Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: Identification of constructed TCF4−/+ U266 and MMSA−1−/+ U266 stable cell lines and MMSA-1 mRNA and protein levels in TCF4−/+ U266 cells 1: U266 cells; 2: TCF4− U266-NC; 3. TCF4− U266 cells; 4: TCF4+ U266-NC; 5. TCF4+ U266 cells

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques: Construct, Stable Transfection

Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: Dual-Luciferase Report Assay results in different U266 cells

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques: Luciferase

Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: MMSA-1 and RAS co-localization and interaction in U266 cells 1: U266 cells; 2: MMSA−1− U266-NC; 3. MMSA−1− U266 cells; 4: MMSA−1+ U266-NC; 5. MMSA−1+ U266 cells

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques:

Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: RAS downstream related molecule expressions in different U266 cells 1: U266; 2: MMSA−1− U266-NC; 3. MMSA−1− U266; 4: MMSA−1+ U266-NC; 5. MMSA−1 + U266

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques:

Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: MMSA-1’s effect on U266 cells proliferation, migration and apoptosis A showed cloning formation assay result; B showed transwell assay result; C showed apoptosis related proteins expression and D showed mitochondrial apoptosis in different U266 cells. 1: U266; 2: U266 + CCCP-1; 3. MMSA−1− U266-NC; 4. MMSA−1− U266-NC + CCCP-1; 5. MMSA−1− U266; 6: MMSA−1− U266 + CCCP-1; 7. MMSA−1+ U266-NC; 8. MMSA−1+ U266-NC + CCCP-1; 9. MMSA−1 + U266; 10. MMSA−1 + U266 + CCCP-1

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques: Migration, Cloning, Tube Formation Assay, Transwell Assay, Expressing

Journal: Annals of Hematology

Article Title: MMSA-1 is regulated by Wnt/TCF4 and involved in multiple myeloma progression and invasion via RAS/RAF signaling pathway

doi: 10.1007/s00277-026-06740-8

Figure Lengend Snippet: Adhesion molecules and angiogenesis factors levels in different U266 cells A showed qRT-PCR result and B showed Western Blot result of adhesion molecules; C showed angiogenesis factors levels. 1: U266 cells, 2: MMSA−1− U266-NC; 3. MMSA−1− U266; 4. MMSA−1+ U266-NC; 5. MMSA−1+ U266

Article Snippet: Human multiple myeloma cell line U266 and 293 T were purchased from Wuhan Procell Life Science&Technology Co., Ltd, both of which have been identified by Short Tandem Repeat (STR).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Molecules

Article Title: Src Mediates Epigallocatechin-3- O -Gallate-Elicited Acid Sphingomyelinase Activation

doi: 10.3390/molecules25225481

Figure Lengend Snippet: Pharmacological inhibition of Src attenuated the cell death inducing effect of EGCG. ( A ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and SKI1 (2.5 μM, from 1 h before EGCG treatment) for 72 h. Cell viability was measured using the trypan blue method ( n = 4). ( B ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and SKI1 (2.5 μM; from 1 h before EGCG treatment) for 1 h. Akt activity was evaluated using the K-LISA kit ( n = 4). ( C ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and SKI1 (2.5 μM; from 1 h before EGCG treatment) for 3 h. cGMP levels were evaluated using competitive immunoassay ( n = 4). ( D ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and SKI1 (2.5 μM; from 1 h before EGCG treatment) for 3 h and ASM activity was evaluated ( n = 3).

Article Snippet: Human multiple myeloma cell line U266 cells were maintained in the Roswell Park Memorial Institute (RPMI) 1640 medium (Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma Aldrich, St Louis, MO, USA) and antibiotics (Meiji, Tokyo, Japan) in a humidified incubator with 5% CO 2 at 37 °C.

Techniques: Inhibition, Activity Assay

Journal: Molecules

Article Title: Src Mediates Epigallocatechin-3- O -Gallate-Elicited Acid Sphingomyelinase Activation

doi: 10.3390/molecules25225481

Figure Lengend Snippet: EGCG induced phosphorylation of Tyr 416 in multiple myeloma cells. ( A ) U266 human multiple myeloma cells were treated with EGCG (10 μM) for 30 min. Phosphorylation levels of Src were evaluated using Western blotting ( n = 3). ( B ) U266 human multiple myeloma cells were pretreated with the anti-67LR antibody or with the isotype control antibody and treated with EGCG (10 μM) for 30 min. Src phosphorylation levels were evaluated using Western blotting ( n = 4).

