Sequences of PCR and real-time PCR primers.

<p>Sequences of PCR and real-time PCR primers.</p

Hallmark function assays of mature hepatocytes.

<p><i>(</i><b><i>A</i></b><i>)</i> Periodic Acid-Schiff staining assay in hepatogenic differentiated cells. A1:HuMSCs; A2: HuMSCs were cultured in hepatocyte differentiation medium for 21 days; A3: HuMSCs were transfected with HNF4α plasmid and cultured until day 21. <i>(</i><b><i>B</i></b><i>)</i> Expression of MHC (HLA-DR and HLA-DQ) assay by flow cytometry analysis. B1,B4:HuMSCs;B2,B5: HuMSCs were cultured in hepatocyte differentiation medium for 21 days transfected with PCDNA3.1 alone;B3,B6: HuMSCs were transfected with HNF4α plasmid and cultured until day 21. <i>(</i><b><i>C, D</i></b><i>)</i> Albumin levels in hepatogenic transfected with HNF4α differentiated cells in comparison to cells cultured in differentiation medium for 21 days transfected with PCDNA3.1 alone by enzyme linked immunosorbent assay (P<0.05) and Flow cytometry analysis (D1: control group D2:HNF4α group).</p

Mechanism of HNF4α-mediated improved hepatic differentiation.

<p><i>(</i><b><i>A</i></b><i>)</i> Detection of liver-enriched transcription factors in induced and HNF4α-transfected HuMSCs by RT-PCR. 0 d: HuMSCs before induction; DM 9 d and 21 d: Cells cultured in hepatic differentiation medium for nine days and 21 days, respectively; HNF4α 12 d and 21 d: Cells transfected with HNF4α nine days after induction (gene expression was determined three and 12 days later, respectively). <i>(</i><b><i>B</i></b><i>)</i> Wnt/β-catenin signaling-related genes were examined by real-time RT-PCR. All of the data are presented as the means ± SD (n = 3), and the fold induction for each gene by HNF4α overexpression was significant (P<0.05).</p

In vitro hepatic differentiation of HuMSCs and HNF4α transfection.

<p><i>(</i><b><i>A</i></b><i>)</i> Morphological changes observed as undifferentiated HuMSCs differentiated into hepatocytes under hepatogenic conditions. A1: Undifferentiated HuMSCs; A2: 1 week postinduction. A3: 2 weeks postinduction. A4–A5: 3 weeks postinduction. Original magnification, ×100 (A1–A4), ×200 (A5). <i>(</i><b><i>B</i></b><i>)</i> Immunocytochemical analysis of hepatocyte-specific marker expression. HuMSCs were cultured in hepatocyte differentiation medium for 21 days (B1, B3) or in growth medium (negative controls; B2, B4). The cells were stained with mouse antibodies against the hepatocyte-specific markers ALB (B1, B2) and AFP (B3, B4) and incubated with Dylight594-conjugated anti-mouse IgG. The cell nuclei were counterstained with DAPI. Original magnification, ×400 (B1–B4). <i>(</i><b><i>C</i></b><i>)</i> Overexpression of HNF4α was detected by immunofluorescence two days after transfection. C1: Green light could be detected from the nuclei of the transfected HNF4α group; C2: Control group transfected with PCDNA3.1. Original magnification, ×400 (C1, C2) <i>(</i><b><i>D</i></b><i>)</i> Overexpression of HNF4α was detected by RT-PCR two days after transfection.</p

Additional file 1 of Ae index is an independent predictor of kidney stone recurrence in overweight and obese patients

Additional file 1: Table S1. Univariate analysis of patient data of overweight and obesity stone recurrence

Effects of MSK1 gene silencing and Thr-581 mutation on cytokine production in LPS-treated astrocytes.

<p><b>A</b>. Effects of siRNA for MSK1 and non-specific siRNA on LPS-induced expression of MSK1, p-MSK1 (Thr581) and iNOS were detected by Western blotting. <b>B</b>. The bar chart shows the ratio of total MSK1 and p-MSK1 to β-actin. <b>C–E</b>. ELISA showed that MSK1 gene silencing by siRNA further promoted the LPS-mediated upregulation of inflammatory cytokines. <b>F</b>. Western blot analysis showed the effect of mutation of Thr-581 to an alanine residue on LPS-induced expression of p-MSK1 Thr-581, p-MSK1 Ser-360, and total MSK1. <b>G</b>. The bar chart shows the ratio of p-MSK1 Thr-581 and p-MSK1 Ser-360 to total MSK1. <b>H–J</b>. ELISA showed the effect of mutation of Thr-581 on LPS-induced TNFα, IL-6, and IL1-β production in activated astrocytes.</p

Expression profile of MSK1 and p-MSK1 (Thr-581 and Ser-360) following LPS intracerebral injection.

<p><b>A</b>. Protein levels of t-MSK1, p-MSK1 Thr-581, p-MSK1 Ser-360 were detected before (control) and after injury. GAPDH was also detected by Western blotting. <b>B</b>. Quantification graphs (relative optical density) of the intensity of staining of p-MSK1 (Thr-581) and total MSK1 to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. <b>C–H</b>. Immunofluorescence staining of MSK1 and p-MSk1 (Thr581) was performed to assess the staining changes for MSK1 and p-MSK1 immunoreactivity in the cortex at day 1 after LPS-injection. <b>I</b>. Negative control. * and ^# indicate significant differences at P<0.05, compared with normal brain cortex. Scale bars: 40 µm (C–F), 20 µm (G–J).</p

Immunolocalization of MSK1 and p-MSK1 (Thr-581) with different cellular markers in cerebral cortex by double immunofluorescence staining.

<p>In the adult rat brain cortex, within 5(red, <b>A</b> and <b>E</b>) and p-MSK1 (Thr581) (red, <b>I</b> and <b>M</b>) and different cell markers (green, <b>B, F, J, N</b>), such as a neuronal marker (NeuN) and an astrocyte marker (GFAP). The yellow color in the merged images represents colocalization of MSK1 or p-MSK1 (Thr581) with different phenotype-specific markers (<b>C, G, K, O</b>). Colocalization of MSK1 and p-MSK1 (Thr581) with different phenotype-specific markers in the normal group are shown in the brain cortex (<b>D, H, L, P</b>). Quantitative analysis of different phenotype-specific marker-positive cells expressing MSK1 (<b>Q</b>) and p-MSK1 (<b>R</b>) (%) in the unit area (mm²) in the normal group and 1 day after injury. *indicates significant difference at P<0.05, compared with the normal group. Error bars indicate SEM. Scale bars: 20 µm (<b>A–P</b>).</p