Figure 1

<p> <i>Mu</i> insertion into <i>EXPB1</i> and its effect on Zea m 1 content of pollen. (a) Cartoon showing the structure of <i>EXPB1</i> and location of the <i>Mu</i> insertion (exons denoted with boxes). Also indicated are the locations of primers used for PCR screening. (b) <i>Mu</i> is inserted near the intron border flanking the fourth exon. (c) Portion of a 2-D gel image of wild type (<i>EXPB1</i>) pollen protein showing the Zea m 1 isoforms, which were identified by immunoblotting. (d) Relative amount of total Zea m 1 protein extracted from pollen of <i>EXPB1/EXPB1</i> and <i>expb1/expb1</i> plants. (Mean±SE; N = 2; t = 9.15; p = 0.035).</p

Figure 2

<p>Pollen viability and pollen performance <i>in vitro</i> and <i>in vivo</i>. (a) Percentage of viable pollen, based on staining with thiazolyl blue (mean±SE, N = 20–22 plants). (b) Micrograph of pollen stained with thiazolyl blue. Viable pollen stained dark purple. (c) Pollen tube growth <i>in vitro</i> (mean±SE, N = 20–22). Bars with different letters of the alphabet differ significantly using Tukey pairwise comparisons with the overall probability adjusted for multiple comparisons.</p

Figure 4

<p>Pollen tube from <i>EXPB1</i> pollen growing through ovary tissue for 22 h after pollination. Ovaries were stained with 0.1% aniline blue for 30 min and then examined under a fluorescence microscope.</p

Figure 3

<p>Transmission rate of <i>expb1</i> as a function of the size of the pollen load from <i>EXPB1/expb1</i> plants (mean±SE, N = 4).</p

MOESM5 of Scutellarin regulates microglia-mediated TNC1 astrocytic reaction and astrogliosis in cerebral ischemia in the adult rats

Additional file 5: Showing Notch-1, NICD, HES-1, Nestin, TNF-α, IL-1β and iNOS expression (red) in GFAP (green) reactive astrocytes at 21 d after MCAO (M) and after scutellarin treatment (M + S). Note that the expression is diminished in MCAO but remained more intense with scutellarin treatment. Scale bars: 50 µm. DAPI-blue

MOESM2 of Scutellarin regulates microglia-mediated TNC1 astrocytic reaction and astrogliosis in cerebral ischemia in the adult rats

Additional file 2: Scutellarin enhanced IL-1β (A1, A2, A3) and iNOS (B1, B2, B3) expression in TNC1 via BV-2-conditioned medium. In TNC1 astrocytes treated with CM, moderate expression of IL-1β and iNOS was detected (A1, B1). The expression was noticeably increased in CM + L (A2, B2) and further enhanced upon incubation with CM + SL for 24 h (A3, B3) with long cytoplasmic processes with expansions projected by TNC1 astrocytes (A3, B3). Scale bars: 20 μm

β-Adrenergic Agonist and Antagonist Regulation of Autophagy in HepG2 Cells, Primary Mouse Hepatocytes, and Mouse Liver

<div><p>Autophagy recently has been shown to be involved in normal hepatic function and in pathological conditions such as non-alcoholic fatty liver disease. Adrenergic signalling also is an important regulator of hepatic metabolism and function. However, currently little is known about the potential role of adrenergic signaling on hepatic autophagy, and whether the β-adrenergic receptor itself may be a key regulator of autophagy. To address these issues, we investigated the actions of the β₂-adrenergic receptor agonist, clenbuterol on hepatic autophagy. Surprisingly, we found that clenbuterol stimulated autophagy and autophagic flux in hepatoma cells, primary hepatocytes and <i>in vivo</i>. Similar effects also were observed with epinephrine treatment. Interestingly, propranolol caused a late block in autophagy in the absence and presence of clenbuterol, both in cell culture and <i>in vivo</i>. Thus, our results demonstrate that the β₂- adrenergic receptor is a key regulator of hepatic autophagy, and that the β-blocker propranolol can independently induce a late block in autophagy.</p></div

Clenbuterol increases autophagic flux <i>in vivo</i>.

<p><b>A–B.</b>) Clenbuterol increases autophagosomal marker LC3-II in the liver of mice treated for 3 days. A further increase is seen with co-administration of chloroquine, indicating an increase in autophagic flux. Asterisk represents significance vs. ctrl, hash represents significance vs. CQ, and ampersand represents significance vs. Clen as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) SQSTM1/P62 levels in the livers of the same mice. Asterisk indicates p<0.05. <b>E–F.</b>) Electron micrographs of the same mice, showing isolation membranes, autophagosomes, and autolysosomes. A significant increase in the number of autophagic vesicles per cell was observed. Asterisk represents p<0.05. Bar = 2 mm.</p

Clenbuterol increases autophagic flux in HepG2 cells and mouse primary hepatocytes.

<p><b>A–B.</b>) Co-treatment of HepG2 with Clenbuterol and Chloroquine shows a greater accumulation of LC3-II compared to control cells. Asterisk represents significance vs. Clen/CQ, and hash represents significance vs. CQ as per Tukey's post-hoc test following one-way ANOVA. <b>C–D.</b>) Clenbuterol increases autolysosomes in HepG2 cells transiently transfected with GFP-RFP-LC3 plasmid. Image taken at 40× magnification. <b>E.</b>) Clenbuterol increases lysosomal acidification (orange/red structures) in HepG2 stained with Acridine Orange. Image taken at 20× magnification. <b>F–G.</b>) Clenbuterol induces SQSTM1/p62 degradation in HepG2 cells. <b>H–I.</b>) Clenbuterol induces SQSTM1/p62 degradation in mouse primary hepatocytes. Error bars represent SEM. Unless otherwise noted, asterisk represents p<0.05 relative to control using Student's T-test.</p

Propranolol inhibits autophagic flux in mouse primary hepatocytes and <i>in vivo</i>.

<p>Propranolol induces accumulation of both LC3-II and p62 in mouse primary hepatocytes (<b>A,B</b>), and in mouse liver (<b>C,D</b>). Asterisk represents p<0.05 as per Student's t-test with respect to control. For all parts, error bars represent SEM.</p