Article Snippet: Human multiple myeloma cell line U266 cells were maintained in the Roswell Park Memorial Institute (RPMI) 1640 medium (Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma Aldrich, St Louis, MO, USA) and antibiotics (Meiji, Tokyo, Japan) in a humidified incubator with 5% CO 2 at 37 °C.

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Molecules

Article Title: Src Mediates Epigallocatechin-3- O -Gallate-Elicited Acid Sphingomyelinase Activation

doi: 10.3390/molecules25225481

Figure Lengend Snippet: Pharmacological inhibition of FAK attenuated the Akt/ASM axis elicited by EGCG. ( A ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and PF-573228 (1 μM; from 1 h before EGCG treatment) for 3 h ( n = 3). ( B ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and PF-573228 (1 μM; from 1 h before EGCG treatment). Akt activity was evaluated using the K-LISA kit ( n = 3). ( C ) U266 human multiple myeloma cells were treated with EGCG (10 μM) and PF-573228 (1 μM; 1 h before EGCG treatment) for 30 min. Src phosphorylation levels were evaluated using western blotting ( n = 4). Data are presented as means ± SEM.

Article Snippet: Human multiple myeloma cell line U266 cells were maintained in the Roswell Park Memorial Institute (RPMI) 1640 medium (Fujifilm, Tokyo, Japan) supplemented with 10% fetal bovine serum (Sigma Aldrich, St Louis, MO, USA) and antibiotics (Meiji, Tokyo, Japan) in a humidified incubator with 5% CO 2 at 37 °C.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Western Blot

Journal: Oncotarget

Article Title: Terpenes from essential oils and hydrolate of Teucrium alopecurus triggered apoptotic events dependent on caspases activation and PARP cleavage in human colon cancer cells through decreased protein expressions

doi: 10.18632/oncotarget.25955

Figure Lengend Snippet: ( A ) Chemical structures of the major compounds of Hydrophobic (TA-1) (i) and Hydrophilic (TA-2) (ii) fractions of Teucrium alopecurus ; ( B ) Phenolic content (i) and antiradical potential of TA-1 and TA-2 using DPPH (ii) and FRAP (iii) assay; ( C ) Antibacterial activity of hydrophobic fraction of Teucrium alopecurus . Plant essential oil (1 μl) were applied per disc (Whatman No 5 mm) onto the seeded top layer of the agar plates containing tested bacteria ( E.coli , S.aureus , B.subtilis and K.pneumoniae ). Essential oil was tested with a Chloramphenicol, and Ertapenem discs as positive controls. DMSO discs were used as negative controls. The plates were incubated at 37°C for 24 h, and zone of inhibition was determined. ( D ) Effect of TA-1 and TA-2 on RAW cell viability. RAW 264.7 cells, seeded in triplicate in 96-well plates, were pre-treated with the concentrations of TA-1 (0.5, 1, 5, 10 pg/mL) (i) and TA-2 (30, 50, 70, 100 μg/mL) (ii) and incubated for 24 h, 48 h and 72 h. Cell viability was analyzed with the MTT assay. ( E ) Effect of TA-1 and TA-2 on tumor cell proliferation. KBM-5, HCT-116, Panc-28, MCF-7, U266, and SCC4 cells (5 × 10 3 cells/well) were seeded in triplicate in 96-well plates, treated with different concentrations of TA-1 (0.5, 1, 5, 10 pg/mL) and TA-2 (30, 50, 70, 100 μg/mL); cell growth was analyzed on days 0, 1, 3, and 5 by tetrazolium salt 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay. The results show the mean ± SD values. These are representative results of three independent experiments.

Article Snippet: The cell lines HCT-116, MiaPaca-2 (Human pancreatic carcinoma), Panc28, KBM-5, SCC4 (human head and neck cancer), MCF7, U266 (human multiple myeloma), HL-60 (human promyelocytic leukemia), RAW 264.7 (mouse monocyte macrophage) and HUVECs were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Bacteria, Incubation, Inhibition, MTT Assay

Journal: Oncotarget

Article Title: Terpenes from essential oils and hydrolate of Teucrium alopecurus triggered apoptotic events dependent on caspases activation and PARP cleavage in human colon cancer cells through decreased protein expressions

doi: 10.18632/oncotarget.25955

Figure Lengend Snippet: TA-1 induces PARP cleavage and caspases activation and suppresses the expression of gene products involved in tumor cell survival, proliferation, and metastasis in ( A ) Panc28- and MIA PACA-2 cells, ( B ) KBM5 and U266 cells, ( C ) and HL60 cells. The cells were treated with the concentrations of TA-1 indicated for 24 h. Whole cell protein extracts were prepared, separated by electrophoresis, and then transferred to the nitrocellulose membrane using the antibodies indicated. These are representative results of three independent experiments.

