Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia. Giardia lamblia is a common cause of waterborne diarrhea due to contamination of water with human or animal feces [Giardia can serve as a unique protozoan model for eukaryotic cell differentiation because its life cycle has been completed with the test tube [G. lamblia undergoes differentiation from a pathogenic trophozoite form into a resistant infectious cyst form [al feces ,2. Althoal feces . Chronical feces –6. Giardest tube ,7. Like yst form ,8. The cyst form –11. Syntyst form –16. Histyst form ,18.Giardia for a stable and high expression of a target gene [Giardia for studies of gene function is also needed. A Cre/loxP system has been developed for genetic manipulations [Giardia [Giardia. A CRISPRi system with catalytically inactive Cas9 has been demonstrated for stable transcriptional repression of flagellar and ventral disc genes in Giardia recently [Plasmodium, Toxoplasma, Trypanosoma, Leishmania, and Trichomonas [Stable transfection systems that are involved in maintenance of episomal plasmids under drug selection, including neomycin and puromycin selection have been developed in get gene ,20. A syulations ,22, but [Giardia . Althoug[Giardia ,25, they[Giardia . Developrecently . CRISPR/chomonas –32.Streptococcus pyogenes for defense against viral or plasmid DNA invasion [The type II CRISPR/Cas9 system is from invasion . Cas9 cainvasion ,35. Afteinvasion . NHEJ geinvasion ,37. HR hinvasion , but enainvasion ,37. Scr7invasion ,40, 41. mlf) genes have been identified only in mammals and Drosophila [Giardia genome with a higher expression level during drug selection [mlf gene is upregulated during encystation [The myeloid leukemia factor (obtained from ATCC), were cultured in modified TYI-S33 medium [5 cells/ml.Trophozoites of 3 medium . Encysta3 medium . Briefly6 cells/ml) during vegetative growth as previously described [6 cells/ml. Cells seeded at this density became confluent within 24h. Confluent cultures were maintained for an additional 8h to ensure that the cultures were in stationary phase (at a density of ~2×106 cells/ml). Cyst count was performed on these stationary phase cultures. Cyst count was also performed on 24h encysting cultures.Cyst count was performed on the stationary phase cultures 12-18 and random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR with primers mlfF and mlfR. Genomic and RT-PCR products were cloned into pGEM-T easy vector (Promega) and sequenced . Comparison of genomic and cDNA sequences showed that the mlf gene contained no introns.Synthetic oligonucleotides used are shown in mlf , cwp1 , cwp2 , and ran  genes was performed using primers mlfF (PCR1F) and mlfR (PCR1R), PCR2F and PCR2R, PCR3 and PCR3R, cwp1F and cwp1R, cwp2F and cwp2R, ranF and ranR, respectively. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the mlf, cwp1, cwp2, and ran genes: mlfrealF and mlfrealR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; ranrealF and ranrealR. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as a relative expression level over control. Student’s t-tests were used to determine statistical significance of differences between samples.Synthetic oligonucleotides used are shown in G. lamblia cell line during vegetative growth or encystation stages using TRIzol reagent (Invitrogen). For RT-PCR, 5 μg of DNase-treated total RNA was mixed with oligo (dT)12-18 and random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR. Semi-quantitative RT-PCR analysis of mlf , mlf-ha, cwp1 , cwp2 , cwp3 , myb2 , wrky , pax1 , cdk2 , gRNA, ran , and 18S ribosomal RNA  gene expression was performed using primers mlfF and mlfR, mlfHAF and HAR, cwp1F and cwp1R, cwp2F and cwp2R, cwp3F and cwp3R, myb2F and myb2R, wrkyF and wrkyR, pax1F and pax1R, cdk2F and cdk2R, MLFgF and MLFgR, ranF and ranR, 18SrealF and 18SrealR, respectively. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes: mlfrealF and mlfrealR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; cwp3realF and cwp3realR; myb2realF and myb2realR; wrkyrealF and wrkyrealR; pax1realF and pax1realR; cdk2realF and cdk2realR; ranrealF and ranrealR; 18SrealF and 18SrealR. Each primer pairs were determined for amplification efficiency ~95% based on the slope of the standard curve. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as a relative expression level over control. Student’s t-tests were used to determine statistical significance of differences between samples.Synthetic oligonucleotides used are shown in mlf gene was amplified with oligonucleotides MLF 5HF and MLF 5NR, digested with HindIII/NcoI and cloned into HindIII/NcoI digested 5’Δ5N-Pac, resulting in MLF5. The 700-bp 3’-flanking region of mlf gene was amplified with oligonucleotides MLF 3XF and MLF 3KR, digested with XbaI/KpnI and cloned into XbaI/KpnI digested MLF5, resulting in MLF53. We used gene synthesis services from IDT to obtain the fragment MLF-guide. The NCBI Nucleotide Blast search was used to avoid the potential off-target effects of guide sequence. The MLF-guide was digested with KpnI/EcoRI and cloned into KpnI/EcoRI digested MLF53, resulting in pMLFko. Cas9 gene was amplified using primers Cas9NF and Cas9XR with the template pCas9_GFP (Addgene), digested with NcoI/XhoI and cloned into NcoI/XhoI digested 5’Δ5N-Pac, resulting in pgCas9dC. We used gene synthesis services from IDT to obtain the fragment Cas9CXK. The Cas9CXK was digested with XhoI/KpnI and cloned into XhoI/KpnI digested pgCas9dC, resulting in pgCas9.Synthetic oligonucleotides used are shown in mlf gene and its 324 bp of 5’- flanking region were amplified with oligonucleotides MLFNF and MLFMR, digested with NheI/MluI, and cloned into NheI/MluI digested pPop2NHA [KpnI/EcoRI and cloned into KpnI/EcoRI digested pBluescript SK, resulting in pgU6. To make pgCas9MLFko, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9 template, and primers Cas9insF and Cas9insR to insert multiple restriction sites, resulting in pgCas9ins. The HR template  and gRNA was amplified with the pMLFko plasmid and primers, mlf53UNF and mlf53UMR, digested with NotI/MluI and cloned into NotI/MluI digested pgCas9ins, resulting in pgCas9MLFko. To make pgCas9MLFko gRNA1, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9MLFko template, and primers U6R and MLFgFnew to change the gRNA, resulting in pgCas9MLFko gRNA1. To make pgCas9MLFko gRNA2, Q5 Site Directed Mutagenesis Kit was used to perform a reaction with a pgCas9MLFko template, and primers U6R and control gF to change the gRNA, resulting in pgCas9MLFko gRNA2.To make construct pPMLF, the pPop2NHA . To makepac gene were selected and maintained with 54 μg/ml (100 μM) puromycin [Giardia trophozoites were transfected with plasmid pMLFko and pgCas9, and then selected in 100 μM puromycin. The MLFko stable transfectants were established after selection. Stable transfectants were maintained at 100 μM puromycin and were further analyzed by Western blot, or DNA/RNA extraction. The replacement of the mlf gene with pac gene was confirmed by PCR and sequencing. The control is G. lamblia trophozoites transfected with double amounts of 5’Δ5N-Pac plasmid and selected with puromycin. For removal of puromycin experiments, puromycin was removed from the medium for each stable cell line for a month to obtain MLFko–pu and control–pu cell line. For establishing MLFkoSC stable transfectants, the same plasmids were used, except that the culture medium in the first replenishment contained 6 μM Scr7 and 100 μM puromycin. For establishing Cas9MLFko stable transfectants, Giardia trophozoites were transfected with plasmid pgCas9MLFko, and selected in 25 μM puromycin. The culture medium in the first replenishment contained 1.5 μM Scr7 and 25 μM puromycin. The Cas9MLFko stable transfectants were established after selection. Stable transfectants were maintained at 100 μM puromycin and were further analyzed by Western blot, or DNA/RNA extraction. The control cell line is G. lamblia trophozoites transfected with 5’Δ5N-Pac plasmid and selected with puromycin. Puromycin was then removed from the medium for each stable cell line to obtain Cas9MLFko–pu cell line. Subsequent analysis was performed after removal of the drug for a month. The control cell line is wild type nontransfected WB trophozoites. The single cell population was obtained by dilution of the Cas9MLFko cell line. The cell density was determined first and then diluted to 1 cell/culture tube. The single cell populations were established within 2 weeks.Cells transfected with pPMLF plasmid containing the uromycin . For CRIWestern blots were probed with anti-HA monoclonal antibody , anti-MLF (1/10000 in blocking buffer) (see below), anti-CWP1 (1/10000 in blocking buffer) , anti-MYmlf gene was amplified using oligonucleotides mlfF and mlfR. The product was cloned into the expression vector pET101/D-TOPO (Invitrogen) in frame with the C-terminal His and V5 tag to generate plasmid pMLF. The pMLF plasmid was freshly transformed into Escherichia coli BL21 Star (DE3) (Invitrogen). An overnight pre-culture was used to start a 250-ml culture. E. coli cells were grown to an A600 of 0.5, and then induced with 1mM isopropyl-D-thiogalactopyranoside (IPTG) (Promega) for 4 h. Bacteria expressing pMLF were harvested by centrifugation and sonicated in 10 ml of buffer A  containing 10 mM imidazole and complete protease inhibitor cocktail (Roche). The samples were centrifuged and the supernatant was mixed with 1 ml of a 50% slurry of Ni-NTA superflow (Qiagen). The resin was washed with buffer B  and buffer C  and eluted with buffer E . Fractions containing MLF were pooled, dialyzed in 