Figure 6. Identifying all toxic shRNAs derived from CD95L and CD95.

(A) Schematic showing the cloned shRNAs covering the ORF of Venus and the ORFs and 3'UTRs of CD95L and CD95. The 3’UTR is displayed as a dashed line because it was not included in the full-length Venus-CD95L/CD95 sensors. (B) Work-flow of pTIP-shRNA library synthesis, shRNA screen and data analysis. (C) Ranked fold reduction of shRNAs spanning Venus and CD95L (ORF and 3'UTR) (left three panels) and Venus and CD95 (ORF and 3'UTR) (right three panels). The ranked lists were separated into the shRNAs derived from Venus (top), the ORFs (center) and the 3'UTRs (bottom). The p-value of enrichment for each ranked set of shRNAs is given. Only the parts of the ranked lists are shown with the downregulated shRNAs. For all six panels, the top section of each panel (boxed in blue) contains the data on shRNAs downregulated after infection of cells and cultured for 9 days without Dox when compared to the composition of the shRNA plasmid library and the bottom half (boxed in orange) contains the data on shRNAs downregulated after culture with Dox for 9 days when compared to the culture without Dox. P-values were calculated using Mann Whitney U tests with a one-sided alternative that the rank was lower. (D) The location of all shRNAs significantly downregulated at least five fold along the sequences of Venus, CD95L ORF, CD95L 3'UTR (left panel) and Venus, CD95 ORF, and CD95 3'UTR (right panel). The top half of each sub panel (blue ticks) shows the shRNAs downregulated after infection and the bottom half (orange ticks) contains the data on shRNAs downregulated after culture with Dox for 9 days. Significance of enrichment in the different subpanels is shown. p-values were calculated according to statistical tests of two proportions. Each data set was compared to the corresponding Venus distribution. Green line: sequence that corresponds to the intracellular domain of CD95L.

Figure 6—figure supplement 1. Toxicity and RNAi of individual shRNA pools.

(A) Top panels: Green object intensity over time of NB7 Venus-CD95L sensor cells infected with the pTIP-Venus shRNA pool (left panel), pTIP-CD95L ORF shRNA pool (center panel), or pTIP-CD95L 3’UTR shRNA pool (right panel) with or without Dox treatment. Bottom panels: Green object intensity over time of NB7 Venus-CD95 sensor cells infected with the pTIP-Venus shRNA pool (left panel), pTIP-CD95 ORF shRNA pool (center panel), or pTIP-CD95 3’UTR shRNA pool (right panel) with or without Dox treatment. Values were calculated from samples done in quadruplicates shown as mean ±SE. (B) Percent confluence over time of parental NB7 cells infected with the pTIP-Venus shRNA pool (top left panel), pTIP-CD95L ORF shRNA pool (top center panel), pTIP-CD95L 3’UTR shRNA pool (top right panel), pTIP-CD95 ORF-shRNA pool (bottom center panel), and pTIP-CD95 3’UTR shRNA pool (bottom right panel) with or without Dox treatment. Values were calculated from samples done in triplicate shown as mean ±SE. P-values were calculated using two-way ANOVA with a factor for Dox treatment and a factor for time. Similar data were obtained when either HCT116 or 293T cells were treated with each of the five shRNA pools (data not shown).

Figure 6—figure supplement 2. Fold change in shRNA representation after infection of NB7 cells and after treatment with Dox.

(A) Change in green fluorescence (top panels) and percent cell confluence (bottom panels) over time of NB7 cells expressing either Venus-CD95 (left panels) or Venus-CD95L (right panels). Cells were infected with the Tet-inducible pTIP-shR6 virus, selected for two days with puromycin and then subjected to an analysis in the IncuCyte Zoom. No Dox was added. Two other inducible constructs (pTIP-shL1 and pTIP-shL3) were tested in the same way and no evidence of leakiness was observed (data not shown), supporting the finding in the shRNA screen that certain shRNA constructs display leakiness while others do not. Values were calculated from samples done in triplicate shown as mean ±SE. (B) Scatterplot showing the fold down of shRNAs after infection of cells and culture for 9 days without Dox when compared to the composition of the shRNA plasmid library (X axis) and the fold down of shRNAs after culture with Dox for 9 days when compared to the culture without Dox (Y axis). The red dots are the shRNAs that were significantly downregulated at least five fold. The number of shRNAs labeled in red in each quartile is given in blue. Two of the shRNAs tested before are labeled in green.