In silico analysis revealed that miR-744-3p targeted 2 distinct signaling cascades which eventually upregulated MMP-9 expression in LSCC. First, miR-744-3p could suppress programmed cell death 4 (PDCD4), a direct suppressor of NF-κB (p65). PDCD4 could also prevent AKT activation and suppress MMP-9 expression. Further, suppressing miR-744-3p expression could restore phosphatase and tensin homolog (PTEN) expression. PTEN could inhibit AKT activation and inhibit MMP-9 expression in LSCC cells. The results revealed that suppressing miR-744-3p was effective to inhibit LSCC metastasis by inactivating AKT/mTOR and NF-κB (p65) signaling cascade. Targeting miR-744-3p could be a valuable therapeutic intervention to suppress the aggressiveness of LSCC.MicroRNA controls cancer invasion by governing the expression of gene regulating migration and invasion. Here, we reported a novel regulatory pathway controlled by miR-744-3p, which enhanced expression of matrix metallopeptidase 9 (MMP-9) in laryngeal squamous cell carcinoma (LSCC). We profiled the differential micoRNA expression pattern in LSCC cell lines and normal epithelial cultures derived from the head and neck mucosa using microRNA microarray. MiR-7-1-3p, miR-196a/b and miR-744-3p were expressed differentially in the LSCC cell lines. Subsequent validation using real-time PCR revealed that high miR-744-3p level was positively correlated with regional lymph node metastasis of LSCC. Real-time cellular kinetic analysis showed that suppressing miR-744-3p could inhibit migration and invasion of LSCC cell lines and reduce the number of lung metastatic nodules in nude mice modules.  Cancer of the larynx originating from the head and neck region is the second most common upper-aerodigestive cancer , 2. HistMicroRNAs are highly conserved non-coding RNAs (~18–25 nucleotides) involved in gene regulation at post-transcription level. The single-stranded mature microRNA can bind to the target mRNA at the 3′ untranslated region (3′UTR) forming a stable duplex by Watson-Crick complementarities at a partial complementary manner. By recruiting and incorporating with the RNA-induced silencing complex (RISC), translation of the target mRNA transcript is hindered and/or the mRNA is subjected to degradation , 8. MicrIn silico analysis showed that miR-744-3p could directly target the mRNA transcript of both programmed cell death 4 (PDCD4) and phosphatase and tensin homolog (PTEN), both of which had been reported to be correlated with LSCC metastasis [In order to identify the key functional microRNA in LSCC, we performed microRNA profiling in LSCC cell lines using microRNA microarray and validated the results in LSCC tissues. We found that deregulation of miR-744- 3p was a common event in LSCC. At present, however, its functions in LSCC remains to be defined. tastasis –13. Redutastasis . PDCD4 ktastasis , 16; PTEtastasis , which atastasis . Both PTtastasis . Our resP < 0.05)  Figure . The micNext, we validated the microarray results by analyzing the aberrant expressed microRNA level  in a cohort of 47 LSCC tissues using QPCR and compared with the paired normal tissues . Let-7a-3p was not detected in all the LSCC tissues and the paired normal epithelia. MiR- 7-1-3p, miR-338-5p and miR-365a-5p were detected in the LSCC tissues and the paired normal epithelia. However, there were no significant difference in the expression level between cancer and the normal tissues (P > 0.05). In the microarray results, miR-34a-3p expression was found in normal epithelial cell lines and was undetectable in the LSCC cell lines. In the validation set using laryngeal tissues, however, miR-34a-3p was significantly upregulated in tumor (P = 0.013). Thus, we shortlisted miR-196a, miR-196b and miR-744- 3p as candidate microRNA and explored their clinical significance by evaluating the statistical association with the clinicopathological parameters of LSCC patients.Three microRNA  were significantly upregulated in the LSCC tissue (P = 0.007). Thus, we proposed that miR-744-3p overexpression could promote migration and invasion of the LSCC cells.All the LSCC cases were grouped into high expression and low expression group using median expression level of each microRNA in LSCC as cut- off points. As shown in Table in vitro data, the miR-744-3p suppressed LSCC cell line was injected into nude mice and the formation of lung metastatic foci were evaluated on day 60 after injection. Mice injected with miR-744-3p suppressed LSCC cells group had lower number of metastatic cancer nodules in comparison with the control group  expressing the miR-744-3p shRNA. Significant reduction in migration and invasion ability was observed in both SNU899 and SNU1076 Figure . To confp Figure .To evaluate the functional target of miR-744- 3p, the target transcript was predicted using miRWalk. In total, 2004 genes were simultaneously predicted by 2 or more databases. Of which, 13 genes had been reported to be downregulated in LSCC tissues . Next, wP = 0.037) and PTEN (P < 0.001) were significantly associated with the regional lymph node metastasis of LSCC patients. Reduced PTEN expression was also found to be significantly associated with the advanced clinical stage (P = 0.036). Furthermore, MiR-744-3p was negatively correlated with PTEN  and PDCD4  in LSCC tissues . Target gene prediction analysis