25 mM HEPES pH 7.9, 40 mM KCl, and 15% glycerol, and stored at -70°C. Protein purity and concentration were estimated by Coomassie Blue and silver staining compared with bovine serum albumin. MLF protein is purified to apparent homogeneity (>95%).The genomic Purified MLF protein was used to generate rabbit polyclonal antibodies through a commercial vendor .6 cells/coverslip) and then fixed and stained [Cells cultured in growth medium or encystation medium for 24 h were harvested, washed in phosphate-buffered saline (PBS), and attached to glass coverslips (2 × 10 stained . Cells wGiardia encystation. The deduced Giardia MLF protein contains 252 amino acids with a predicted molecular mass of ~29.71 kDa and a pI of 6.81. It has one MLF1IP domain (residues 9 to 170) as predicted by Pfam (or residues or 59 to 170 by NCBI Conserved Domain Database) (Drosophila MLF (residues 36 to 199). The Drosophila MLF1IP domain contains a 14-3-3 binding motif, which may involve in interaction with the transcription factor Dref [Giardia MLFIP domain has no 14-3-3 interaction motif, RSXSXPRXSXSX [Giardia MLF is moderately similar to the human MLF1 and MLF2 and Drosophila MLF  . The MLFtor Dref , but theXPRXSXSX . Sequenchila MLF . The fulmlf gene is controlled by its own promoter and contains an HA epitope tag at its C terminus , during vegetative growth and encystation using anti-HA antibody  as compared with strategy 1 (nt 115–134)  but also with very low homology. Scr7 may inhibit Giardia DNA repair since Scr7 can inhibit Giardia growth  as compared with strategy 1 (nt 115–134) . We also performed single cell dilution for the all-in-one plasmid system and found that the obtained two clonal populations have a similar disruption efficiency of 73% and 78% as compared with their original cultures (70%)  , suggest or nonessential (growth) requires successful knockout of mlf gene. A complete knockout of an essential gene may lead to cell death, so it is hard to determine the gene function for that case. We have tried to knockdown 12 genes, including mlf by our CRISPR/Cas9 system, but only have obtained successful growth of 4 mutant cell lines after gene disruption under drug selection. We only generated the knockdown but not knockout mutants for these 4 genes. It is possible that the other 8 genes whose mutants cannot be obtained could be essential genes. Combining the CRISPR/Cas9 system and tetracycline inducible system can allow an adjustable knockdown of a target gene in the future [Because of tetraploid genome and two nuclei, it could be hard to disrupt a gene completely in . This search identified similarity of Giardia MLF to Drosophila MLF human MLF1 and MLF2 in the GenBank database. Sequence of MLF proteins is analyzed by ClustalW 1.83, including Drosophila MLF human MLF1 and MLF2 , and Giardia MLF (orf number 16424 in Giardia genome database). The gray boxes indicate the MLFIP domain of Giardia MLF predicted by pfam (http://pfam.sanger.ac.uk/). (B) Schematic representation of the Giardia MLF protein. The gray box indicates the MLFIP domain, as predicted by pfam. The residue numbers are shown.(A) Alignment of the MLF proteins. Specific sequence similarity search was performed against the GenBank database on NCBI's Web site using the BLASTP algorithm ((PDF)Click here for additional data file.S2 Figmlf-ha, mlf, cwp1, cwp2, cwp3, myb2, ran, and 18S ribosomal RNA genes, respectively. Similar levels of the ran mRNAs and 18S ribosomal RNA were detected. (B) Overexpression of MLF increased the levels of MLF proteins. The 5’Δ5N-Pac and pPMLF stable transfectants were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-MLF antibody. The result is the same as in mlf gene with the pac gene in the MLFko cell line confirmed by PCR2 and sequencing. Genomic DNA was isolated from MLFko and control cell lines cultured in growth medium. PCR was performed using primers specific for pac  Replacement of the mlf gene with the pac gene in the MLFko cell line confirmed by PCR3 and sequencing. Genomic DNA was isolated from MLFko and control cell lines cultured in growth medium. PCR was performed using primers specific for pac (PCR3), which are PCR3F for bold region 1 and PCR3R for bold region 2, to verify the integration of pac gene into the correct region in genomic DNA. The sequence results obtained from the PCR3 product are shown as underlined letters. Capital letters indicate the coding sequence for pac gene, which starts at ATG and stops at TGA. This indicates the replacement of the mlf gene with the pac gene. The region used to clone the mlf 3’ region into the pMLFko plasmid for HR is shown in red, which is also between the sequence of MLF3XF and MLF3KR. The underlined and lower case letters, which are upstream and outside of the red region of MLF3XF and MLF3KR, indicate that HR occurred in the sequence of mlf 3’ region and that the pac gene was integrated in the genomic DNA. Replacement of the mlf gene with the pac gene in the MLFkoSC and Cas9MLFko cell line was also confirmed by PCR3 and sequencing with the same sequencing results. (E) RT-PCR analysis of gene expression in the MLFko cell line during encystation. The control and MLFko cell lines were cultured in encystation medium and then subjected to RT-PCR analysis using primers specific for mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively. Similar levels of the 18S ribosomal RNA were detected. The ran mRNAs slightly decreased.(A) RT-PCR analysis of gene expression in the MLF-overexpressing cell line. The 5’Δ5N-Pac and pPMLF stable transfectants were cultured in growth medium and then subjected to RT-PCR analysis. PCR was performed using primers specific for  PCR2 in , which a(PDF)Click here for additional data file.S3 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko cell line during vegetative growth. The control and MLFko cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively.(A) Cyst formation decreased by MLF knock down in the MLFko cell line during vegetative growth. The control and MLFko cell lines were cultured in growth medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S4 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko–pu cell line. The control–pu and MLFko–pu cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Quantification of MVs in MLFko–pu and control–pu cell lines using Imaris software. *, P <0.05 (n = 200–300 cells/condition). (B) Knock down of (PDF)Click here for additional data file.S5 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFko–pu cell line during encystation. The control–pu and MLFko–pu cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis, as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the MLFko–pu cell line during encystation. The control–pu and MLFko–pu cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S6 Fig6 cells/ml in growth medium containing 33μM Scr7 for 24 h and then subjected to cell count. An equal volume of Me2SO was added to cultures as a negative control. The sum of total cells is expressed as a relative level over control. Values are shown as means ± S.E. of three independent experiments. *, P <0.05. (B) Partial replacement of the mlf gene with the pac gene in the MLFkoSC cell line confirmed by PCR. The pgCas9 and pMLFko constructs were transfected into trophozoites as described in mlf (PCR1), pac (PCR3), cwp1, cwp2, and ran genes, respectively, as described in mlf gene in the MLFkoSC cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFkoSC cell line during vegetative growth. The control and MLFkoSC cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Treatment with Scr7 decreased cell growth. The wild-type non-transfected WB cells were subcultured at an initial density of 1×10(PDF)Click here for additional data file.S7 Figmlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the MLFkoSC cell line during encystation. The control and MLFkoSC cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S8 Figs of the pMLFko plasmid is the same as in mlf (PCR1), pac (PCR2) and ran genes, respectively, as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf (PCR1), pac (PCR2) and ran genes, respectively, as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Diagram(PDF)Click here for additional data file.S9 Figmlf gene decreased the levels of CWP1, MYB2, and other proteins in the MLFkoSC cell line relative to the MLFko cell line during encystation. The MLFko and MLFkoSC cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, cwp2, cwp3, myb2, wrky, pax1, cdk2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) MLF knock down decreased cyst formation in the MLFkoSC cell line relative to the MLFko cell line during encystation. The MLFko and MLFkoSC cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S10 Figmlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in mlf gene in two single clone populations of the Cas9MLFko cell line confirmed by PCR. Two single clone populations, clones 1 and 2, of the Cas9MLFko cell line were obtained using the single cell dilution method. Genomic DNA was isolated from Cas9MLFko clone 1, clone 2, and control cell lines cultured in growth medium. PCR was performed using primers specific for mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in two single clone populations of the Cas9MLFko cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the Cas9MLFko cell line during encystation. The control and Cas9MLFko cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Methods” and (PDF)Click here for additional data file.S11 Figmlf gene, has a different sequence compared with pgCas9MLFko in mlf gene with the pac gene in the Cas9MLFko gRNA1 cell