bmscs hs 5  (ATCC)

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: HuL001 reduces CAF-like differentiation, glycolytic activity, and secretion of IL-6 and VEGF in MM-educated BMSCs. ( A – C ) NOD.Cg-Prkdc scid Il2rg tm1Vst /Vst mice bearing KMS-11/BTZ xenografts were treated with vehicle or HuL001 (30 mg/kg, i.p., twice weekly) starting at ~100 mm 3 . FAP and HK2 were assessed in five representative tumors per group, with GAPDH as a loading control. Panels ( B , C ) show the quantified FAP and HK2 levels corresponding to panel ( A ). ( D ) Experimental design: HS-5 BMSCs (adherent culture) were directly co-cultured with KMS-11/BTZ cells (suspension culture) at a 1:2 ratio with or without either HuL001 (10 µg/mL) or human IgG1 (10 µg/mL) for 5 days. Subsequently, suspended KMS-11/BTZ cells were removed, and adherent MM-educated BMSCs (referred as CAFs) were collected for immunoblot analysis or secretion assays. ( E , F ) Representative immunoblots from one of three independent biological experiments. Panel ( E ) shows FAP and HK2, and panel ( F ) shows FSP1, PDGFRβ, and α-SMA. ( G , K ) Densitometric quantification of FAP and HK2 immunoblot signals corresponding to the experiments represented by panel ( E ), based on three independent biological experiments. ( H – J ) Densitometric quantification of FSP1, PDGFRβ, and α-SMA immunoblot signals corresponding to the experiments represented by panel ( F ), based on three independent biological experiments. Quantified protein levels were normalized to GAPDH, and relative expression levels were calculated by comparison with un-educated BMSCs, which were set to 1.0. ( L – N ) After removal of suspended KMS-11/BTZ cells, adherent BMSCs were incubated with fresh culture medium for 2 h, and the secretion of ( L ) lactate, ( M ) IL-6, and ( N ) VEGF was measured. The results were normalized to the cell number in each group. Data in panels ( G – N ) are presented as the mean ± standard deviation from at least three independent biological experiments. Statistical analyses were performed using two-sided unpaired Student’s t -test or one-way analysis of variance with Tukey’s post hoc test, as appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Activity Assay, Control, Cell Culture, Suspension, Western Blot, Expressing, Comparison, Incubation, Standard Deviation

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: HIF-1α contributes to glycolytic remodeling and modulates FAP expression in MM-educated BMSCs. HS-5 BMSCs were transfected with HIF-1α siRNA or control siRNA (scramble) for 3 days, and then co-cultured with KMS-11/bortezomib cells at a BMSC:MM ratio of 1:2. After removing the suspended MM cells, the adherent BMSCs were analyzed for ( A ) HIF-1α and HK2 at day 1 and ( B ) FAP at day 5, or ( F ) were incubated in fresh medium for 2 h to assess lactate secretion. Panels ( A , B ) show representative immunoblots from one of three independent biological experiments, with GAPDH used as a loading control. Panels ( C , D ) show densitometric quantification from the same experimental set represented in panel ( A ), based on three independent biological experiments. Panel ( E ) shows densitometric quantification from the same experimental set represented in panel ( B ), based on three independent biological experiments. The signals were normalized to GAPDH, and relative expression levels were calculated by comparison with un-educated BMSCs (MM−, scramble), which were set to 1.0. The results in panels ( C – F ) are presented as the mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance with Tukey’s post hoc test. * p < 0.05, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Expressing, Transfection, Control, Cell Culture, Incubation, Western Blot, Comparison, Standard Deviation

Journal: Cancers

Article Title: Extracellular Enolase-1 Promotes CAF-Associated Stromal Reprogramming via the Plasmin/TGF-β Axis in Multiple Myeloma

doi: 10.3390/cancers18091467

Figure Lengend Snippet: Extracellular ENO1 promotes cancer-associated fibroblast-like differentiation of BMSCs through the plasmin/TGF-β axis. ( A , G , J ) HS-5 BMSCs were cultured in 50% CM derived from KMS-11/bortezomib cells (MM CM) with or without ENO1 protein, TXA (10 mM) and the TGF-β receptor kinase inhibitor SB431542 (10 nM) for 1 day. ( B ) HS-5 BMSCs were transfected with hexokinase 2 small interfering RNA or scramble control, cultured in 50% MM CM for 3 days, and then treated with ENO1 protein for 1 additional day before immunoblot analysis. In panels ( A , B , G , J ), the left panels show representative immunoblots for FAP, whereas the right panels show densitometric quantification of FAP immunoblot signals from the same experimental sets. FAP signals were normalized to GAPDH, and relative expression levels were calculated by comparison with untreated cells, which were set to 1.0. Quantification in panels ( A , B , G ) was based on two independent biological experiments, whereas quantification in panel ( J ) was based on three independent biological experiments. Data in the right panels of ( A , B , G , J ) are presented as the mean ± standard deviation. ( C – F , I ) HS-5 BMSCs were cultured in 50% MM CM with or without ENO1 protein, HuL001 (100 µg/mL), hIgG1 (100 µg/mL), and TXA (10 mM) for 2 days. Supernatants were collected to measure ( C ) lactate, ( D ) interleukin-6, ( E ) vascular endothelial growth factor and ( F , I ) active transforming growth factor-β1. The results were normalized to the cell number in each group. ( H ) HS-5 BMSCs were cultured in 50% MM CM with or without ENO1 protein, HuL001 (100 µg/mL), and hIgG1 (100 µg/mL), followed by measurement of plasminogen receptor activity. Data in panels ( C – F , H , I ) are presented as the mean ± standard deviation from three independent biological experiments. Statistical analyses were performed using one-way analysis of variance with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. Original Western blot images corresponding to the blots shown in this figure are provided in .

Article Snippet: The immortalized human BMSCs HS-5 (cat. no. CRL-3611) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were authenticated using STR profile analysis at ATCC.

Techniques: Cell Culture, Derivative Assay, Transfection, Small Interfering RNA, Control, Western Blot, Expressing, Comparison, Standard Deviation, Activity Assay