Fig 1.
Schematic presentation of the CRISPR/Cas9-Knock out screen.
Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.
Fig 2.
Volcano plot showing significantly enriched genes.
The volcano plot shows gene expression changes between cells surviving treatment with usually lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [5,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.
Fig 3.
LRP1 knockout prohibits binding and uptake of PMT.
MEF (circles) and MEF-LRP1-/- cells (squares), respectively were incubated with increasing concentrations of PMT(C1165S)DTa for 48h and cell viability was analyzed as % of untreated controls. Shown is the mean of three independent experiments (A). Cells were incubated with increasing concentrations of Alexa488 labeled PMT (PMT488), washed and bound fluorescence analyzed by FACS shown as mean of three independent experiments (B).
Fig 4.
LRP1 knockout or competitive inhibition of LRP1 inhibit modification of PMT.
MEF and MEF-LRP1-/- cells were incubated in the presence of 5nM PMT for the indicated time intervals, washed and lysed. Lysates were analyzed for toxin-induced modification of Gαq by an antibody detecting GαQ209E, for expression of LRP1 and for tubulin as loading control (top). MEF and MEF-LRP1-/- cells were incubated in the presence of 1 or 10 nM PMT in the presence or absence of GST or GST-RAP (1 μM), respectively, washed and lysed. Lysates were analyzed for toxin-induced modification of Gαq, for expression of LRP1 and for tubulin as loading control (c, bottom). Statistics: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 5.
Solid-phase binding assay of PMT to clusters 2, 3, and 4 of LRP1.
Graph depicts the binding curves of cluster 2 (dots), 3 (squares), and 4 (triangles) to plates coated with His-PMT. The equilibrium-binding affinity (KD) is presented inside the graph. Data are shown as mean with STDEV.
Fig 6.
Expression of LRP1 or LRP1 cluster 4 in MEF-LRP1-/- cells restores the ability of PMT to intoxicate cells.
MEF-LRP1-/- cells were transduced with retroviruses encoding LRP1 (top) or LRP1-cluster 4 (bottom), incubated in the presence of 5 nM PMT for the indicated time intervals, washed and lysed. Lysates were analyzed for toxin-induced modification of GαQ, for expression of LRP1 and for tubulin as loading control.
Table 1.
Oligonucelotides used in this study.
Table 2.
Antibodies used in this study.