Additional file 1: of Antiviral and virucidal activities of Duabanga grandiflora leaf extract against Pseudorabies virus in vitro

Supplementary document DMSO Cytotoxicity assay. (JPG 40 kb

Additional file 1: Figure S1. of Combination of VP3 and CD147-knockdown enhance apoptosis and tumor growth delay index in colorectal tumor allograft

Morphological and histological features of CT26 tumor. A) Exposed CT26-induced tumor becoming a spheroid measuring 2–2.5 cm in diameter at day 25 post-treatment. The tumor mass is well-defined and vascularized. Right panel, cross section of the tumor. B) Photomicrographs of H&E-stained tumor sections. The tumor was dissected at day 25 post-treatment for histological analysis. Top, peripheral region showing intact tumor cells, and, bottom, inner region showing tumor cells and a necrotic core. 100× magnification. C) TUNEL assay were evaluated on CT26 tumor sections. Apoptotic cells indicate by TUNEL-positive are stained dark brown while viable cells stained green color. Top, peripheral region showing intact tumor cells and apoptotic cells, and, bottom, inner region showing apoptotic cells, viable tumor cells and a necrotic core. 100× magnification. (TIF 636 kb) (TIF 635 kb

DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN.

<p>MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.</p

Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment.

<p>(A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p<0.001, ns: non-significant compared to the normal control.</p

Levels of MDM2 and p53 proteins expressed in HT-29 cells.

<p>(A) Level of proteins in cells treated with (a) crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) 25 μM (7.1 μg/ mL) of FKB at different time interval. (c) 25 μM (6.75 μg/mL) of APN at different time interval. (B) Level of MDM2 and p53 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT 29 cells treated with (a) Hexane and chloroform extracts (b) FKB, and (c) APN. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; **p<0.01; ***p<0.01; ns: non-significant compared to the DMSO control. DC: DMSO used as negative control at a final concentration of 0.1%.</p

Extraction and isolation of ethyl acetate extract of <i>Dillenia suffruticosa</i>.

<p>Sequential solvent extraction using solvents with increasing polarity (hexane< dichloromethaneD. <i>suffruticosa</i>. The extract obtained was subjected to isolation using column chromatography and thin layer chromatography by using different solvent systems.</p

List of genes with their respective primers for GeXP multiplex analysis.

<p>Forward universal primer sequence (5’-AGGTGACACTATAGAATA-3’); Reverse universal primer sequence (3’-GTACGACTCACTATAGGG-5’).</p><p>List of genes with their respective primers for GeXP multiplex analysis.</p

Expression level of the apoptotic-related genes in MCF-7 cells treated with EADs as determined by GeXP analysis.

<p>EADs upregulated the expression of <i>Bax</i> and <i>p21</i> and downregulated the expression of <i>Bcl-2</i> and <i>caspase-9</i>. The expression of genes was normalized against beta actin and compared to the control. The data are represented as relative expression of genes in bars±SD of at least three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p

Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.

<p>(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk ^a indicates statistically significantly different from the untreated control (P<0.05).</p

Chemical structure of the compounds isolated from EADs.

<p>Structures of the isolated compounds were elucidated using 1^H and 13^C NMR spectroscopy. The isolated compounds were identified as kaempferide (<b>1</b>), kaempferol (<b>2</b>), protocatechuic acid (<b>3</b>), gallic acid (<b>4</b>), 3-epimaslinic acid (<b>5</b>) and β-sitosterol-3-O-β-D-glucopyranoside (<b>6</b>).</p