line confirmed by PCR. Puromycin was kept in the Cas9MLFko gRNA1 and control cell lines as described in mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in the Cas9MLFko gRNA1 cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko gRNA1 cell line during vegetative growth. The control and Cas9MLFko gRNA1 cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf gene with the pac gene in the Cas9MLFko gRNA2 cell line confirmed by PCR. Puromycin was kept in the Cas9MLFko gRNA2 and control cell lines as described in mlf (PCR1), pac (PCR2), and ran genes, respectively, as described in mlf gene in the Cas9MLFko gRNA2 cell line confirmed by real-time PCR. Real-time PCR was performed using genomic DNA and primers specific for mlf, cwp1, cwp2, and ran genes, respectively, as described in (A) Diagrams of the pgCas9MLFko gRNA1 or 2 plasmid are the same as in (PDF)Click here for additional data file.S12 Figmlf gene in the Cas9MLFko–pu cell line confirmed by real-time PCR. Puromycin was removed from the Cas9MLFko cell line to obtain the Cas9MLFko–pu cell line as described in mlf, cwp1, cwp2, and ran genes, respectively, as described in mlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko–pu cell line. The control and Cas9MLFko–pu cell lines were cultured in growth medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko–pu cell line. The control and Cas9MLFko–pu cell lines were cultured in growth medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Partial disruption of the (PDF)Click here for additional data file.S13 Figmlf gene decreased the levels of MLF and CWP1 proteins in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium and then subjected to SDS-PAGE and Western blot analysis as described in mlf, cwp1, and cwp2 gene expression by MLF knock down in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium and then subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in (A) Cyst formation decreased by MLF knock down in the Cas9MLFko–pu cell line during encystation. The control and Cas9MLFko–pu cell lines were cultured in encystation medium for 24h (Enc) and then subjected to cyst count as described under “Materials and Methods” and (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The existence of extended post-reproductive lifespan is an evolutionary puzzle, and its taxonomic prevalence is debated. One way of measuring post-reproductive life is with post-reproductive representation, the proportion of adult years lived by females after cessation of reproduction. Analyses of post-reproductive representation in mammals have claimed that only humans and some toothed whale species exhibit extended post-reproductive life, but there are suggestions of a post-reproductive stage for false killer whales and Asian elephants. Here, we investigate the presence of post-reproductive lifespan in Asian elephants using an extended demographic dataset collected from semi-captive timber elephants in Myanmar. Furthermore, we investigate the sensitivity of post-reproductive representation values to availability of long-term data over 50 years.We find support for the presence of an extended post-reproductive stage in Asian elephants, and that post-reproductive representation and its underlying demographic rates depend on the length of study period in a long-lived animal.The extended post-reproductive lifespan is unlikely due to physiological reproductive cessation, and may instead be driven by mating preferences or condition-dependent fertility. Our results also show that it is crucial to revisit such population measures in long-lived species as more data is collected, and if the typical lifespan of the species exceeds the initial study period. The evolution of extended post-reproductive lifespan is a long-term puzzle in evolutionary biology, where extended refers to post-reproductive lifespan that is not an artefact of individual variation in somatic and reproductive senescence rates . SpeciesTo distinguish extended post-reproductive lifespan from individual variation in senescence, measures that do not depend on any one individual are preferable to those calculated off specific individuals (e.g. expected lifespan vs maximum recorded lifespan) or individuals meeting specific criteria , 13. IndOrcinus orca), narwhals (Monodon monoceros), beluga whales (Delphinapterus leucas), and short-finned pilot whales  . WhOur second aim was to quantify how values of PrR and its underlying demographic variables may change depending on the length of study. There was large variation in post-reproductive representation through time Fig. , which wOne cannot consider PrR alone though, as its calculation relies heavily on other demographic variables. Whilst sample size is not directly incorporated into the PrR equation, it can have a large effect on the survival and fertility series. For example, one birth will have a greater impact on the fertility rate if the sample size at that age is smaller, which may be important for slow reproducers such as elephants. The same is true for the sampling of old individuals to determine, for example, the maximum lifespan or expected survival to a given age. The number of female elephants used to calculate the demographic series rose steadily, from 998 individuals in 1960 to 3802 individuals in 2018  have changed considerably with the length of the longitudinal data collection , and has remained unchanged in the last 4 years. Note that even though eM has not increased much . Howeveoduction , our resOur results also highlight an important issue in assessing PrR for long-lived species: the need for sufficiently long follow-up in longitudinal datasets. Here, we find a PrR of 0.162, higher than the value calculated in a previous assessment with the same population (0.128) [It is therefore important for future studies to consider when a species is assessed if they are calculating post-reproductive lifespan for long-lived species in which the study period is shorter than the typical adult lifespan. This issue can be alleviated by highly accurate age estimation for individuals of all ages, though there can be difficulties with verifying such estimation methods. For example, size cannot be used for accurate age estimation if growth is determinate and individuals have reached the asymptote of growth , 50. FurDue to the rarity of early reproductive cessation, we still do not know whether its evolution requires a specific driver or drivers. It may be that the prolonged post-reproductive lifespans of Asian elephants are currently driven by behaviour or body condition, rather than reproductive physiology, and may therefore be in an evolutionary transition; an extended post-reproductive life may be a prerequisite of an early end to reproductive capabilities. To properly tackle the puzzle provided by the evolution of early reproductive cessation and post-reproductive lifespan, we first need to know the taxonomic prevalence of these traits, underscoring the importance of long-term studies on known-age individuals.Additional file 1. Dataset containing the lx and mx series of the population.Additional file 2. Dataset containing the changing lx and mx series of the population (1960-2018).


Graft-versus-host disease (GVHD) is one of the most complex complications after allogeneic stem cell transplantation. Current standard of grading system is based on clinical symptoms in skin, liver and intestinal. However, it’s difficult to differ GVHD and its extent just by clinical manifestation. Here we retrospectively analyzed cell immune function in patients implemented allogeneic stem cell transplantation in Ningbo first Hospital from Jan 2013 to Jan 2018.the data are collected from 51 patients . The average NK cell percentage was 39.31% in severe GVHD (Grade III-IV), was 16.98% in mild GVHD (GradeI-II), while was 21.15% in No GVHD group. The statistical analysis showed difference among each grade. Further analysis was performed in Antithymocyte globulin (ATG) treated group and control group. We showed NK Cell percentage was sharply different in ATG treated group: 47.34% in severe GVHD, 11.98% in mild GVHD group, while 18.3% in no GVHD group. However, in control group, the average percentage of NK cells was 23.27% in severe GVHD, was 23.22%in mild GVHD group, while was 21.13% in no GVHD group.The data supports that ATG can prevent GVHD by increasing NK cell percentage. The percentage of NK cell seemed to be a useful probe to evaluate the severity of GVHD in allogeneic stem cell transplantation patients using ATG in pretreatment. Graft-versus-host disease (GVHD) poses as a major complication following allo-genetic hematopoietic stem cell transplantation . GVHD occurs in both acute and chronic forms, which can lead to morbidity and mortality . Allo-reAntithymocyte globulin (ATG) is a polyclonal antibody against fresh human thymocytes derived from rabbits, horses, or pigs. It has been used as a T cell-depleting agent in stem cell transplantation and organ transplantation, and has been found to decrease the incidence of GVHD . Due to The use of ATG may alter the immune cell repertoire in vivo sharply, which may provide clues for the prediction GVHD development and severity. Although the criteria for the clinical manifestations of GVHD, it remains difficult to predict the severity of GVHD in some cases. We speculate that the microenvironment of the graft recipient may vary with the use of ATG, resulting in variations in the onset and degree of severity in GVHD. It may therefore be possible to predict GVHD by monitoring changes in immune cell subsets after transplantation preceded by the use of ATG as part of the