Article Snippet: The cell lines HCT-116, MiaPaca-2 (Human pancreatic carcinoma), Panc28, KBM-5, SCC4 (human head and neck cancer), MCF7, U266 (human multiple myeloma), HL-60 (human promyelocytic leukemia), RAW 264.7 (mouse monocyte macrophage) and HUVECs were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activation Assay, Expressing, Electrophoresis, Membrane

Journal: Nature Communications

Article Title: Regulation of bone homeostasis by MERTK and TYRO3

doi: 10.1038/s41467-022-33938-x

Figure Lengend Snippet: a – k After intraosseous femoral injection of U266 myeloma cell line, NSG mice were treated with vehicle or 60 mg/kg R992 BID. a Assessment of Igλ light chain paraprotein in the peripheral blood by ELISA (week 3 n = 8/6, week 5 n = 5/4, and week 8 n = 5/5). b Bone marrow CD138 + myeloma plasma cell infiltration in the contralateral leg by FACS ( n = 5/4). c μCT analysis of bone loss by calculating the change in bone volume (BV/TV) in relation to healthy control mice ( n = 7/8). d 3D reconstructions of microcomputed tomography (μCT) recordings of the metaphyseal proximal region of the femur of healthy and myeloma-bearing mice. e Representative pictures of TRAP Hematoxylin staining (osteoclasts stained in red) showing myeloma infiltration in the bone marrow (#1), osteoblasts visible as cuboidal or polygonal mononuclear cells on the endosteal bone surface (arrows in green) (#2), osteolysis (#3), histologically detectable signs of bone repair in osteolysis zones induced by R992 (arrows in green pointing to osteoblasts, arrows in red pointing to osteoclasts) (#4), and detection of TRAP-positive mononuclear cells indicating osteoclast precursor cell recruitment (#5) (arrows in yellow) ( n = 5). f Histomorphometric analysis of osteoblast number per bone perimeter ( n = 7/8). g Determination of serum bone formation marker P1NP by ELISA ( n = 8/8). h Histomorphometric analysis of osteoclast number per bone perimeter. i Determination of serum osteoclast marker TRAP5b by ELISA ( n = 8/8). j Histomorphometric analysis of the number of TRAP + mononuclear cells in the bone marrow ( n = 7/8). k Kaplan–Meier curve showing overall survival ( n = 13/13, p = 0.030 Mantel–Cox test). Median survival was 73 vs. 88 days. Data were means ± SEM. Statistical significance was determined by a two-tailed unpaired t -test unless otherwise stated.

Article Snippet: U266 (obtained from DSMZ, no. ACC 9), RPMI8226 (obtained from DSMZ, no. ACC 402), H460 (the gift from Klaus Pantel), and MDA-MB-231 (the gift from Klaus Pantel) cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Tomography, Staining, Marker, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

doi: 10.1074/jbc.m110.158378

Figure Lengend Snippet: FIGURE 1. -T3 inhibits constitutively active STAT3 in U266 cells. A, chemical structure of -T3. B, -T3 suppresses phospho-STAT3 levels in a dose-dependent manner. U266 cells (2 106/ml) were treated with the indicated concentrations of -T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of protein were resolved on 7.5% SDS-polyacrylamide gel, electrotransferred onto nitrocellulose membrane, and probed for phospho-STAT3. C, -T3 suppresses phospho-STAT3 levels in a time-dependent manner. U266 cells (2 106/ml) were treated with the 60 M -T3 for the indicated time points, after which Western blotting was done as described above. D, -T3 suppresses STAT3 DNA binding in a dose-dependent manner. U266 cells (2 106/ml) were treated with the indicated concentrations of -T3 for 6 h and analyzed for nuclear STAT3 levels by EMSA. E, U266 cells (2 106/ml) were treated with 60 M -T3 for the indicated durations and analyzed for nuclear STAT3 levels by EMSA. F, nuclear extracts from U266 cells were incubated with STAT3 antibody and an unlabeled STAT3 oligonucleotide probe. Nuclear extracts from myeloid leukemia (KBM-5) cells were taken alone. They were then assayed for STAT3 DNA binding by electrophoretic mobility shift assay. G, -T3 inhibits translocationofSTAT3tothenucleus.U266cells(1105/ml)wereincubatedwithorwithout60M-T3for6h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. The same slides were counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. H, U266 cells (2 106/ml) were treated with the indicated concentrations of -T3 for 6 h and analyzed for nuclear STAT3 levels by Western blot.

