Figure 3

<p>A: Cleavage of NORE1A and RASSF1A requires calcium and is sensitive to calpain inhibitors. NORE1A, tagged N-terminally with the FLAG tag, was expressed in HEK293 cells and adsorbed on FLAG beads (lanes 1–4). RASSF1A, tagged N-terminally with the FLAG tag, was expressed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003997#pone-0003997-g001" target="_blank">Figure 1 (lanes 5–8)</a>. Equal amount of beads containing NORE1A, or RASSF1A were incubated for 30 min at 37°C with H358 cells extract with no additions (lanes 2, 6) or with 1 µM of calpain inhibitor ALLN (lanes 3, 7) or with 50 mM EDTA (lanes 4, 8). NORE1A and RASSF1A, eluted from beads with the FLAG peptide without cleavage, was used as controls (lanes 1 and 5, respectively). After incubation, NORE1A-containing beads were extensively washed. Samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies. Asterisk denoted IgG heavy chain eluted from FLAG beads. B, Cathepsin B and Cathepsin K are not responsible for NORE1A cleavage by H358 and H460 cell lysates: NORE1A, tagged N-terminally with the FLAG tag, was expressed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003997#s4" target="_blank">Methods</a> and adsorbed on FLAG beads. Equal amounts of NORE1A were incubated for 30 min at 37°C with H358 cells extract (lanes 2–5) or H460 cell extract (lanes 6–9) with no additions (lanes 2, 6) or with ALLN (1 µM, lanes 3, 7) or with inhibitor of Cathepsin B (10 µM, lanes 4, 8) or with inhibitor of Cathepsin K (2 µM, lanes 5, 9). NORE1A eluted from beads with the FLAG peptide was used as control (lane 1). After incubation, beads were extensively washed and proteins retained on them were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies.</p

Figure 4

<p>A, The addition of a calpain inhibitor ALLN to cultured cells prevents NORE1A degradation: NORE1A was re-expressed in H157 cells (lanes 1, 6), H358 cells (lanes 3, 8) and H460 cells (lanes 5, 10) using a vector with a drug-resistance marker with subsequent selection for the drug. Lanes 2, 7 represent H358 parental cells and lanes 4, 9 - H460 parental cells. Cells were cultured for 3 days without inhibitors (top panel) or with 1 µM ALLN (bottom panel). Cell extracts, equalized by α-tubulin, were probed with 10F10 antibody by Western Blotting. At 1 µM concentration ALLN did not induce apoptotic response in H358 and H460 cells (data not shown). B, NORE1A expression at the mRNA level in H358 and H460 cells: NORE1A cDNA was re-expressed in H358 and H460 cells using a vector with a drug-resistance marker. Total mRNA was extracted from pools of drug-resistant cells emerged after selection as indicated and analyzed by RT-PCR for NORE1A expression. Note that these cells did not express NORE1A protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003997#pone-0003997-g004" target="_blank">Fig 4A, lanes 3 and 5</a>). NORE1A, cDNA of vector used for transduction was included as a positive control. M, DNA molecular weight markers. C: Extracts of human tumors are capable of degrading NORE1A. Human lung tumors from patients 3, 9, 10 and 8 and matching normal lung tissues were extracted into buffer for cell extraction and normalized by total amount of cellular protein. Equal amount of extracts from tumors (T) and normal tissues (N) were mixed with FLAG-tagged NORE1A, immunopurified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003997#s4" target="_blank">Methods</a>. After thirty minute incubation at 37°C, electrophoretic sample buffer was added to 1× concentration, samples were boiled and subjected to Western blotting with anti-FLAG antibodies. As positive control for NORE1A degradation, NORE1A incubated with H460 cell extract (lane 2) was used.</p

Cleavage of NORE1A and RASSF1A by an activity expressed in some tumor cell lines.

<p>Full-length NORE1A and RASSF1A, tagged at the N-terminus with the FLAG tag, were expressed in HEK293 cells, immunopurified on FLAG beads and beads were eluted with FLAG-peptide. NORE1A (lanes 1–4) or RASSF1A (lanes 5–8) were incubated with the Buffer A (lanes 1, 5) or H358 cell lysate (lanes 2, 6), or H460 cell lysates (lanes 3, 7), or A549 cell lysates (lanes 4, 8) for 30 minutes at 37°C. After incubation, samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies.</p

NORE1A cleavage occurs at the N-terminus of the protein.