myeloabaltive regimen.loCD62hi in mice, CD44lowCCR7highCD45RA in human) can induce GVHD, effector T cells (CD44highCD62Llo in mice or CD44loCCR7hiCD45RO in human) do not induce GVHD, which could be explained by memory T cell apoptosis or exhaustion following alloantigen exposure [Previous studies have found that the frequency of Tregs is inversely correlated with GVHD development , 10 whilexposure , 12. Theh cells) requires more than 1 year [h cell deficiency renders patients susceptible to infection aside from the concurrent lack of protective immunoglobulins. Furthermore, the rapid recovery of donor-derived alloreactive T cells significantly increases the incidence of GVHD [As one of the major causes of mortality after allo-HSCT aside from relapse of the primary disease, GVHD has been found to be promoted by T cell-induced cytokine storm. Although total T cell number can be recovered approximately 3 months after allo-HSCT, the recovery of CD4 helper T cells  with or without ATG treatment. We recorded the changes of immune cell subsets in the recipients after the use of ATG, and analyzed the correlation between these changes and the occurrence and severity of GVHD. Specifically, focused on NK cells and CD4This study enrolled 51 patients (17-68 years) diagnosed with acute myeloid leukemia(AML)/acute lymphocyte leukemia(ALL)/myelodysplastic syndrome(MDS)/chronic myeloid leukemia(CML)/aplastic anemia(AA)/ paroxysmal nocturnal hemoglobinuria(PNH) who received allogeneic hematopoietic stem cell transplantation in Ningbo First Hospital between Jan 2013 to Jan 2018 . Cells were stained with antibody cocktail and analyzed using a FASCanto flow cytometer equipped with FACSDiva software (BD Biosciences). Absolute cell counts were determined using Flow-Count Fluorospheres . The antibodies included FITC-conjugated anti-CD62L (clone MEL-14), R-PE–conjugated anti-CD45.1 (clone A20), cy-chrome-conjugated anti-CD44 (clone IM7), and the corresponding isotype controls ; FITC-conjugated anti–H-2Db (clone CTDb), PE-conjugated anti-CD4 (clone CT-CD4), B220 (clone RA3-6B2), PE-Texas red–conjugated anti-CD4 (clone RM4–5), tricolor-conjugated anti-CD4 (clone CT-CD4), CD8a (clone CT-CD8a), and corresponding isotype controls . After each graft collection, samples were stained using the multitest kit . A panel of directly conjugated monoclonal antibodies (Mabs) was used to separate distinct T-cell subsets and homeostatic characteristics of each subset. Activated CD4 T and NK cells were defined as CD4 + CD25+, CD3-CD56 + CD16+, respectively. The panel design included: CD3FITC/CD56CD16PE/CD19APC/CD45PE-Cy7; CD3FITC/CD25PE/CD4PerCP-Cy5.5/CD45PE-Cy7 and CD38FITC/CD8PE/HLADR PerCP-Cy5.5/CD3APC/CD45PE-Cy7 .A total of 51 patients were analyzed in current study. The median age was 42 years (range 17–68 years), 86.3% (44/51) had hematologic malignancies , 13.7% (7/51) were diagnosed with nonhematologic malignancies, which including 5 AA patients and 2 PNH patients. Myeloablative conditioning was performed in 46 patients (90.2%), peripheral blood stem cells were used as the only source of stem cell in 27 patients (52.9%). Furthermore, 27 patients (52.9%) underwent transplantation from HLA-matched donors, including 22 from siblings, 5 from the bone marrow bank, while the remaining 24 patients (47.1%) received haplo-matched transplantation. Main clinical characteristics of the patients included in the study were summarized in Table Among HLA-matched sibling and 5 AA transplant recipients in the ATG-treated group , all patients received a small dose (2.5 mg/kg) of ATG in the conditioning regimen. In addition, all patients in the haplo-HSCT transplantation group received ATG in the pretreatment. Furthermore, 5 patients received the reduced dose regimen while the remaining 46 patients received busultfan/cyclophosphamide  or modified BUCY (34/51). Filgrastim-mobilized peripheral blood stem cells. All 51 patients reached complete chimerism by day 30. The median neutrophil cell recoverytime was 13 days (10–30) while the median platelet recovery time was 15 days (9–221).In all patients, GVHD prophylaxis included cyclosporine, mycophenolate mofetil, and methotrexate. For GVHD treatment, prednisone or methylprednisolone were added. For severe GVHD, CD25 or anti-IFN-α antibody were used. In retrospective analysis, GVHD were graded using the classic criteria. Severe GVHD were defined as grade III and IV while mild GVHD were defined as grade I and II. In addition, due to the uncertainty in the GVHD grading for lethal versus non-lethal GVHD , the grading of GVHD was corrected in the retrospective analysis.Patients enrolled in this study had a total GVHD occurrence of 52.9% 27/51). In the ATG-treated group, the incidence of GVHD was 53.3% (16/30) which included 6 patients with lethal GVHD (20%) and 10 surviving patients from GVHD (33.3%). In the control group, the incidence of GVHD was 52.3% (11/21), which included 3 patients with lethal GVHD , 8 patients with mild GVHD, and the remaining 10 patients did not develop GVHD. Statistical analysis showed no significant difference between the grades of GVHD in the two groups , activated CD4+ T cells (CD4 + CD25+) cells were analyzed each month after transplantation. Retrospective study was used to collect the time point before GVHD happened in patients diagnosed with aGVHD. In patients without GVHD, data from 3 months post- transplantation were used for analysis.In order to predict GVHD development, T cell subsets were compared within different grades of GVHD. Results showed that NK Cell percentage were noticeably different in ATG used group: in severe GVHD is 47.34%, in mild GVHD group was11.98%, while in no GVHD group is 18.3%. However, in the control group, data indicated the median NK cell percentage in severe GVHD is 23.27, 23.22% in mild GVHD group, and 21.13% in the group without GVHD. While there was no difference across different grades of GVHD in the absolute cell counts for CD4+ T cell subsets, CD4 + CD25 + T cell subsets, NK cell appeared to be a useful probe to indicate the severity of GVHD. However, there was some difference between the severe GVHD and mild GVHD groups in terms of CD8 T cell and NK cell subsets. In NK cell subsets, there was difference between severe GVHD and group without GVHD, although there was no difference between mild GVHD and group without GVHD. In terms of the CD8 T cell subset, there was difference between mild GVHD and group without GVHD while no difference was found between severe GVHD and no GVHD (Fig. Furthermore, we analyzed T cell and NK cell functions, T cell subsets including CD4+ T cells, CD8+ T cells and CD4 + CD25 + T cells. Our data showed that NK cells were correlated with GVHD occurrence in the ATG group while no correlation found between NK cells and GVHD in the control group. In addition, although our results indicate that GVHD incident was slightly higher in the ATG group, there was no statistical significance.The standard GVHD grading system was proposed by Dr. Tomas . HoweverIn this study, percentage instead of absolute count number was used to analysis lymphocyte subsets. Due to the different condition regimens, T cell repertoire recovery can vary. In addition, the absolute T cell number recovery period were different, which cannot be used to predict T cell subset recovery. However, during T cell reconstitution, the percentage of different T cell subsets change proportionally, hence different T cell subsets can be compared this way.The optimal balance between GVHD and GVL appears to be an excellent parameter to achieve long-term survival after allo-genetic stem cell transplantation. Previous study has showed that NK cells can eliminate leukemic cells, restore immunity early after stem cell transplant, while at the same time minimal graft versus host disease . The useNK cells are an important arm of the innate immune system and play an important role in mediating anti-tumor immunity of the recipient. Compared with conventional T cells, NK cells have many advantages. For example, allogeneic NK cells can be used as effector cells without the requirement for HLA matching and they do not cause GVHD .Previous study showed an increase in NK cells may induce mild to middle degree of GVHD, if clinicians can intervene at the optimal time point to control GVHD, patients may benefit from the intervention. However, exact higher cell number may connect with poor prognosis of GVHD. NKT cell subsets may have different function in GVHD producing. Hu et al. showed that in GVHD patients, NKG2A + subset was decreased compared to control subsets. However, during recovery from GVHD, the number of NKG2A increased . MonitorOur study showed that ATG can prevent GVHD by increase NK cell percentage. Monitoring the change of NK cell percentage after allogeneic stem cell transplantation can predict the severity of GVHD in the ATG used group. Our study also indicated in the control group, there was no significant difference between severe GVHD and mild GVHD in terms of NK cell percentage. Furthermore, other T cell subsets including CD4 T cells, CD8 T cells, Tregs, and CD8 + CD38+ T cells were unable to indicate GVHD incidence. The dynamic monitoring of lymphocyte subsets in post-transplant patients using ATG as a pretreatment scheme, especially the monitoring of NK cells, can be of great significance in clinic, and the occurrence can be predicted before the clinical occurrence of lethal GVHD symptoms, thus reducing the mortality rate of after transplantation.Additional file 1: Figure S1. NK cells were defined as CD3-CD56 + CD16+, Treg cells were defined as CD4 + CD25 +.