Article Snippet: Cell Lines—We obtained the human multiple myeloma (MM) cell lines U266, MM.1R, and MM.1S (dexamethasone-sensitive) and MIA PaCa-2, PC3, DU-145 cells, from the American Type Culture Collection (Manassas, VA).

Techniques: Membrane, Western Blot, Binding Assay, Incubation, Electrophoretic Mobility Shift Assay, Immunocytochemistry

Journal: Journal of Biological Chemistry

Article Title: γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

doi: 10.1074/jbc.m110.158378

Figure Lengend Snippet: FIGURE 2. -T3 down-regulates IL-6-induced phospho-STAT3. A, -T3 sup- presses phospho-STAT3 levels in various cells. DU-145, PC3, and MiaPaCa-2 cells (1106/ml)weretreatedwith60M-T3for6h,afterwhichwhole-cellextracts were prepared, and protein was resolved on 7.5% SDS-polyacrylamide gel, elec- trotransferred onto nitrocellulose membrane, and probed for phospho-STAT3. B and C, -TP has no effect on activation of STAT3. U266 cells (2 106/ml) were treated with the indicated concentrations of either -TP or -T3 for 6 h, after which the whole-cell extracts were prepared, and phospho-STAT3 levels were detected by Western blot. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. D, -T3 suppresses IL-6-induced phospho-STAT3 levels in a dose-dependent manner. MM.1s cells (2 106) were treated with IL-6 (10 ng/ml) for the indicated times. Whole-cell extracts were prepared, and phospho-STAT3 level was detected by Western blot. E, -T3 sup- pressesIL-6-inducedphospho-STAT3levelsinatime-dependentmanner.MM.1s cells (2 106) were treated with 60 M -T3 for the indicated times and then stimulated with IL-6 (10 ng/ml) for 30 min. Whole-cell extracts were then pre- pared and analyzed for phospho-STAT3 by Western blotting.

Article Snippet: Cell Lines—We obtained the human multiple myeloma (MM) cell lines U266, MM.1R, and MM.1S (dexamethasone-sensitive) and MIA PaCa-2, PC3, DU-145 cells, from the American Type Culture Collection (Manassas, VA).

Techniques: Membrane, Activation Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

doi: 10.1074/jbc.m110.158378

Figure Lengend Snippet: FIGURE 3. -T3 down-regulates constitutively active Src, Jak1, and Jak2. A, -T3 suppresses phospho-Src levels in a dose-dependent manner. U266 cells (2 106/ml) were treated with indicated doses of -T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose mem- branes, and probed with phospho-Src antibody. The same blots were stripped and reprobed with Src antibody to verify equal protein loading. B, -T3 suppresses phospho-JAK1 expression in a dose-dependent manner. U266 cells (2 106/ml) were treated with indicated doses of -T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 7.5% SDS-PAGE, electrotransferred onto nitrocellulose mem- branes, and probed with phospho-JAK1. C, -T3 suppresses phospho-JAK2 expression in a dose-dependent manner. U266 cells (2 106/ml) were treated with indicated doses of -T3 for 6 h, after which whole-cell extracts were prepared, and 40 g of those extracts were resolved on 7.5% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with phos- pho-JAK2. The same blots were stripped and reprobed with respective non- phosphorylated protein antibodies to verify equal protein loading.

Article Snippet: Cell Lines—We obtained the human multiple myeloma (MM) cell lines U266, MM.1R, and MM.1S (dexamethasone-sensitive) and MIA PaCa-2, PC3, DU-145 cells, from the American Type Culture Collection (Manassas, VA).

