A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

A hybrid Pum1 fusion protein with a MED15 CC domain is a potent enhancer of mutant ATXN1 aggregation in cell-based assays.

<p>(A) Schematic representation of the hybrid Pum1-MED15CC fusion protein. (B) Effects of mCherry-tagged proteins Pum1-MED15CC, Pum1, MED15, S100B, STUB1 and luciferase on spontaneous YFP-ATXN1Q82^NT aggregation in human neuroblastoma SH-EP cells. The formation of polyQ-containing ATXN1 aggregates was quantified by fluorescence imaging after 48 h. Data is normalized to the total mCherry fluorescence intensity. The proteins MED15, STUB1 and Pum1-MED15CC enhanced YFP-ATXN1Q82^NT aggregation compared to Pum1 or the luciferase control protein (100%). A suppression of YFP-ATXN1Q82^NT aggregation was observed with the control protein S100B. Data is shown as mean ± SD from three independent experiments. Experiments were performed in triplicates. Student's t-test was used for statistical comparisons, p<0.05. (C) Confocal microscopy images of COS-1 cells co-transfected with pairs of plasmids encoding YFP-ATXN1Q82^NT and mCherry-tagged Pum1 or Pum1-MED15CC. Nuclei were stained with Hoechst 33342 (blue). YFP-ATXN1Q82^NT aggregates were stained in green and mCherry-Pum1 or mCherry-Pum1-MED15CC in red color. Both Pum1 and Pum1-MED15CC proteins co-localize with YFP-ATXN1Q82^NT proteins.</p

The N-terminal CC domain in MED15 enhances polyQ-mediated ATXN1 aggregation in cell-free assays.

<p>(A) Effects of His-tagged modifier proteins MED15 and Pum1 on spontaneous ATXN1Q82 aggregation <i>in vitro</i>. SDS-insoluble ATXN1Q82 aggregates were detected by filter assay. The protein MED15 enhanced spontaneous ATXN1Q82 aggregation while Pum1 showed the opposite effect. The protein GST was used as a control. Formation of SDS-insoluble ATXN1Q82 protein aggregates was detected by filter assay using the anti-ATXN1 antibody SA4645. (B) Quantification of ATXN1Q82 aggregates on filter membranes was performed using the AIDA densitometry software. The ATXN1Q82 immunoreactivity of samples obtained with the control protein GST was set to 100% (48 h). Error bars represent SD of three independent experiments. (C) Schematic view of the cloning strategy for the generation of the MED15CC fragment for <i>in vitro</i> aggregation experiments. A Gateway compatible entry plasmid encoding the N-terminal coiled-coil domain of MED15 (MED15CC) was constructed. (D) Far-UV CD spectra of the GST-tagged MED15CC fusion protein and GST. Both proteins adopt a typical alpha-helical conformation. A ratio of mean residue ellipticities ([θ]222/[θ]208) of 1.078948 indicates that MED15CC has a coiled-coil conformation. (E) The GST-tagged MED15CC fusion protein stimulates spontaneous ATXN1Q82 aggregation in cell-free assays. The formation of SDS-insoluble ATXN1Q82 aggregates was detected by filter retardation assays using the anti-ATXN1 antibody SA4645. (F) Quantification of insoluble ATXN1Q82 aggregates retained on filter membranes was performed using the AIDA densitometry software. The immunoreactivity of insoluble ATXN1Q82 aggregates in control samples with GST (48 h) was set to 100%. Error bars represent SD of three independent experiments.</p

Effects of MED15ΔCC on mutant ATXN1 aggregation and cytotoxicity in cell-based assays.

<p>(A) Schematic representation of the MED15ΔCC fusion protein. (B) Effects of mCherry-tagged proteins MED15, MED15ΔCC and luciferase on spontaneous YFP-ATXN1Q82 aggregation in neuroblastoma SH-EP cells. The formation of YFP-ATXN1Q82 aggregates was quantified by fluorescence imaging after 48 h. Data were normalized to the total mCherry fluorescence intensities. Data is shown as mean ± SD from three independent experiments. Experiments were performed in triplicates. Student's t-test was used for statistical comparisons, p<0.05. (C) Effects of FLAG-tagged fusion proteins MED15 and MED15ΔCC on YFP-ATXN1Q82-induced cellular toxicity in COS-1 cells. YFP-ATXN1Q82 cytotoxicity was increased in the presence of full-length MED15 but not in the presence of the truncated fragment MED15ΔCC. Cells transfected with the control plasmid pFLAG were used as a control (100%). (D) Confocal microscopy images of COS-1 cells co-transfected with pairs of plasmids encoding YFP-ATXN1Q82 and mCherry-tagged MED15 or MED15ΔCC. Nuclei were stained with Hoechst 33342 (blue).</p

Reduced levels of modifier proteins influence the cytotoxicity of pathogenic ATXN1 in model systems.