This study aimed to explore the underlying mechanism of linc01014 in oesophagus cancer gefitinib resistance. Gefitinib‐resistant oesophagus squamous cell carcinoma (ESCC gefitinibR) cell lines were constructed by using different gefitinib treatment in FLO‐1, KYAE‐1, TE‐8 and TE‐5 cell lines and confirmed by MTS50 and proliferation assays. Expression of linc01014 was overexpressed/silenced in FLO‐1 cells followed by gefitinib treatment, and then, the apoptosis‐associated markers Bax and Bcl‐2, and PI3KCA in PI3K signalling pathway were determined using Western blotting. MST50 and morphology analyses showed that ESCC gefitinibR cell lines presented obvious gefitinib resistance than their parental ESCC cell lines. ESCC gefitinibR cell lines showed significantly higher proliferation abilities than their parental ESCC cell lines after treating with gefitinib. Overexpression of linc01014 significantly inhibited the apoptosis of FLO‐1 cells induced by gefitinib and silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib. Silencing linc01014 could significantly increase the gefitinib chemotherapy sensitivity of oesophagus cancer via PI3K‐AKT‐mTOR signalling pathway. ESCC gefitinib‐resistant cell lines were successfully constructed.Overexpression of linc01014 significantly decreased the apoptosis of FLO‐1 cells induced by gefitinib.Silencing linc01014 obviously promoted the apoptosis of FLO‐1 cells induced by gefitinib.1Oesophageal cancer is one of the most leading cause of mortality cancer worldwide, and oesophageal squamous cell carcinoma (ESCC) is the main type for oesophageal cancer arising from oesophageal epithelial cells.Epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase, plays critical role in several solid cancers and its chemotherapy resistance regulation.Long non‐coding RNAs (lncRNAs) is a class of transcripts without coding capacity at a length of >200 nt and has reported to play critical roles in the pathogenesis and drug resistance of ESCC.In this study, the role of lncRNA linc01014 involved in gefitinib resistance was explored in ESCC cell lines. According to these investigations, we hope to provide some new insights in solving gefitinib resistance in ESCC and improving the prognosis of patients with ESSC.22.12 in atmosphere. Cells were transfected with linc01014 mimic, si‐linc01014 or negative control (NC) using lipofectamine 2000 (Invitrogen) according to manufacturer's protocol.Human ESCC cell lines KYAE‐1, TE8, TE5 and FLO‐1 were purchased from the Chinese Type Culture Collection, Chinese Academy of Sciences . All cell lines were cultured in PRIM 1640 medium (Gibco) supplemented with 10% fetal bovine serum  and 100 U/mL penicillin/streptomycin (Sigma) at 37°C in a humidified incubator with 5% CO2.2Gefitinib‐resistant cell lines of KYAE‐1, TE8, TE5 and FLO‐1 were constructed via repeatedly subcultured with an increasing concentration of gefitinib (Sigma) starting from 0.1 μM/mL to final concentration of 10.2 μM/mL. Finally, the gefitinib‐resistant cell lines were collected and used for the following investigations.2.33/well and maintained in PRIM 1640 medium contained with 10% FBS overnight. Then, cells were treated with gefitinib in dimethyl sulphoxide  at concentrations of 0, 1, 10, 20 and 30 μmol/L for 48 hours. Concentration of gefitinib that caused 50% inhibition on the MTS activity (MTS50) was determined as previously reported. For proliferation detection, cells were seeded in 96‐well plates at a density of 1 × 103/well and maintained in PRIM 1640 medium contained with 10% FBS overnight. Then, cells were treated with MTS50 determined gefitinib concentration for 0, 48 and 72 hours. Subsequently, cell proliferation was determined. Each experiment was performed in triplicate, and average value was calculated as the results.The cytotoxic effect of gefitinib on ESCC cell lines and the proliferation abilities of these cell lines under different treatments were detected by the MTS ‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulphopheny)‐2H‐tetrazolium]) assay using CellTiter‐96‐AQueous One Solution Cell Proliferation Assay reagent (Promega). Briefly, cells were seeded in 96‐well plates at a density of 5 × 102.4Morphology of cells after treating with or without gefitinib was pictured under light microscope (Olympus CKX41) at 24, 48 and 72 hours at 200× magnification. The morphological appearance of cells was compared at each time‐point.2.5−ΔΔCt method with GAPDH as the internal control. All premier sequences were purchased from RiBio .After treatment, total RNA in cells was isolated using TRIzol reagent (Takara) according to manufacturer's protocol. cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) according to manufacturer's instruction. Then, the expression of linc01014 was determined using SYBR Green Master Mix (Applied Biosystems) with a total of 2 μg cDNA as a template on a BioRad CFX96 Sequence Detection System (Bio‐Rad company). Relative expression of linc01014 was analysed using 22.6After treatment, cells were lysated with RIPA lysis buffer (Millipore) supplemented with protease inhibitors (Sigma). Protein concentrations were measured using BCA method (Pierce) according to the manufacturer's protocol (Beyotime). Then, proteins were boiled with loading buffer and subjected for 10% SDS electrophoresis followed by transferring to polyvinylidene fluoride membranes (Millipore). Subsequently, membranes were blocked with 5% milk at room temperature for 1 hour. Following this, membranes were incubated with a 1:1000 solution of primary antibodies  at 4°C overnight. Then, membranes were incubated with the secondary antibody at room temperature for 1 hour and visualized using ECL method (Pierce).2.7t test or one‐way ANOVA analysis. P < .05 was considered statistically significant.SPSS 19.0 (SPSS Incorporation) was used to perform statistical analyses in this study. Data in this study were presented with mean ± standard deviation (SD). Comparisons among groups were analysed using Student's 33.1The sensitivity of ESCC and ESCC gefitinibR cells to gefitinib was determined by MTS viability assay and cell morphology. The MTS assay showed that FLO‐1‐gefitinibR cell lines showed remarkably higher relative MTS activity than FLO‐1 cells at concentration of 0.10‐10.0 μmol/L, indicating that FLO‐1‐gefitinibR cell lines presented a higher gefitinib resistance that FLO‐1 cells  was used to treat FLO‐1 cells after altering the expression of linc01014. The result showed that overexpression of linc01014 could significantly increase the expression of Bcl‐2 and PI3CA, but decrease the expression of Bax, indicating that linc01014 could inhibit the apoptosis of FLO‐1 cells induced by gefitinib. However, silencing the expression of linc01014 could significantly increase the expression of Bax, but decrease the expression of Bcl‐2 and PI3KCA, indicating that silencing linc01014 could promote the apoptosis of FLO‐1 cells induced by gefitinib , indicating that linc01014 might play a potential role in regulating the PI3K/AKT/mTOR signalling pathway via PIK3CA. In the current study, we have identified that overexpression of linc01014 could significantly increase the expression of PIK3CA and Bcl‐2, but decrease the expression of Bax. In addition, inhibiting the expression of linc01014 could obviously inhibit the expression of PIK3CA and Bcl‐2, but induce the expression of Bax after treated with gefitinib. These findings suggested that linc01014 could significantly regulate the activity of PI3K/Akt/mTOR signalling pathway via regulating the expression of PIK3CA to altering the sensitivity of ESCC cells to gefitinib.Linc01014 is newly identified PIK3CA corresponding lncRNA (There were still some limitations in this study. First, because of the limited clinical samples, the expression of linc0104 in ESCC and gefitinib‐resistant ESCC tissue samples was not explored, which might impair the significance of our study. Moreover, lncRNAs have many regulatory mechanisms to modulate the pathogenesis of disease, but because of the shortage of research funds, the exact mechanism of linc0104 in regulating gefitinib resistance in ESCC was not further explored. A deepened exploration about linc0104 in gefitinib resistance is still required in the further.In conclusion, linc0104 might play a critical role in the pathogenesis of ESCC. Silencing of linc01014 could significantly promote the sensitivity of ESCC cell lines to gefitinib and increasing the apoptosis of ESCC cells via PI3K/AKT/mTOR signalling pathway. These finding might be of importance in understanding and treatment of ESCC gefitinib resistance in clinic.All of the authors have no conflict of interest in this research.Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.All of the authors have consented to publish this research.


