Additional file 1 of Fused expression of Sm1-Chit42 proteins for synergistic mycoparasitic response of Trichoderma afroharzianum on Botrytis cinerea

Additional file: Figure S1. Construction of chimeric protein engineered strains of T. afroharzianum. (A) Sm1 and Chit42 overlap fragments for chimeric protein and TaSm1 and MaChit42 overexpression vectors construction; (B) PCR verification of chimeric protein and TaSm1 and MaChit42 engineered strains by using hygromycin primer; (C) and (D) were PCR verification of chimeric protein and TaSm1 and MaChit42 engineered strains using by differential primer pairs (PC between trpC promoter and Chit42; CS between Chi42 and Sm1; ST between Sm1 and trpC terminator; PS between trpC promoter and Sm1; SC between Sm1 and Chit42; CT between Chit42 and trpC terminator); (E) Southern blot analysis of chimeric protein and TaSm1 and MaChit42 engineered strains; (F) qPCR results of Sm1 gene expressing in T. afroharzianum with different culture medium (PDA and PD)

Additional file 3 of Fused expression of Sm1-Chit42 proteins for synergistic mycoparasitic response of Trichoderma afroharzianum on Botrytis cinerea

Additional file: Figure S3. Hydrophobicity modulation ability of TaSm1, MaChi42, and SCf expressing in T. afroharzianum. (A) Pictures and (B) box plot of a water droplet in the surface of T. afroharzianum wild-type (T30), OE:TaSm1, OE:MaChi42, and OE:SCf strains. Hydrophobicity of spores suspension of T. afroharzianum wild-type (T30), OE:TaSm1, OE:MaChi42, and OE:SCf strains in glass (C) and PET (D) slides

Additional file 4 of Fused expression of Sm1-Chit42 proteins for synergistic mycoparasitic response of Trichoderma afroharzianum on Botrytis cinerea

Additional file: Table S1 Primers used in this study

Additional file 2 of Fused expression of Sm1-Chit42 proteins for synergistic mycoparasitic response of Trichoderma afroharzianum on Botrytis cinerea

Additional file: Figure S2. Sm1 gene expressing in the process of T. afroharzianum engineered strains interact with B. cinerea

ER1626 inductive effect on cell apoptosis of MCF-7 and Ishikawa.

<p>Cells were grown in 6-well plates and apoptosis assay were performed following incubation with ER1626 (10⁻⁷, 10⁻⁶ and10⁻⁵M) for 24 h. Treated cells were processed with annexin V-FITC Apoptosis kit and analyzed in flow cytometery. <b>A</b> Typical pictures of the apoptotic cells in MCF-7; <b>B</b> Typical apoptosis pictures of Ishikawa cells; <b>C</b> Apoptosis ratios (the number of apoptotic cells in treatment groups to that of control group).</p

Effect of ER1626 on HUVEC cell migration and tube formation.

<p>HUVEC cells were cultured in 6-well plates overnight, creating a straight scratch on each confluent monolayer and incubating with ER1626 (10⁻⁷, 10⁻⁶ and10⁻⁵M) or vehicle. After 0, 24, 48 and 72 h, images of five randomized vision of scratches were captured and the width of scratches was measured. Cell migration ratio was calculated following the formula: Cell migration ratio was presented in the curve diagram. <b>A</b> Images of the scratch in HUVEC cells; <b>B</b> The curve diagram of HUVEC cell migration. HUVEC cells were re-suspend in 96-well plate previously coated with 80 µl of commercial matrigel per well and incubated with ER1626 (10⁻⁷, 10⁻⁶ and 10⁻⁵M) for 8 h. Five randomly pictures of the enclosed networks of complete tubes were photographed under the microscope and the tube number was reckoned. The inhibition ratio of ER1626 was counted as the percentage of intact networks number in treated cells to those of the control cells (100%) and shown in the bar graph. <b>C</b> Images of VEGF-stimulated tube formation in HUVEC cells. a Control; b ER1626(10⁻ M); c ER1626(10⁻⁶M); d ER1626(10⁻⁵M); <b>D</b> Inhibition ratio of ER1626 on tube formation in HUVEC cells. *<i>p</i><0.05, **<i>p</i><0.01 compared with the control.</p

Reduction of VEGF secreted by MCF-7 and Ishikawa cells.

<p><b>A</b> MCF-7 and Ishikawa cells were planted respectively in 6-well dishes overnight and incubated with ER1626 (10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶ or 10⁻⁵M) or control for 24 h. Elisa kit was employed to detect the VEGF production in the cultured medium. Inhibition ratio was expressed as the percentage of VEGF product in treated cells to those of the control cells (100%). <b>B</b> The relative VEGF level in ER1626-treated cultured system versus the relative ERα level in ER1626-treated MCF-7 and Ishikawa cells. *<i>p</i><0.05, **<i>p</i><0.01 compared with the control.</p

The expression of ERα and ERβ protein in ER1626-treated MCF-7 and Ishikawa cells.

<p><b>A</b>. Cells were respectively in 6-well plates and maintained in specific medium supplemented with 5% FBS for 24 h prior to incubation with ER1626 (10⁻⁷, 10⁻⁶ and 10⁻⁵M) or vehicle for another 24 h. Treated cells were lysed in RIPA buffer and cell lysate was electrophoresed. Immunoblotting was performed for ERα, ERβ and the loading control β-actin. <b>B</b> The intensity of the band of ERs protein was normalized and expressed as relative fold change in MCF-7 cells. <b>C</b> The intensity of ERs protein was expressed in Ishikawa cells.*<i>p</i><0.05,^#<i>p</i><0.05 compared with their corresponding control.</p

ER1626 inhibits the angiogenesis of chick embryos.

<p>A window was carefully created through the pore of 5-day chicken embryo. The window was covered with plastic wrap to form a fake-pore after the shell membrane on CAM was detached gently. Chicken embryos were adapted for one day more in incubater. Filter paper disks (5×5 mm) saturated with 10 µl of ER1626 (5×10⁻⁴ or 5×10⁻³M) or 2-Methoxyestradiol (5×10⁻³M) or 0.1% DMSO were lightly placed on CAMs. They were then incubated for 48 h and CAMs under paper disks were harvested and photographed. Compare the blood vessel on CAMs. <b>A</b> Control (0.1% DMSO); <b>B</b> 2-ME2 (16 µg); <b>C</b> ER1626 (2.3 µg); <b>D</b> ER1626 (23 µg).</p