<p>(A) Comparison of modifier gene effects on YFP-ATXN1Q82^NT-induced toxicity in cell-based cDNA overexpression and siRNA knock-down experiments. Most modifiers in cDNA overexpression studies show opposing effects on YFP-ATXN1Q82^NT toxicity than modifiers in siRNA knock-down experiments. Error bars indicate SD from three independent experiments performed in triplicates. Student's t-tests were used for statistical comparisons, p<0.05. (B) Reduced levels of modifier proteins influence the toxicity of pathogenic ATXN1 in transgenic flies. RNAi knockdown of MED15 reduces ATXN1Q82-induced retinal degeneration, while knockdown of niki (homologue of human NEK8) increases the toxicity of the polyQ disease protein. The effects of modifiers on <i>Drosophila</i> eyes are visualized by loss of pigmentation (upper panel, optical microscopy), alterations in morphology of ommatidia (middle panel, electron microscopy) and bristle disorganization (lower panel, 10× magnifications of electron microscopy images). <i>Drosophila</i> eyes are shown from representative animals: a) wild-type (+/+), b) <i>GMR</i>-GAL4;UAS-ATXN1Q82/+;UAS-Luciferase RNAi, c) <i>GMR</i>-GAL4;UAS-ATXN1Q82/+; UAS-MED15 RNAi, and d) <i>GMR</i>-GAL4;UAS-ATXN1Q82/+;UAS-niki RNAi.</p

Identification of ATXN1 toxicity and aggregation modifiers using cell-based assays.

<p>(A) Relative caspase 3/7 activity change induced by overproduction of proteins YFP-ATXN1Q30^NT or YFP-ATXN1Q82^NT in COS-1 cells compared to YFP overproducing cells. Caspase 3/7 activity was significantly increased in cells producing pathogenic YFP-ATXN1Q82^NT compared to non-pathogenic YFP-ATXN1Q30^NT (Student's t-test, ** p<0.01, n = 3). Error bars indicate SD. (B) Detection of SDS insoluble protein aggregates by filter retardation assay. The protein YFP-ATXN1Q82^NT but not YFP-ATXN1Q30^NT forms SDS-stable protein aggregates in COS-1 cells. Equal amounts of total protein were loaded. FLAG control protein was set to 100%. (C) Western blot detection of YFP-ATXN1Q30^NT and YFP-ATXN1Q82^NT fusion proteins in total COS-1 cell extracts, supernatant and pellet samples. The pellet samples were dissolved in 8 M urea. Proteins were detected using an anti-GFP monoclonal antibody (Sigma). Actin protein levels were used as a loading control; Abbreviations: TL - total lysate; P – pellet; Sup – supernatant. (D) Quantification of YFP-tagged ATXN1 fusion proteins using the AIDA densitometry software. Abbreviations: TL - total lysate; P – pellet; Sup – supernatant. (E) Effects of modifier proteins on YFP-ATXN1Q82^NT-induced cellular toxicity. 12 proteins were defined as enhancers and 9 as suppressors of YFP-ATXN1Q82^NT toxicity. (F) Effects of modifier proteins on YFP-ATXN1Q82^NT aggregation monitored by filter retardation assays. 15 of 21 YFP-ATXN1Q82^NT toxicity modulators also influence polyQ-mediated protein aggregation. Data is shown as mean ± SD from three independent experiments, which were performed in triplicates. Student's t-tests were used for statistical comparisons, p<0.05. (G) A comparison of modifier effects on YFP-ATXN1Q82^NT cytotoxicity and aggregation.</p

The modulator proteins MED15 and Pum1 interact with wild-type and mutant ATXN1.

<p>(A) Schematic representation of a LUMIER co-immunoprecipiation assay. The protein A (PA)-Renilla luciferase (RL)-tagged bait (PA-RL-Y) and the firefly luciferase (FL)-tagged prey proteins (FL-X, modifier) were co-produced in HEK293 cells. After co-immunoprecipitation from cell lysates the interaction between bait and prey fusion proteins was monitored by quantification of firefly luciferase activity in protein complexes. Quantification of Renilla luciferase activity in precipitated protein complexes indicates the immunoprecipitation of bait protein. Abbreviations: PA – protein A tag; RL – Renilla luciferase; FL – firefly luciferase; X – prey protein; Y – bait protein; TF – transfection of cells; IP –immunoprecipitation; LA – luminescence assay; LS – luminescence signal. (B) The human proteins U2AF2, MED15 and Pum1 interact with both wild-type and mutant ATXN1 fusion proteins in LUMIER co-immunoprecipitation assays. The R-op and R-ob binding ratios (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002897#pgen.1002897.s008" target="_blank">Figure S8</a>) of >1.5 indicate that the proteins U2AF2, MED15 and Pum1 specifically interact with ATXN1Q30 or ATXN1Q82 in mammalian cells.</p

Specific functional categories and structural domains are over-represented among YFP-ATXN1Q82^NT toxicity modifiers.

<p>(A) Functional annotation of YFP-ATXN1Q82^NT toxicity modifiers was carried out using the EASE program. Genes encoding proteins involved in protein folding, RNA binding, translation, ribosome function, membrane fusion and protein biosynthesis are over-represented among YFP-ATXN1Q82^NT toxicity modifiers compared to the human genome (Fisher's exact test, p<0.05). (B) Proteins with coiled-coil domains are significantly enriched among YFP-ATXN1Q82^NT toxicity enhancers but not among suppressors (p = 0.0093; Chi-square test). Coiled-coil domains in modifier proteins were predicted using the COILS program (probability 0.8–1). Numbers of coiled-coil domain containing proteins were compared to the human proteome (73,427 proteins, Swiss-Prot database). (C) The human MED15 protein contains a conserved N-terminal Q-rich region with short polyQ tracts. The graphs represent the amounts of glutamines (Qs) in a window of 50 amino acids (blue line) or the amount of consecutive polyQ sequences (>5 Qs) in a window of 50 amino acids (red line). Abbreviations: hs – <i>Homo sapiens</i>, mm – <i>Mus musculus</i>, dm – <i>Drosophila melanogaster</i>, ce – <i>Caenorhabditis elegans</i>. (D) The human Pum1 protein contains conserved Q-rich regions but does not contain short polyQ tracts. For species abbreviations see C.</p