Ovarian cancer (OC) is one of the leading causes of female cancer death. Recent studies have documented its extensive variations as a disease entity, in terms of cell or tissue of origin, pre-cancerous lesions, common mutations, and therapeutic responses, leading to the notion that OC is a generic term referring to a whole range of different cancer subtypes. Despite such heterogeneity, OC treatment is stereotypic; aggressive surgery followed by conventional chemotherapy could result in chemo-resistant diseases. Whereas molecular-targeted therapies will become shortly available for a subset of OC, there still remain many patients without effective drugs, requiring development of groundbreaking therapeutic agents. In preclinical studies for drug discovery, cancer cell lines used to be the gold standard, but now this has declined due to frequent failure in predicting therapeutic responses in patients. In this regard, patient-derived cells and tumors are gaining more attention in precise and physiological modeling of in situ tumors, which could also pave the way to implementation of precision medicine. In this article, we comprehensively overviewed the current status of various platforms for patient-derived OC models. We highly appreciate the potentials of organoid culture in achieving high success rate and retaining tumor heterogeneity. Ovarian cancer (OC) is the most devastating gynecologic cancer. Even after development of many surgical techniques and chemotherapies, its overall five-year survival rate is still as low as 47% . The proTP53 is exclusively mutated in HGSC in a proportion as high as 95% [RAS and BRAF are frequently mutated in LGSC [KRAS mutation is also implicated in mucinous carcinoma [ARID1A, PIK3CA, TERT promoter mutations [PTEN, PIK3CA, ARID1A, and CTNNB1 mutations [With many histological variations, OC has been regarded as a highly heterogeneous disease. Indeed, epithelial OC that comprises nearly 90% of OC is usually classified into many histologically distinct subtypes. Major subtypes include serous carcinoma, endometrioid carcinoma, clear cell carcinoma, and mucinous carcinoma. Recently, epithelial OC are more loosely classified into two general categories by integrating the results from histopathological, molecular biological, and genetic analyses, to underscore biological properties of OC. Type 1 cancers grow slowly and contain low-grade serous carcinoma (LGSC), mucinous carcinoma, endometrioid carcinoma, clear cell carcinoma, and transitional carcinoma. Type 2 cancers progress rapidly and contain high-grade serous carcinoma (HGSC), undifferentiated carcinoma, and carcinosarcioma . Research as 95% , RAS and in LGSC . KRAS muutations ,11,12, autations . HoweverBRACA1 or BRCA2 [Despite the highly heterogeneous nature of OC, standard treatment of ovarian cancer is stereotypically composed of aggressive surgery followed by platinum-taxane chemotherapy. Of the four major subtypes, clear cell carcinoma and mucinous carcinoma tends to be refractory to chemotherapy ,15. Moreor BRCA2 , and molor BRCA2 . Nonetheor BRCA2 . HoweverSuch patient-derived models, especially primary cell culture, have not been intensively developed for OC thus far, unlike for cancers of other vital organs. It is not clear whether this is because of any technical difficulties specific to OC or researchers simply did not attempt to obtain patient-derived material for OC. In this article, we comprehensively overview the current status of various patient-derived platforms  and illuCancer cell lines are special types of cells that acquire infinite proliferation potential on plastic dishes. They can expand as a monolayer sheet, with simple media that typically contains fetal bovine serum (FBS). For their high usability as adherent cells, cancer cell lines have long contributed to many areas of scientific research, including cell biology, biochemistry, physiology, and drug discovery, let alone cancer research . To bettOn the other hand, there are several drawbacks in cell lines. Firstly, they tend to be passaged too many times. As the success rate for establishing cell lines from primary tumor tissues is generally low, only a subset of cell lines has been extensively used, thereby cultured over a significantly long period of time or many passages. With the high potential of FBS-based cultures to introduce mutations and genome instability, cell lines could undergo drastic changes in terms of morphology and biological properties, presumably by selection of some specific clones. Consequently, even cell lines under the same name could harbor significant diversity between laboratories . DiversiTP53 was intact, although this is a hallmark mutation in HGSC. On the other hand, it detected mutations in ARID1A, BRAF, PIK3CA, and PTEN, which are commonly mutated in other subtypes of OC. Besides, OVCAR-3 and CAOV3 were not among the top-ranking HGSC cell lines based on integrative molecular profiling, although they in fact possessed TP53 mutations and substantial copy number change. IGROV1 had a hypermutated phenotype, which is frequently observed in endometrioid carcinoma rather than in HGSC. Strikingly, the 12 cell lines evaluated as best candidates for HGSC models accounted for only 1% of the Pubmed literature on the 47 analyzed cell lines. Morphological characterization of 39 OC cell lines also led to a similar conclusion that questioned SK-OV-3, S2780, and IGROV1 as representative models for HGSC [More than 50 OC cell lines have been so far established and are currently available for research. As predicted, discrepancies in histology or mutation profiles between cell lines and original tumors were documented in a study that investigated in detail the validity of 47 OC cell lines derived from various OC subtypes . Among tfor HGSC . These rfor HGSC , suggestTumorigenicity assay was conducted by inoculating 17 OC cell lines into the subcutis, peritoneal cavity, and ovaries of nude mice, revealing that the tumorigenicity varied among inoculation sites and cell lines . For exaPatient-derived xenografts (PDXs) are usually generated by directly engrafting tumor fragments into immunodeficient mice. Not only surgically resected tumors, but also samples from biopsy, ascites, and pleural effusion can be used. Inoculation sites are most commonly subcutaneous tissues. Depending on the tumor types and purposes of the experiments, orthotopic engraftment or intraperitoneal injection could be an option. Established PDXs can be serially engrafted to multiple mice without a culture step in between. Starting from primary tumors, the tumor take rate of PDX is generally higher than the success rate of establishing cancer cell lines. One possible explanation is that microenvironments reconstituted by interactions between subcutaneous tissues and tumor fragments might be physiological, accelerating tumor development faster than in cell lines, with only epithelial cells in an artificial condition. PDXs have been established from various types of cancer . In mostnull (NSG) mice, which could further involve a significant cost. For that reason, use of PDXs for high-throughput drug screening might be limited. Secondly, PDXs often require long latency to be established [+ cells isolated from blood of the same patient were intravenously inoculated to reconstitute a functional human immune system [On the other hand, there are downsides to PDXs. Firstly, maintaining tumors in immunocompromised mice might be costly and require a more specialized skill in serial passage in mice, compared to simple monolayer cultures of cell lines. Besides, mice with more severely compromised immunity than nude mice might be eventually required to increase the tumor take rate. These mice include non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and NOD/SCID/IL2Rγablished , and couablished . In prine system . PDXs wiThe first xenograft model for OC was described back in 1977, in which tumor tissue was engrafted into subcutaneous tissue of nude mice . SeveralThe responses to platinum chemotherapy in OC PDXs were highly correlated with those in the donor patients ,51,52,53As mentioned earlier, establishment of cell lines from primary cultures of patient-derived samples has only a low efficiency. For example, only 10 breast cancer cell lines were established out of 135 resected primary tumors . Other rIntriguingly, 25 novel cell lines were recently established from primary OC with significantly high efficiency, by using culture media and conditions optimized to each histological subtype . More imSpheroids are sphere-like cell aggregates, which are usually maintained as floating three-dimensional structures. Among various spheroid culture methods so far documented , the sta+ CD133+ stem cells derived from cancerous ascites of OC patients [For OC patients, drug resistance and peritoneal dissemination accompanied by cancerous ascites are two major factors that could profoundly affect prognosis. Given the morphological similarity between tumor cell aggregates within cancerous ascites and tumor spheroids in culture, it was natural that researchers initiated spheroid culture studies of OC with cancerous ascites samples. As was observed in other types of cancer, only a subset of ascites-derived cancer cells exhibited stem-cell like properties, which indeed developed tumors in immune-deficient mice . Resectepatients . ThroughFor spheroid culture, standard preparation of tumor samples begins by obtaining singly dissociated cells. However, this was originally optimized for hematopoietic cells or neuronal cells, by which stem cell biology has considerably evolved. In sharp contrast, destruction of cell-cell adhesion could readily induce anoikis for epithelial cells, which significantly lowers the success rate of spheroid formation for many types of tumors of epithelial origin. To address this issue, only partially digested colorectal cancer (CRC) tissues were selectively subjected to spheroid formation, resulting in substantial improvement of the success rate of primary culture . This ap+ could infinitely proliferate in vitro [An organoid is an emerging concept that is literally a mini-organ exclusively composed of epithelial cells. Without the aid of a stromal niche, even a single normal stem cell can differentiate into all the lineages of the organ, while stem cells self-renew, thereby reconstituting in vivo homeostasis of the whole organ. In 2009, it was first demonstrated that murine intestinal stem cells marked by LGR5in vitro . This stin vitro ,82,83. Iin vitro , developin vitro , and epiin vitro . Organoids can be subjected to genetic engineering by viral introduction of shRNA and cDNA. With highly efficient lentiviral gene transduction into murine organoids , we demoOrganoid culture techniques can be applied to various patient-derived samples, such as resected tissue, biopsy, ascites, and pleural effusions. Obvious advantages of organoid culture over PDX and spheroid culture include that it allows propagation of normal cells and precancerous cells, and a higher success rate for cancer cells. In CRC, the success rate of organoid culture was significantly higher than that of PDX or spheroid culture . Given tEstablishment of tumor-derived organoids from various cancer types has been reported, namely colon , pancreaOn the other hand, there are several shortcomings in organoid culture. Firstly, tumor-derived organoids lack stroma, immune cells, and blood vessels. To address this issue, some co-culture systems involving pancreatic ductal adenocarcinoma-derived organoids and murine pancreatic stellate cells was established to investigate interactions between cancer cells and cancer-associated fibroblasts . RecentlUnlike many other types of cancer, only a few studies have documented organoid cultures of OC . Drug reTo better understand the biological features of OC, thorough elucidation of interactions between tumor cells and the microenvironment is a critical issue. However, standard organoid culture consists of only epithelial cells and lacks such interactions. To address this issue, 3D organotypic models that reproduce a similar situation to that in vivo have been developed . SpecifiUntil very recently, use of patient-derived OC as preclinical models has been quite limited because the success rate of primary cultures was low, and even if cell lines are established, features often differ from those of the original tumors. Recent remarkable advances in 3D culture technique have allowed us to reconstitute many features of the original tumors in an in vitro setting. In the light of rapid implementation of precision medicine, which largely depends on genomic information of each cancer, establishment of patient-derived cell-based assays will be of critical relevance. Among various platforms, the organoid might be the most powerful tool in high-throughput drug screening and establishment of xenografts. Now that OC organoids are becoming available, its nation-wide banking will be readily started as a valuable resource for OC researches. Collectively, OC organoids per se and organoid-derived PDX will likely contribute to the development of novel therapies as well as elucidation of its pathogenesis.


