells before and after lentiviral MYC-transduction (U266/U266-MYC) compared to endogenous expression levels in INA-6. MYC expression significantly elevates GAC and ASCT2 protein expression in U266 cells. (b) Oligomycin and 2-deoxyglucose (2-DG) treatment of INA-6, U266 and U266-MYC. Comparison of ATP-generation from glycolysis (2-DG incubation) and oxidative phosphorylation (oligomycin treatment) revealed that INA-6 cells synthesize ATP via glycolysis independent of oxidative phosphorylation as 2-DG reduces measurable ATP. In contrast, U266 produce ATP via mitochondrial respiration, which can be blocked using the ATP-synthase inhibitor oligomycin. INA-6 and U266 cells use contrary mechanisms to support ATP generation. U266-MYC cells are more sensitive to glycolysis inhibition and less sensitive to inhibition of the oxidative phosphorylation pathway (n=3). (c) Western Blot of MM cell lines incubated with or without glucose and L-glutamine in the culture medium for 24 hours. All HMCLs tested showed minimal changes in both MYC expression and generation of cleaved active caspase 3 when glucose is removed from the media. Removal of glutamine however diminishes MYC expression and increases cleaved active caspase 3 levels. For U266-MYC both withdrawal conditions reduce MYC protein while caspase 3 is cleaved.

Article Snippet: Quantitative transcriptome analysis using random-primed cDNA library from endogenous cell lines INA-6, U266 and the generated MYC-positive U266-CDS-MYC cells (GATC Biotech).

Techniques: Western Blot, Expressing, Transduction, Comparison, Incubation, Phospho-proteomics, Inhibition