Substrate Engineering Enabling Fluorescence Droplet Entrapment for IVC-FACS-Based Ultrahigh-Throughput Screening

In vitro compartmentalization-based fluorescence-activated cell sorting (IVC-FACS) is a powerful screening tool for directed evolution of enzymes. However, the efficiency of IVC-FACS is limited by the tendency of the fluorescent reporter to diffuse out of the droplets, which decouples the genotype and phenotype of the target gene. Herein we present a new strategy called fluorescence droplet entrapment (FDE) to solve this problem. The substrate is designed with a polarity that enables it to pass through the oil phase, react with the enzyme and generate an oil-impermeable and fluorescent product that remains entrapped inside the droplet. Several FDE substrates were designed, using two distinct substrate engineering strategies, for the detection of phosphotriesterases, carboxylesterases, and glycosidases activities. Model screening assays in which rare phosphotriesterase-active cells were enriched from large excesses of inactive cells showed that the enrichment efficiency achievable using an FDE substrate was as high as 900-fold: the highest yet reported in such an IVC-FACS system. Thus, FDE provides a means to tightly control the onset of the enzymatic reaction, minimize droplet cross-talk, and lower the background fluorescence. It therefore may serve as a useful strategy for the IVC-FACS screening of enzymes, antibodies, and other proteins

The effect of Z-LEHD-FMK and Z-DEVD-FMK on thimerosal-induced apoptosis in C2C12 myoblast cells.

<p>C2C12 cells were treated with Z-LEHD-FMK, an inhibitor of caspase-9, or with the caspase-3 inhibitor Z-DEVD-FMK at a concentration of 50 µM for 24 h and then incubated with or without thimerosal (250 nM) for 48 h. <b>A.</b> After treatment, cells were stained with Annexin V-FITC/propidium iodide followed by flow cytometry. <b>B.</b> After treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M of the values from three independent experiments. **P<0.01 vs. single treatment with 250 nM thimerosal.</p

Wortmannin, a specific inhibitor of PI3K, enhanced the sensitization of C2C12 myoblast cells to thimerosal-induced apoptosis.

<p><b>A.</b> C2C12 myoblast cells were treated with thimerosal (125 nM, 250 nM and 500 nM) for 24 or 48 h. Cells were lysed, and the expression of Akt and pAkt^Ser473 were assayed by western blot analysis. GAPDH was used as a loading control, and the blots were quantified by densitometry. <b>B.</b> C2C12 myoblast cells were treated with wortmannin at concentrations of 2.5, 5.0 or 10 µM for 24 h. Expression of total Akt and pAkt^Ser473 was assayed by western blot analysis and densitometry. <b>C.</b> Cells were co-treated with thimerosal (250 nM) and wortmannin (5 µM) for 24 h and stained with annexin V-FITC/propidium iodide. The columns illustrate the flow cytometric results. <b>D.</b> After co-treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against Akt, pAkt^Ser473, cytochrome c, cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M. of values from three independent experiments. **P<0.01 vs. single treatment with 250 nM thimerosal; ## P<0.01 vs. untreated cells.</p

Effects of thimerosal on proliferation of C2C12 myoblast cells.

<p>C2C12 myoblast cells were treated with thimerosal (125, 250 and 500 nM) for 24, 48 or 72 h. <b>A.</b> C2C12 myoblast cell viability determined by WST-1. <b>B.</b> Cell cycle distribution of C2C12 myoblast cells analyzed by flow cytometry. <b>C.</b> The percentage of cells in the various phases of the cell cycle. All data are reported as the mean±S.E.M. of three independent experiments. **P<0.01 compared with control.</p

Activation of PI3K/Akt signaling inhibited thimerosal-induced apoptosis in C2C12 myoblast cells.

<p><b>A.</b> C2C12 cells were treated with mIGF-I at concentrations of 25, 50, or 100 ng/mL for 24 h. Expression of total Akt and pAkt^Ser473 was assayed by western blot analysis followed by densitometry. <b>B.</b> Cells were co-treated with thimerosal (250 nM) and mIGF-I (50 ng/mL) for 48 h and stained with Annexin V-FITC/propidium iodide followed by flow cytometry. <b>C.</b> After co-treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against Akt, pAkt^Ser473, cytochrome c, cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M of values from three independent experiments. **P<0.01 vs. single treatment with 250 nM thimerosal.</p

Overexpression of survivin inhibited thimerosal-induced apoptosis in C2C12 myoblast cells.

<p><b>A.</b> C2C12-survivin with overexpression of survivin, C2C12-puro with control vector and normal C2C12 cells (Mock) were subjected to western blot analysis with antibody against survivin followed by densitometric quantification. **P<0.01 vs. C2C12-puro <b>B.</b> C2C12-survivin and C2C12-puro were treated with or without thimerosal (250 nM) for 48 h and stained with Annexin V-FITC/propidium iodide followed by flow cytometry. <b>C.</b> After treatment with or without thimerosal, proteins from C2C12-survivin and C2C12-puro were analyzed by immunoblotting with antibodies against survivin, cytochrome c, cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M of the values from three independent experiments. **P<0.01 vs. single treatment with 250 nM thimerosal.</p

Thimerosal induced apoptosis in C2C12 myoblast cells.

<p>C2C12 myoblast cells were treated with thimerosal (125, 250 and 500 nM) for 24 or 48 h. <b>A.</b> After treatment, cells were stained with Annexin V-FITC/propidium iodide and measured by flow cytometry. The columns illustrate the flow cytometric results. <b>B.</b> After exposure to thimerosal for 48 h, cells were stained with Hoechst 33258 and observed under a fluorescence microscope. The magnification was ×200. <b>C.</b> After treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against cytochrome c, cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M. of values from three independent experiments. **P<0.01 vs. control.</p

Primary data

Primary data(table1, table2 and table3 in the maintext were extracted from these data, it also contains screening data for tyr99 saturation mutagenesis

Thimerosal decreases the expression of survivin via the PI3K/Akt pathway.

<p><b>A.</b> C2C12 myoblast cells were treated with thimerosal (125 nM, 250 nM or 500 nM) for 24 or 48 h. Cells were lysed, and the expression of survivin was assayed by western blot analysis and densitometry. GAPDH was used as a loading control. <b>B.</b> C2C12 cells were treated with wortmannin at concentrations of 2.5, 5.0 and 10 µM for 24 h. The expression of survivin was assayed by western blot analysis followed by densitometry. <b>C.</b> Cells were co-treated with thimerosal (250 nM) and mIGF-I (50 ng/mL) for 48 h, and survivin expression was quantitated by western blotting and densitometry. Data are means±S.E.M of the values from three independent experiments.</p

SiRNA against survivin enhanced the sensitization of C2C12 myoblast cells to thimerosal-induced apoptosis.

<p><b>A.</b> After 48 h transfection with non-targeted (NC) or survivin (S1, S2 and S3) siRNA or mock transfection without siRNA (MOCK), the expression of survivin was assayed by western blot analysis and densitometry. **P<0.01 vs. non-targeted (NC) <b>B.</b> After transfection with non-targeted (NC) or survivin S1 siRNA for 48 h, cells were treated with or without thimerosal (250 nM) for 24 h and stained with Annexin V-FITC/propidium iodide followed by flow cytometry. <b>C.</b> After treatment of siRNA and thimerosal, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against survivin, cytochrome c, cleaved caspase-9, cleaved caspase-3 and GAPDH followed by densitometric quantification. Data are means±S.E.M of the values from three independent experiments. **P<0.01 vs. single treatment with 250 nM thimerosal; ## P<0.01 vs. untreated cells.</p