To address this challenge, we introduce a novel algorithm for the systematic inference of protein kinase pathways, and applied it to published mass spectrometry-based phosphotyrosine profile data from 250 lung adenocarcinoma (LUAD) samples. The resulting network includes 43 TKs and 415 inferred, LUAD-specific substrates, which were validated at >60% accuracy by SILAC assays, including “novel’ substrates of the EGFR and c-MET TKs, which play a critical oncogenic role in lung cancer. This systematic, data-driven model supported drug response prediction on an individual sample basis, including accurate prediction and validation of synergistic EGFR and c-MET inhibitor activity in cells lacking mutations in either gene, thus contributing to current precision oncology efforts.To understand drug combination effect, it is necessary to decipher the interactions between drug targets—many of which are signaling molecules. Previously, such signaling pathway models are largely based on the compilation of literature data from heterogeneous cellular contexts. Indeed,  Lung adenocarcinoma (LUAD) is a leading cause of cancer related deaths in United States, representing 40% of 225,500 new lung cancer cases every year, and has a 5-year survival rate of only 16% –7. AmongWT patients have been shown to respond to Afatinib -2,5 diphenyl tetrazolium bromide (MTT) assay for short term sensitivity analysis . For colony formation assays, cells were treated with either an EGFR inhibitor  or a c-MET/ALK inhibitor , either individually or in combination (see IC50 (concentration resulting in 50% cell death). Next, cells were treated with 1 uM of Erlotinib and varying concentrations of Crizotinib to identify combinations resulting in IC50 and used the combination index (CI) statistic [To validate pVIPER-predicted, cell line specific EGFR/c-MET synthetic lethality, we selected a panel of 14 cell lines, 11 of which were predicted to be synergistically dependent on EGFR/c-MET , as well as 3 negative controls with no predicted synergistic or individual dependencies on the two TKs . To measure sensitivity to these agents, we used two different and complementary assays, including: (a) colony formation assay to assess long term sensitivity  to either individual inhibitors  or synergistic sensitivity to the combination (HCC78E+C and H2023 E+C)  or both inhibitors  in isolation, thus making the assessment of synergistic drug sensitivity difficult. In addition, three EGFRWT cell lines harboring BRAF  or KRAS (H2122) mutations were also highly sensitive to Erlotinib as a single agent, as predicted by pVIPER, despite the fact that KRAS pathway mutations are mutually exclusive with EGFR mutations and predictive of Erlotinib resistance . Finally, none of these cell lines was predicted to be sensitive to ALK inhibitors, suggesting that Crizotinib sensitivity is mediated by c-MET specific dependencies. Of the negative controls, only one (H2170) showed high sensitivity to Erlotinib. Taken together, 8/11 cell lines (73%) predicted as sensitive to the inhibitors were validated long term colony formation assays, while only 1/3 negative controls showed sensitivity to these agents (33%).Across all 11 cell lines tested in colony formation assay, 8 showed significant sensitivity  and 1 borderline synergy (CI = 0.82), showing the consistency between two assays. However, for two cell lines, H1666 and H2170 (a negative control), results were inconsistent between long term colony formation and MTT assays. For both H1666 and H2170 cell lines, colony formation and MTT assay to showed sensitivity to Erlotinib alone, where colony formation assay has complete abrogation of colonies at 1 μM of Erlotinib, and later had IC50 = 1.25 μM and 3.7 μM for H1666 and H2170 respectively. However, in combination therapy, MTT assay showed antagonism (CI >1), despite the fact that colony formation assay still showed complete abrogation which could be either the difference in the nature of the two assays or associated to the accumulation of new mutations in these cell lines. However, this is just hypotheses and needs to be verified by further experiments such as sequencing of these cell lines pre-and post-treatment.To evaluate the short-term interaction between EGFR and c-MET, we performed MTT assay across 11 cell lines  including 2 negative control cell lines (H520 and H2170). Similar to colony formation assay, we found synergistic sensitivity to EGFR and c-MET inhibitors in 6/9 cell lines (67%), with 5 cell lines showing strong synergy (CI ≤ 0.8) , cell lines responded to Erlotinib in isolation, while when predictions were based on phosphosites EGFR1197 and c-MET1003 , cells exhibited bona fide synergistic sensitivity to the two inhibitors, with the only possible exception of Calu-3, which showed synergistic sensitivity in MTT assays and additive sensitivity to both inhibitors in colony formation assays. Conversely, when predictions were not based on either phosphotyrosine, cells exhibited no sensitivity to the individual inhibitors or the combination . Thus, predictions based on these two phosphosites produced no false positives  and only 2 false negatives (H2170 and H2122), resulting in an error rate of only 2 out of 14 cell lines .In previous section, we assessed the pVIPER predictions after consolidating the result at protein level. Following the results from MTT and colony formation assays, we reanalyzed the pVIPER predictions at the phosphopeptide level. Interestingly, this revealed that whenever synergistic EGFR/c-MET dependencies were predicted from phosphosite EGFR1197 and c-MET1003, cell lines did not respond to the inhibitors, either individually or in combination. For these two peptides, we found their common substrates to be hyper phosphorylated in the sensitive cell lines  compared to the specific substrates of each of them, whereas cell line responding only to EGFR inhibitors showed more hyper phosphorylation of EGFR only substrates . Cell lines resistant to both EGFR and c-MET inhibitors either showed no change in the phosphorylation status or hypo-phosphorylation compared to the normal samples . Hence, either the common substrates of EGFR and c-MET, or the phosphorylation status of EGFR1197 and c-MET1003 could potentially be used as biomarkers for predicting therapy with the dual inhibitors. However, this conclusion is based on a very limited number of observations and lacks the statistical power. This finding needs a re-evaluation/validation using larger cohort of samples to establish an appropriate biomarker for combination therapy.This finding is in agreement with the previously reported finding that the level of tyrosine-1197 phosphorylation on EGFR is predictive of sensitivity . IntriguFig 6). We inferred EGFR dependency in 12 patients. Of these, 5 harbored EGFR mutations, while the remaining 7 patients had not been tested for this mutation, showing a high consistency between our predictions and the genetic predisposition for sensitivity to EGFR inhibitors. In the entire cohort, there were only 3 patients with EGFR mutations that were not identified as EGFR dependent by pVIPER, resulting in an overall sensitivity of 62.5% (5/8). However, it is well known from literature that >50% of patients harboring EGFR mutations do not respond to Erlotinib [Mut state. Similarly, our analysis identified candidate ALK dependencies in 4 patients. Of these one had an established TFG-ALK fusion, whereas the others had not been tested for ALK fusion events.Similar to cell lines, when applied on patient data , pVIPER rlotinib  suggestiAcross all patient samples, we observed Discoidin Domain Receptor-1 (DDR1) to be the most frequent addiction point, which was not predicted for any of the 46 cell lines. One reason for the difference is that DDR1 is collagen dependent and there may be differences in the 3D structure of the tumor and the cell lines growing on the plate. An independent study  in a cohIn this paper, we developed pARACNe to infer Tyrosine Kinase (TK) signaling network using published global phosphoproteomic data from lung cancer. The network prediction was validated using SILAC experiments, with high accuracy. Interrogation of the predicted TK-substrate network generated biologically meaningful hypotheses, followed by experimental validations illustrating the effectiveness of predicted kinase inhibitor combination, EGFR and c-MET combination inhibitors, in treating lung cancer cell lines. Furthermore, Master Regulator Analysis using patient proteomics data provides implications for using targeted agent combinations to treat patients based on their proteomic profile data.pARACNe, addresses critical issues that prevented the direct application of the original ARACNe algorithm on phosphoproteomic profile data. Briefly, the new algorithm addresses critical computational challenges presented by LC-MS/MS and spectral counting data, while incorporating enzymatic signaling characteristics into the algorithm design. In particular, pARACNe is designed to handle three critical issues resulting from the use of LC-MS/MS assays, including the highly sparse nature of phosphopeptide abundance data, the large amount of noise and missing data, and the degenerate peptides-to-protein mapping. The algorithm could be applied to proteomics measurements after preprocessing of the original data. For example, where there are datasets from different programs, they could be first transformed into rank based on total peptides identified. When mapping across datasets, one can fill in missing data using 0 –this type of data could be properly handled by the Iterative Quantile