Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Complement inhibition decreases early fibrogenic events in the lung of septic baboons

doi: 10.1111/jcmm.12667

Figure Lengend Snippet: Immunolocalization and semi-quantitative analysis of fluorescence intensity in lung tissue stained for proteins contributing to fibrosis via transforming growth factor-beta (TGF-β) signalling. (A) Log ratio representation of qPCR analysis of TGF-B1, SMAD2 and CTGF transcripts in Escherichia coli versus healthy controls and animals treated with compstatin at T0 or T+5, versus untreated septic animals. Expression values were normalized by housekeeping gene β-actin. Data are shown as mean ± S.E.M., n = 3; one-way anova with Dunnett's multicomparison test. ** P < 0.01, *** P < 0.001 as compared to E. coli versus Control ratio. (B) Immunostaining for TGF-β, phospho-Smad2/Smad3, and CTGF (columns) in the lungs of septic baboons ( E. coli ) versus compstatin-treated baboons at T+5 ( E. coli + CS T+5) (rows). Magnification: bars, 50 μm. (C) Histogram representations of mean fluorescence intensity (MFI) of images collected for the above-mentioned proteins as detailed in Figure . Data are shown as means ± S.E.M.; one-way anova with Dunnett's multicomparison test. *** P < 0.001 as compared to the E. coli group. (D) Phospho-antibody array analysis. MFI of the antibody spots are presented as means ± S.E.M. of six replicates per experimental condition with unpaired two-tailed t -tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The expression profile of profibrotic signalling proteins was analysed with Jak/Stat Pathway Phospho and TGF-β signalling antibody arrays (U.S. Biomax, Inc.).

Techniques: Fluorescence, Staining, Expressing, Immunostaining, Ab Array, Two Tailed Test