20 ms pulses  (Bio-Rad)

Journal: American Journal of Stem Cells

Article Title: TGF-B1-over-expressed adipose stem cells-derived secretome exhibits CD44 suppressor and anti-cancer properties via antagonistic effects against SMAD4 in breast cancer cells

doi:

Figure Lengend Snippet: TGF-B1 transfected AD-MSCs and transfection efficiency. As a result of measuring the absorbance values at 560/750 nm wavelength with MTT, the IC50 dose that killed half of the cells on the 7th day, which is half the selection time, was determined as the minimum inhibitory concentration. Considering the absorbance and proliferation curves, it was found that the 6.25 µg/ml hygromycin dose was the appropriate dose for AD-MSCs in terms of minimum inhibitory concentration (A, B). Transfected cells are seen under an inverted light microscope and fluorescence microscope after GFP plasmid transfection with different parameters for transfection optimization. At 1100 v, 40 ms, and 1 pulse electroporation parameters, it was observed that there was GFP expression in more cells and at higher intensities compared to transfections made with other parameters (C). Microscope objective: 10x, Light microscope images scale bar: 100 µm, fluorescence microscope images scale bar: 200 µm. Culture images of AD-MSCs without hygromycin application just before TGF-B1 gene transfection and 24 hours and 48 hours after transfection. Microscope objective: 10x, scale bar: 200 µm (D). Viability analysis of AD-MSCs before transfection and at 0, 24, and 48 hours after transfection (E). Measurement of TGF-B1 protein expression by flow cytometry and western blot analysis before and 24 and 48 hours after transfection to determine transfection efficiency (F, G). ***: P<0.001. All experiments were performed in triplicate.

Article Snippet: Transfection was performed by 1100 v, 20 ms, and 1 pulse electroporation parameters (Invitrogen, USA).

Techniques: Transfection, Selection, Concentration Assay, Light Microscopy, Fluorescence, Microscopy, Plasmid Preparation, Electroporation, Expressing, Flow Cytometry, Western Blot

Journal: Nature Metabolism

Article Title: Impaired phosphocreatine metabolism in white adipocytes promotes inflammation

doi: 10.1038/s42255-022-00525-9

Figure Lengend Snippet: a . Representative immunofluorescence microphotographs of adipocyte progenitors stained with Hoechst and antibodies targeting DPP4. Scale bar=200 μm. b-e . In vitro differentiated human adipocytes were transfected with scrambled non-silencing (si C ) or CKB -targeting (si CKB ) (three-four replicates/condition). Effects on gene expression of adipogenic/thermogenic markers (experiment repeated twice) (B), adiponectin secretion (five replicates/condition) (C), cellular triglyceride levels (four replicates/condition) (D) and lipid droplet morphology (from experiments in panel D) (E) were determined. Scale bar in panel E = 10 μm. Data in bar charts are shown as mean ± SEM. f . PCr and Cr levels in in vitro differentiated human adipocytes transfected with scrambled non-silencing (si C ) or CKMT2 -targeting (si CKMT2 ) oligonucleotides (n = 5 replicates/group). Data are shown as mean ± SEM. *p = 0.024 for PCr and 0.045 for PCr/Cr, respectively by Student’s two-sided t-test. g . Gene set enrichment analysis (GSEA) of genes correlating with CKB expression in human WAT and regulated by si CKB transfection in human in vitro differentiated adipocytes. Hallmark gene sets from MsigDB were used to calculate the enrichment. Details on the analyses are found in the Methods section. The data are presented as dot plots. The color of the dot indicates the normalized enrichment score (NES) for each pathway. The size of the dot represents the negative log 10 adjusted p-value for the enrichment of each pathway. h . CKB and CCL2 mRNA levels in in vitro differentiated human adipocytes, derived from a female donor, transfected with si C ) or si CKB (four replicates/condition). Data are shown as mean ± SEM. *p = 0.024, ****p < 0.0001 by Student’s two-sided t-test. i . Same experiment as in panel H but displaying CCL2 secretion (four replicates/condition). Data are shown as mean ± SEM.****p < 0.0001 by Student’s two-sided t-test. Abbreviations: Adj. p-val.=adjusted p-value, Cr=creatine, NES = normalized enrichment score, PCr=phosphocreatine, WAT = white adipose tissue.

Article Snippet: Short interfering oligonucleotides (siRNAs) were introduced by electroporation using a Neon Transfection System (1,300 V, 20 ms, 2 pulses) 100 µl Kit (Invitrogen) in human in vitro differentiated adipocytes at day eight of differentiation.

Techniques: Immunofluorescence, Staining, In Vitro, Transfection, Expressing, Derivative Assay

Journal: Nature Metabolism

Article Title: Impaired phosphocreatine metabolism in white adipocytes promotes inflammation

doi: 10.1038/s42255-022-00525-9

Figure Lengend Snippet: a . Maximal respiration of in vitro differentiated human adipocytes determined by Seahorse Mito stress analysis using 1-2 μmol/L of FCCP, after transfection of the cells with si C or si CKB (12 replicates/condition). Data are shown as mean ± SEM. ****p < 0.0001 by Student’s two-sided t-test. b . ATP/ADP levels in in vitro differentiated human adipocytes transfected with si C or si CKB after 1 hour of incubation with 1.5 μmol/L of FCCP (eight replicates per condition). Data are shown as mean ± SEM. ****p < 0.0001 by Student’s two-sided t-test. c . Quantification of Mitotracker TM red fluorescence intensity in in vitro differentiated human adipocytes transfected with si C or si CKB (four replicates/condition, repeated twice). Data are shown as mean ± SEM. d-e . In vitro differentiated human adipocytes were transfected with si C or si CKB (two replicates per blot). Protein levels of OXPHOS components (D) and TOM20 (mean values) (E) were determined together with CK-B and GAPDH by western blot to estimate mitochondrial abundance. f . Representative immunofluorescence microphotographs of in vitro differentiated adipocytes transfected with si C or si CKB and stained with antibodies targeting TOM20 and Hoechst. Scale bar=50 μm. Abbreviations: ATP5A = Mitochondrial membrane ATP Synthase, AU = arbitrary units, FCCP = Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, C + = positive control, mitochondrial extract from rat heart supplied with the antibody as a positive control, MTCO1 = Mitochondrially Encoded Cytochrome C Oxidase I, NDUFB8 = NADH:Ubiquinone Oxidoreductase Subunit B8, SDHB = Succinate dehydrogenase B, UQCRC2 = Cytochrome b-c1 complex subunit 2, mitochondrial.

Article Snippet: Short interfering oligonucleotides (siRNAs) were introduced by electroporation using a Neon Transfection System (1,300 V, 20 ms, 2 pulses) 100 µl Kit (Invitrogen) in human in vitro differentiated adipocytes at day eight of differentiation.

Techniques: In Vitro, Transfection, Incubation, Fluorescence, Western Blot, Immunofluorescence, Staining, Positive Control