Discretization process in pARACNe. Future improvement of pARACNe includes design of a new schema to directly compute Mutual Information from continuous MS/MS data with many missing value produced by multiplexing platforms.The new method, Notably, pARACNe is significant and powerful as of its global scale and context-specificity in discovering global signaling cascading relationships, which were missing by previous methods. For example, methods proposed by Linding et al.  combine To be noted, as the LC-MS/MS experiments used here was generated based on Tyrosine-kinase enrichment, which is only about ~2% of whole phosphoproteome. pARACNe is shown only on TK-substrates network. The current methodology could be extended to whole phosphoproteomic data based signaling network reconstruction where the data is available. In addition to label free based LC-MS/MS proteomics data used in this work, label-based approaches, such as ITRAQ or TMT, could generate higher throughput whole proteomic profiles which might require future redesign of ARACNe to incorporate both kinases and phosphatases in regulating their downstream substrates. It is reasonable to expect that a version of ARACNe developed specifically to dissect signaling networks should work at least as well as its transcriptional counterpart. Since the relationship between the mRNA abundance of a gene encoding a transcription factor (TF) and the activity of the corresponding protein is much looser than that between the abundance of a phospho-isoform of a kinase and its enzymatic activity.Even though research has attempted to identify addiction points based on gene expression data , predictThe previously published phosphoproteomic data used to reconstruct signaling network was download from . This daSince phospho-tyrosine represents less than 1% of the cellular phosphoproteome, as determined by tandem mass spectrometry (MS/MS), and is difficult to analyze by conventional methods, immuno-affinity purification was performed with a phospho-tyrosine antibody to enrich for phospho-tyrosine containing peptides prior to tandem mass spectrometry. All tumors were identified as NSCLC based on standard pathology. Only those tumors with greater than 50% of cancer cells were considered for further analysis. NSCLC cell lines were grown overnight in low serum to reduce background phosphorylation from culture conditions.http://www.phosphosite.org), a comprehensive resource of known phosphorylation sites.Tandem MS profiling identified 3920 tyrosine phosphorylation sites on approximately 2600 different proteins. 85% of these sites appeared to be novel when compared against PhosphoSite  are then approximated by the corresponding relative frequencies of occurrence I between datasets X and Y is expressed asARACNe is originally designed for gene expression data, where expression of genes is usually continuous and non-sparse. Quantitative data obtained from label-free LC-MS/MS by data-dependent acquisition via spectral counting is discrete and very sparse, with many phosphopeptides counts not observed for multiple peptides in each sample causing the current version of ARACNe to be not suitable for this data, which thus required major modifications to handle discrete data. To handle discrete abundances, we modified the mutual information computation approach from a kernel density estimation based method to a Naïve based estimation of mutual information, which is a histogram based technique. Brieflykij denotes the number of measurements where X lies in ai and Y in aj and N total number of samples.Here Accuracy of mutual information is dependent on correct numbers of bins, M. To find the optimal number of bins we applied ARACNe on the whole dataset by varying M from 1 to 20 and testing the connections in predicted sub-network against the set of known connections (gold standard) from databases (phosphoDB) .. The basic idea is to divide the number of N data points into M with each bin containing equal number of data point. If the data point(s) with the same value falls into consecutive bin(s), we put those data point(s) into current bin and repartition the remaining points into remaining number of bins. We keep iterating this till we finish either the bins or there are no more data points to bin. For example, in Fig 1C step (3), we initially partition N points into 4 bins. The data points with 0 value does not fit into first bin and falls into subsequent bins, so we assign all data points with 0 value into first bin and repartition the remaining points into 3 bins. We keep on doing this process till we achieve 4 bins.In case of continuous data, partitioning can be achieved by dividing the range of data into M equally spaced distance bins. Our data being discrete, equally spaced partitioning was not possible. So, to overcome this problem, we used an iterative approach of partitioning  which gave us precision of 14% and sensitivity of 24%. This precision is an underestimate of real precision as in the gold standard many interactions are not present.To evaluate initial performance and decide number of bins, we computed the network among all tyrosine kinases and substrates, parsed the sub-network between 49 tyrosine kinases and 114 substrates which were present in PhosphoSite database and compared the results with the connections present in database. From our analysis, we found that M = 10 to be an optimal number To discover the master regulator in various cell lines, we interrogated the network obtained from pARACNe using a novel algorithm, VIPER  , designeSkin, was obtained by t-test analysis by comparing each cell line against all normal samples. First, we associate each kinase with positive and negative activity targets, by computing the correlation between each kinase and its predicted substrates and selecting only those substrates which had a significant correlation . Second, for each kinase we computed an activity by measuring the enrichment of the Skin signature in predicted substrates list, separately for both positive and negative correlated, (Skin-enrichment). Enrichment was computed by Gene Set Enrichment Analysis (GSEA). Since very small percentage of kinases are found to have negative correlation, we did not use those interactions to evaluate enrichment.VIPER requires a network model and signature of the phenotype transition . Here, the signature, 2. Cell lines were fingerprinted using the Perplex 1.2 system . Fingerprints were compared to those generated at ATCC and/or our internal database.All cell lines were grown in RPMI-1640 with 5% fetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5% COueous One Solution Cell Proliferation Assay, Promega, Madison WI) were performed as previously described in [IC50 (Inhibitory Concentration of 50%) value. The assays were repeated at least 3 times and the IC50s are the average of all replicates.Short term MTT assays (CellTiter 96 AQribed in . SpecifiLong term colony formation assays were performed in triplicate in 6-well plates. Cells were added to media containing drug and incubated for 1–2 weeks such that control wells (no drug) contained colonies of 50–70 cells each. At such time media was removed and all wells stained with a solution containing 0.5% crystal violet and 6% glutaraldehyde for 1 hour. The plates were then rinsed, dried, and colonies were manually counted.EGFR SILAC experiment was performed in H3255 cell line by treating samples with Gefitinib. c-MET SILAC experiment was performed in c-MET-driven gastric cancer cell line, MKN45, by using c-MET inhibitor Su11274. For both genes, cells were treated with inhibitors for 3 and 24hr. For control, cells were grown in same conditions but were not treated with the drug. For our comparison we combined the peptides, which were differentially obtained between treated and untreated samples, for 3 and 24 hr. More details about the experiment can be obtained from Guo et al .In PhosphoSite database, there were 282 connections between 49 tyrosine kinases and 114 substrates.S1 FigTo select optimal bin number in pARACNe algorithm, precision and recall curves for various number of bins were computed. Black curve is when no binning of data is done. When using 10 bins, the algorithm achieved the best performance.(PDF)Click here for additional data file.S1 TableA three-column table. Column one and two are the two interacting peptides. Column one represents the gene symbol with dollar sign ($) and numbers specify the sites of phosphorylation in the protein. Column two is the substrate, representing a gene symbol followed by underscore and a number of phosphorylation sites. Column three is the mutual information between the two peptides in column one and column two.(XLSX)Click here for additional data file.S2 TableA three column table includes kinase, mutual information and substrate. Column two is the maximum mutual information when aggregating the peptide network into protein level network.(XLSX)Click here for additional data file.S3 TableThe results for colony formation assay. First column is the cell line, second column is the treatment used, and third column is the mean number of colonies (from triplicates) after normalizing to the control group for each cell line.(XLSX)Click here for additional data file.S4 Table50 for Erlotinib, third Column is the IC50 for Crizotinib, fourth column is the IC50 of Crizotinib when cell cells were treated with 1 uM of Erlotinib and varying concentrations of Crizotinib and fifth column is the combination index (CI).The results for MTT assay. First column is the cell line, second column is the IC(XLSX)Click here for additional data file.