Techniques: SDS Page, Expressing

Journal: Journal of Biological Chemistry

Article Title: γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

doi: 10.1074/jbc.m110.158378

Figure Lengend Snippet: FIGURE 4. -T3 regulates STAT3 through induction of SHP-1. A, pervana- date reverses the phospho-STAT3 inhibitory effect of -T3. U266 cells (2 106/ml) were treated with pervanadate (50 M) and 60 M -T3 for 4 h, after which Western blotting was done as described above. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. B, -T3 induces the expression of SHP-1 protein in U266 cells. U266 cells (2 106/ml) were treated with indicated concentrations of -T3 for 6 h, after which whole-cell extracts were prepared, and 40-g portions of those extracts were resolved on 10% SDS-PAGE, electrotrans- ferred onto nitrocellulose membranes, and probed with SHP-1 antibody. The same blots were stripped and reprobed with -actin antibody to ver- ify equal protein loading. C, effect of SHP-1 knockdown on -T3-induced expression of SHP-1. SCC4 cells (1 105/ml) were transfected with either scrambled or SHP-1-specific siRNA (50 nM). After 48 h, cells were treated with 60 M -T3 for 6 h, and whole-cell extracts were subjected to Western blot analysis for SHP-1, p-STAT3, and STAT3. D, effect of SHP-1 knockdown on the expression levels of constitutively active JAK1, JAK2, and Src. SCC4 cells (1 105/ml) were transfected with either scrambled or SHP-1-spe- cific siRNA (50 nM). After 48 h, cells were treated with 60 M -T3 for 6 h, and whole-cell extracts were subjected to Western blot analysis. E, knock- down of SHP-1 inhibits the apoptotic effect of -T3. SCC4 cells (1 105/ml) were transfected with either scrambled or SHP-1-specific siRNA (50 nM). After 48 h, cells were treated with 25 M -T3 for 24 h, and the percentage of apoptosis was analyzed by the Live/Dead assay. F, human pancreatic tissues from animals treated with vehicle and -T3 (400 mg/kg body weight) for 4 weeks were homogenized and analyzed by Western blot for p-STAT3, STAT3, and SHP-1 expression levels as indicated under “Experi- mental Procedures.” The blots were stripped nd reprobed with -actin antibody to verify equal protein loading. *, p 0.01.

Article Snippet: Cell Lines—We obtained the human multiple myeloma (MM) cell lines U266, MM.1R, and MM.1S (dexamethasone-sensitive) and MIA PaCa-2, PC3, DU-145 cells, from the American Type Culture Collection (Manassas, VA).

Techniques: Western Blot, Expressing, SDS Page, Knockdown, Transfection, Live Dead Assay

Journal: Journal of Biological Chemistry

Article Title: γ-Tocotrienol but Not γ-Tocopherol Blocks STAT3 Cell Signaling Pathway through Induction of Protein-tyrosine Phosphatase SHP-1 and Sensitizes Tumor Cells to Chemotherapeutic Agents

doi: 10.1074/jbc.m110.158378

Figure Lengend Snippet: FIGURE 5. -T3 suppresses STAT3-regulated antiapoptotic gene products, induces apoptosis, and potentiates chemotherapeutic agents. A, -T3 suppresses STAT3-regulated antiapoptotic gene products. U266 cells (2 106/ml) were treated with 25 M -T3 for the indicated time intervals, after which whole-cell extracts were prepared, and 40-g portions of those extracts were resolved on 10% SDS-PAGE; the membrane was sliced according to molecular weight, and the gel was probed using antibodies against cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF. The same blots were stripped and reprobed with -actin antibody to verify equal protein loading. B, -T3 induces caspase-3-dependent PARP cleavage. U266 cells were treated with 60 M -T3 for the indicated times, and whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blotting against caspase-3 antibody and PARP antibody. The same blots were stripped and reprobed with -actin antibody to show equal protein loading. C, -T3 causes significant accumulation of cells in the sub-G1 phase. U266 cells (2 106/ml) were synchronized by incubation overnight in the absence of serum and then treated with 25 M -T3 for the indicated times, after which the cells were washed, fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. D, effects of -T3 on the proliferation of multiple myeloma cells. MM cells were plated in triplicate, treated with the indicated concentrations of -T3 for the indicated days, and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. E, -T3 potentiates the apoptotic effect of thalidomide (Thal) and Velcade (Val). U266 cells (1 106/ml) were treated with 25 M -T3 and 10 ng/ml thalidomide or 20 nM bortezomib alone or in combination for 24 h at 37 °C. Cells were stained with a Live/Dead assay reagent for 30 min, then analyzed under a fluores- cencemicroscope,and20randomfieldswerecounted.C,Control;T,Thalidomide;V,Velcade;*,p0.01;**;p 0.05 versus control.

Article Snippet: Cell Lines—We obtained the human multiple myeloma (MM) cell lines U266, MM.1R, and MM.1S (dexamethasone-sensitive) and MIA PaCa-2, PC3, DU-145 cells, from the American Type Culture Collection (Manassas, VA).

Techniques: SDS Page, Membrane, Molecular Weight, Western Blot, Incubation, Staining, Flow Cytometry, MTT Assay, Live Dead Assay, Control