Journal: Journal of Translational Medicine

Article Title: Comparing atmospheric and hypoxic cultured mesenchymal stem cell transcriptome: implication for stem cell therapies targeting intervertebral discs

doi: 10.1186/s12967-018-1601-9

Figure Lengend Snippet: hBMMSCs cultured in atmospheric and hypoxic conditions display similar identity. Sample-matched hBMMSCs cultured in atmospheric or hypoxic conditions for 3 passages from the time of isolation were tested for the expression of surface markers characteristic of MSCs (CD90, CD105, CD73 and CD49b) and the absence of expression of hematopoietic markers (CD34 and CD45) by flow cytometry. a , b Representative dot plots of atmospheric cultured ( a ) and hypoxic cultured ( b ) hBMMSCs are presented. Positive for CD90, CD105, CD73 and CD49b whereas quadrant Q3-1 display cells that are double negative for CD34 and CD45. c , d Quantification of the percentage of cells that are CD34/CD45 double negative ( c ) and CD90/CD105 double positive ( d ) in hBMMSCs cultured in atmospheric and hypoxic conditions. Data represent mean ± SD, n = 3 independent sample-matched human BMMSCs. Two-tailed paired Student’s t-test: non-significant (NS) p value > 0.05

Article Snippet: Currently, BioRestorative Therapies has been authorized to commence (by the FDA) a Phase II clinical trial using hypoxic cultured bone marrow-derived mesenchymal stem cells to treat chronic lower back pain.

Techniques: Cell Culture, Isolation, Expressing, Flow Cytometry, Two Tailed Test

Journal: Biochemical Journal

Article Title: The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

doi: 10.1042/BCJ20190647

Figure Lengend Snippet: ( A ) CHO-LDM-V5 cells were treated with 10 µg/ml cycloheximide (CHX), 20 µg/ml cholesterol/CD (Chol) or 1 µg/ml 25-hydroxycholesterol (25HC) for 8 h. ( B ) CHO-LDM-V5 cells were treated in 5% (v/v) LPDS or NBS based media with or without statin (5 µM compactin and 50 µM mevalonate) for 16 h. ( C ) HeLaT and HepG2 cells were exposed to 21% (normoxic) or 2% (hypoxic) oxygen conditions for 4 h; the lysates were obtained from Novus Biologicals. ( D ) CHO-LDM-V5 cells were treated with or without 500 µM DPTA NONOate (DPTA) for the indicated time. Protein levels were analysed by Western blotting with V5, endogenous LDM and vinculin antibodies. Except in ( C ), data are presented as mean ± SEM from at least three independent experiments ( A n = 3, B n = 5, D n = 4), where ** P < 0.01. Relative protein levels were measured using ImageStudio Lite (version 5.2) and normalised to the vehicle (Veh) or control condition in each blot, which was set to 1 ( A–C ) or 100% at each time point ( D ).

Article Snippet: Normoxic and hypoxic lysates from HeLaT and HepG2 cells were obtained from Novus Biologicals.

Techniques: Western Blot, Control

Journal: Biochemical Journal

Article Title: The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6

doi: 10.1042/BCJ20190647

Figure Lengend Snippet: ( A ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM of the indicated siRNA (M6: MARCH6), then mRNA levels were measured using qRT-PCR and normalised to the housekeeping gene PBGD . mRNA levels were normalised to the control condition which was set to 1. Data are presented as mean + half range from an experiment performed in triplicate. ( B ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM of the indicated siRNA (M6: MARCH6). ( C ) CHO-LDM-V5 cells were transfected for 24 h with 25 nM control or MARCH6 siRNA, then treated with or without 500 µM DPTA NONOate (DPTA) for 2 h. ( D ) HepG2 cells were transfected for 24 h with 25 nM control or MARCH6 siRNA, then treated with or without 10 µg/ml cycloheximide (CHX) for 8 h. ( E ) CHO-LSS-myc, CHO-LDM-V5, CHO-EBP-V5 or CHO-DHCR24-V5 cells were transfected for 24 h with 25 nM control or MARCH6 (M6) siRNA. Bands for the ectopic cholesterol synthesis enzymes migrated close to the expected size; LSS-myc: 83 kDa, LDM-V5: 57 kDa, EBP-V5: 26 kDa, DHCR24-V5: 60 kDa. Protein levels were analysed by Western blotting with V5, myc, endogenous SM, endogenous LDM and vinculin antibodies. Data are presented as mean + SEM from at least three independent experiments ( B n = 3–7, C n = 6, D n = 3, E n = 3–6), where * P < 0.05 and ** P < 0.01. Relative protein levels were measured using ImageStudio Lite (version 5.2) and normalised to the control condition which was set to 1.

Article Snippet: Normoxic and hypoxic lysates from HeLaT and HepG2 cells were obtained from Novus Biologicals.

Techniques: Transfection, Quantitative RT-PCR, Control, Western Blot