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. 2013 Jan 15;288(9):6386–6396. doi: 10.1074/jbc.M112.410092

FIGURE 1.

FIGURE 1.

Genome-wide screen for modulation of ApoA-I secretion from HepG2 cells. A, HepG2 cells were transiently transfected with siRNA directed toward human ApoA-I as a positive control or an NTC siRNA that served as a reference for 100% expression of ApoA-I mRNA. Cells were cultured for 72 h, and culture media were collected during the final 24 h for measurement of ApoA-I protein by ELISA. Repression of ApoA-I mRNA was confirmed by quantitative PCR. Data are presented as the mean ± S.E. (error bars). Statistical analysis using one-way ANOVA followed by comparison with NTC by Dunnett's method was performed (*, p < 0.01). B, HepG2 cells were transiently transfected with siRNA directed toward human ADRA1A as a positive control or an NTC siRNA as a reference for 100% expression of ADRA1A mRNA. Cells were cultured for 72 h, and culture media were collected during the final 24 h for measurement of ApoA-I protein by ELISA. Repression of ADRA1A mRNA was confirmed by quantitative PCR, and repression of ADRA1A protein was determined by Western blot analysis. GAPDH protein levels served as a loading control. Data are presented as the mean ± S.E. (error bars). Statistical analysis using one-way ANOVA followed by comparison with NTC by Dunnett's method was performed (*, p < 0.01). C, three-plate (Plt), 3-day replication studies were conducted using siRNA targeting either ADRA1A to serve as a positive control for ApoA-I protein secretion (maximum signal; circles) or ApoA-I as a control for reduction of ApoA-I protein secretion (minimum signal; diamonds) or the NTC to represent no change in ApoA-I protein secretion (middle signal; plus symbols). Individual data points are represented to illustrate the variance and reproducibility of the experimental conditions. D, human siRNA libraries targeting 21,789 genes were transfected into HepG2 cells to identify genes that are involved in production of ApoA-I protein. The data are represented as Z-score on the y axis, and the number of siRNA targets is represented on the x axis. E, testing scheme for selecting genes of interest from the whole genome screen illustrates that of 21,789 target genes assessed 432 genes were identified for follow-up. Gene class categories, including the number of genes, that may regulate ApoA-I secretion are listed. GPCR, G-protein-couple receptor; GNF, The Genomics Institute of the Novartis Research Foundation.