Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery

doi: 10.1186/s13046-019-1027-0

Figure Lengend Snippet: N-EV and H-EV treatment promote macrophage M2 polarization by delivering miR-21-5p that targets PTEN. a , western blot analysis of PTEN protein expression level in induced macrophages. H/i-miR-EV, monocytes were induced with the presence of EV secreted by miR-21-5p-inhibited, hypoxia pre-challenged MSCs; H-EV + i-miR, monocytes were transfected with miR-21-5p inhibitor-expressing vector before induction with the presence of H-EV. Macrophages induced without MSC-EV were used as negative control (NC). b, c , flow cytometry determining the percentage of CD163 + CD206 + cells among total CD68 + cells after induction. N-EV + O/E PTEN or H-EV + O/E PTEN, monocytes were transfected with PTEN overexpressing vector before N-EV or H-EV treatment, respectively. d – f , western blot detecting Akt and STAT3 protein expression as well as their activating phosphorylation (p-Ser473 for Akt and p-tyr705 for STAT3) in macrophages after induction. g – i , ELISA evaluating IL-10, TGF-β and VEGF-α in macrophage culture medium after induction. Macrophages induced with the presence of N-EV were used as negative control in b – i . Tukey’s test was used for statistical analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Article Snippet: Protein level of N-cadherin, E-cadherin, and Vimentin (NBP1–48309, NBP2–19051 and NBP1–31327, respectively, Novus Biologicals), CD9 and CD81 (NBP2–22187 and NB100–65805, Novus Biologicals) Arginase-1 and iNOS (P05089 and MAB9502, R&D Systems), PTEN (4C11A11, BioLegend, San Diego, USA), PDCD4 (NBP2–26138, Novus Biologicals, Littleton, USA), RECK (MA5–14781, Invitrogen), AKT (ab8805, Abcam, Cambridge, USA), STAT3 (NBP2–22471, Novus Biologicals), GAPDH (NB300–221, Novus Biologicals) as well as Ser473 phosphorylation of Akt protein (649,001, BioLegend) and Tyr705 phosphorylation of STAT3 (AF4607, R&D Systems) in cell lysate was analyzed by western blot.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Negative Control, Flow Cytometry, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Effects of Tofacitinib on Muscle Remodeling in Experimental Rheumatoid Sarcopenia

doi: 10.3390/ijms241713181

Figure Lengend Snippet: TOFA decreases STAT3 but not STAT1 signaling. ( A ) Densitometric analysis of pSTAT1 and pSTAT3 protein expression in gastrocnemius. ( B ) Densitometric analysis of SOCS1 and SOCS3 protein expression in gastrocnemius. Data are normalized to endogenous control (α-tubulin) and expressed as arbitrary units (A.U.). Representative cropped blots of two animals of each group are shown: healthy, e-RA, and e-RA+TOFA, respectively. Data are shown as the mean and SEM ( n = 7 rabbits per group). *** p < 0.001 vs. healthy. e-RA: experimental rheumatoid arthritis; TOFA: tofacitinib.

Article Snippet: After blocking in 3% skimmed milk (1 h, RT), membranes were incubated overnight at 4 °C with anti-myostatin/GDF8 antibody (0.4 μg/mL; R&D Systems, AF788, Minneapolis, MN, USA), pSTAT3 (2.5 μg/mL; R&D Systems, MAB4607), pSTAT1 (5 μg/mL; Affymetrix eBioscience, 14-9008, San Diego, CA, USA), SOCS1 (1 μg/mL; Abcam, ab9870, Cambridge, UK), SOCS3 (4 μg/mL; Abcam, ab14939), Pax7 (2.8 μg/mL, Developmental Studies Hybridoma Bank, AB 528428, Iowa City, IA, USA), Myogenin (2 μg/mL; Abcam, ab82843), and MyoD1 (1 μg/mL; Abcam, ab16148).

Techniques: Expressing, Control

Journal: Cancer Management and Research

Article Title: Long noncoding RNA PVT1 promotes hepatoblastoma cell proliferation through activating STAT3

doi: 10.2147/CMAR.S213707

Figure Lengend Snippet: PVT1 activates STAT3 and promotes cell cycle progression. ( A ) Western blotting analysis of p-STAT3 and STAT3 in HepG2 and HuH-6 cells stably expressing Lenti-vec or Lenti-PVT1. ( B ) Western blotting analysis of p-STAT3 and STAT3 in HepG2 and HuH-6 cells stably expressing shCtrl or PVT1 shRNA (shPVT1 #1 and shPVT1 #2). The representative images (top) and quantification of p-STAT3 level relative to STAT3 (bottom) are shown. ( C-D ) qRT-PCR analysis of mRNA levels of cyclin D1, myc, and cyclin B1 in HepG2 and HuH-6 cells shown as in ( A-B ). ( E-F ) The cell cycle distribution of HepG2 and HuH-6 cells shown as in ( A-B ) was evaluated by FACS analysis. Data are mean ± SD (n=3). ** P <0.01; * P <0.05 versus control. Abbreviations: STAT3, signal transducer and activator of transcription 3; FACS, fluorescence activated cell sorter.

Article Snippet: The primary antibodies against p-STAT3 (Tyr705) (mouse monoclonal, NBP2-24463) and STAT3 (mouse monoclonal, MAB1779) were purchased from Novus Biologicals.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Control, Fluorescence

Journal: Cancer Management and Research

Article Title: Long noncoding RNA PVT1 promotes hepatoblastoma cell proliferation through activating STAT3

doi: 10.2147/CMAR.S213707

Figure Lengend Snippet: PVT1 promotes hepatoblastoma cell proliferation through activating STAT3-mediated cell cycle progression. ( A-E ) HepG2 and HuH-6 cells stably expressing Lenti-vec or Lenti-PVT1 were treated with vehicle control or STAT3 inhibitor stattic. ( A ) The levels of p -STAT3 and STAT3 were determined by Western blotting analysis. The representative images (top) and quantification of p -STAT3 level relative to STAT3 (bottom) are shown. ( B ) The mRNA levels of cyclin D1, myc, and cyclin B1 were determined by qRT-PCR analysis. ( C ) The cell cycle distribution was evaluated by FACS analysis. #, P <0.01, Lenti-PVT1 versus Lenti-vec; &, P <0.01, Lenti-PVT1 plus stattic versus Lenti-PVT1 plus vehicle control. ( D-E ) The cell proliferation of HepG2 ( D ) and HuH-6 ( E ) cells was monitored by CCK-8 assay during the course of continuous cell culture. Data are mean ± SD (n=5). ** P <0.01; NS, not significant.

Article Snippet: The primary antibodies against p-STAT3 (Tyr705) (mouse monoclonal, NBP2-24463) and STAT3 (mouse monoclonal, MAB1779) were purchased from Novus Biologicals.

Techniques: Stable Transfection, Expressing, Control, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Cell Culture