<p>A: Full-length NORE1A (lanes 1, 2) or its fragments aa 1–364 (lanes 3, 4) or aa 1–190 (lanes 5, 6), tagged N-terminally with the FLAG tag, immunopurified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003997#pone-0003997-g001" target="_blank">Figure 1</a>, were incubated with the lysis buffer (lanes 1, 3, 5) or with H358 cell lysate (lanes 2, 4, 6) for 30 minutes at 37°C. After incubation, samples were subjected to gel electrophoresis followed by Western blotting with anti-FLAG antibodies. Asterisks denote non-specific bands. B: NORE1A fragments, amino acids 18–190, 43–190 and 78–190 were synthesized in vitro using Promega TnT T7 Quick for PCR DNA kit in the presence of ³⁵S-Methionine. Each fragment contained an additional methionine as start codon and two extra methionines at the C-terminus to facilitate detection. An aliquot of translation mixture was incubated with 4× excess (v/v) of H358 cell extract or with Buffer A for 1 hour at 37°C, resolved on SDS gel and transferred to Immobilon. Shown is the autoradiogram of the membrane obtained by Phosphoimager.</p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-4

En infected with retrovirus expressing GFP or GFP-NORE1A effector domain (aa 191–363). Two days later, cells were fixed, processed for flow cytometry and analyzed as described in Methods. The percentage of GFP-positive cells in each phase of the cell cycle +/- mean is shown.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-2

. An aliquot of cells was used to examine efficiency of C19ORF5 depletion (, upper panel) and NORE1A expression level (, lower panel) while other cells growing on coverslip were fixed, processed for immunofluorescence and imaged as described in Methods (). In , each panel shows NORE1A, microtubules (stained with α-tubulin), centrosomes (stained with pericentrin) and a superimposed image. Arrows indicate centrosomes. Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-1

S were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules using the microtubule cosedimentation assay. : HEK293 cell extract was immunodepleted of the C19ORF5 protein by incubation with 4G1 antibody followed by Protein A/G plus agarose, or mock-depleted. Equal amounts of C19ORF5-depleted and mock-depleted extracts were probed for C19ORF5 (upper panel). The ability of purified FLAG-NORE1A to interact with microtubules after preincubation with C19ORF5-immunodepleted extract (lanes 1–2), mock-immunodepleted extract (lanes 3–4), or purified FLAG-C19ORF5 (lanes 5–6) was examined as described in the Methods. . C19ORF5 protein was depleted by RNA interference in A549 cells expressing GFP-tagged NORE1A. Equal amounts of cell extracts were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules as described in Figure 1 (two middle panels). Lower panel, the ability of GFP moiety, expressed alone in A549 cells, to bind to microtubules was examined.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-3

Day cells were infected with retrovirus encoding GFP-NORE1A. Two days later, cells were sorted using GFP and Cy3 markers, allowed to recover overnight, lysed and equal amounts of cell extract were probed for expression of MAP1B protein (, upper panel) and GFP-NORE1A expression level (, lower panel). Lysates of GFP and Cy3- positive cells were examined for the ability of GFP-NORE1A to interact with microtubules as described in Figure 1 . : A549 cells expressing GFP-NORE1A were transfected with anti-MAP1B siRNA pool or control siRNA. Cells were fixed, processed for immunofluorescence, and imaged as described in Methods. Each panel shows NORE1A, microtubules (stained with α-tubulin), centrosomes (stained with pericentrin) and a superimposed image. Arrows indicate centrosomes. Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Additional file 2: Table S1. of Seed-effect modeling improves the consistency of genome-wide loss-of-function screens and identifies synthetic lethal vulnerabilities in cancer cells

Sequences of sgRNAs used against HMX3 and PKN3. (DOCX 13 kb

Additional file 1: Figure S1. of Seed-effect modeling improves the consistency of genome-wide loss-of-function screens and identifies synthetic lethal vulnerabilities in cancer cells

Correlation based on shESs in high data quality cell lines. Figure S2. Examples of seed essentiality (seedES) calculations in an artificial dataset. Figure S3. Rank correlation (ρ) for high data quality cell lines based on shES and seedES over all shRNA family sizes. Figure S4. Reproducibility of the seed essentiality scores with increasing shRNA family size of seed sequences. As shown in Fig. 3, we added the gray trace indicating the correlation based on the average of correlations from all positions. Figure S5. Heatmap of average Spearman correlation of seedES scores with increasing family size, between the matched cell lines, by considering different positions along the shRNA molecule as the seed sequence. Figure S6. As shown in Fig. 5, the number of overlapping SL partners of major cancer driver genes observed in both datasets, before and after cleaning, where the cleaning was based on removal of shRNAs with a high tendency for off-target seed effects (defined by SPS and TA properties of seed sequences; Fig. 4). Figure S7. GARP-based geneES for PKN3 and HMX3 before and after cleaning in PIK3CA mutant and wild-type (WT) cell lines, separately for the Achilles 2.4 and COLT-cancer datasets. Figure S8. Density plots of geneES scores for all the genes and gold-standard constitutive core essential (CCE) genes. Gene-specific phenotypes were calculated based on gespeR and GARP scores in both Achilles and COLT-Cancer datasets, respectively. Figure S9. A stepwise procedure for cleaning genome-wide shRNA datasets. Figure S10. Baseline reproducibility between the Achilles 2.4 and BFG genome-wide shRNA screens. Figure S11. Reproducibility of Achilles 2.4 and BFG genome-wide screens at the level of shRNAs, on-target genes, and off-target seeds. Figure S12. Reproducibility of seed essentiality scores with increasing shRNA family size of seed sequences in additional datasets. Figure S13. Reproducibility of Achilles 2.4 and BFG datasets after accounting for seed sequence properties. (DOCX 1588 kb