revealed that PDCD4 and PTEN 3′-UTR harbored the binding sites for miR-744-3p seed sequence. In the Ago2-IP/IgG-IP, PDCD4 and PTEN mRNA levels were reduced when the endogenous expression of miR-744-3p were suppressed by shRNA  protein levels were significantly reduced. The phosphorylated IKKα/β and IκB-α level remained unchanged in the miR-744-3p suppressed LSCC cell lines Figure .P < 0.001, Figure P = 0.011, Table MMP-9 expression was significantly upregulated in the LSCC tissues (P < 0.05).As activated AKT and NF-κB (p65) could promote MMP-9 upregulation in head and neck cancers, we hypothesized that the miR-744-3p modulated MMP- 9 expression in LSCC. As shown in Figure As a famous tumor suppressor gene, PDCD4 was described as inhibitor of cancer cell migration and metastasis in different cancer forms including LSCC. Low PDCD4 expression was observed to be significantly related with poor differentiation and lymph node metastasis in LSCC patients , 13. HowIn the PDCD4 overexpressing clones, phosphorylated AKT and phosphorylated NF-κB (p65) levels were significantly reduced, while PDCD4 protein level was significantly increased Figure . The phoP < 0.05).Everolimus is an mTOR inhibitor. Exposing LSCC to everolimus will lead to a reduction of the downstream gene regulated by mTOR signaling cascade. LSCC exposed to everolimus showed reduction in MMP-9 expression Figure . MiR-744As there was currently no data on the pathological functions of miR-744-3p, we first examined the tissue expression level and evaluated the statistical association with the clinicopathological characteristics of our LSCC patients. The association between high miR-744-3p expression and regional lymph node positive suggested that miR-744-3p could possibly play a part in controlling LSCC migration and invasion. To establish the causal relationship, we established the miR-744-3p suppressed LSCC cell lines and measured the changes in migration and invasion property of LSCC cells using real-time cellular kinetic assays and animal models. Our results confirmed that the expression level of miR-744-3p could have a functional impact on the migration and invasion propensity of LSCC cells.MMP-9 expression is regulated by the upstream regulator PDCD4 and PTEN. MMP-9 could shape the development of epithelial–mesenchymal transition (EMT) phenotype in LSCC , 36. FurOur data showed that decreased PDCD4 was associated with the nodal status of LSCC patients indicating the role in LSCC migration and invasion. PDCD4 could suppress MMP-9 expression by inactivating AKT/mTOR signaling or reducing NF-κB (p65) activation , 39. In The phosphatidylinositol 3 kinase (PI3K) / protein kinase B  and mammalian target of rapamycin (mTOR) signaling cascade is the most commonly altered signaling cascade in head and neck carcinoma. PI3K  is a family of intracellular lipid and serine/threonine kinases. It functions by phosphorylating the downstream phosphatidylinositols and phosphoinositides at the 3′-hydroxyl group which triggers a cascade of signaling event. Upon receiving external stimulus, PI3K is recruited to the plasma membrane by the activated receptor and catalyzes the formation of phosphatidylinositol 3, 4, 5- bisphosphate (PIP3) from phosphatidylinositol 4, 5- bisphosphate (PIP2). PIP3 will activate the serine/threonine protein kinase Akt  and activate its activity by phosphorylating threonine 308 (by 3-phosphoinositide dependent kinase) or serine 473 (by rapamycin-insensitive mTOR complex/mTORC2) after binding AKT with its PH domain , 42, 43.Everolimus is a selective inhibitor of mTOR approved by FDA for use in cancer treatment. As miR- 744- 3p could simultaneously target dual signaling channels responsible for MMP-9 activation, we hypothesized that suppressing miR-744-3p could have a therapeutic impact on inhibiting MMP-9 expression and should have a better efficacy than everolimus. In comparison with everolimus, MMP-9 expression was significantly reduced in miR-744-3p suppressed LSCC cell lines indicating that targeting miR-744-3p had a therapeutic impact in preventing MMP-9 upregulation in LSCC.Collectively, our data suggested that miR-744-3p was an oncogenic microRNA in LSCC. On one hand, miR-744-3p suppressed the mTOR upstream regulatory element PTEN in the PI3K/AKT/mTOR regulatory axis, which controlled MMP-9 transcription. On the other hand, miR-744-3p activated another MMP-9 regulatory axis by provoking the signaling cascade controlled by NF-κB p65 via PDCD4 suppression  were obtained from Korean Cell Line Bank . Normal Forty-one paired LSCC and the corresponding normal epithelia were obtained from the First Affiliated Hospital, Sun Yat-sen University, Guangdong, China. Six paired LSCC tissues were obtained from Department of Surgery, Queen Mary Hospital, The University of Hong Kong. Consent of tissue donation for research purpose was obtained before tissue collection. Tissues were collected from patients with no prior treatment including radiotherapy and/ or chemotherapy. All the tissues were snap-frozen in liquid nitrogen and were stored at −80°C until use. The protocol was approved by the human research ethics committee of Sun Yet-sen University (reference number: 2013_23) and University of Hong Kong (reference number: UW 10-142).P < 0.05 and 1.5-fold difference were considered as deregulated microRNA in LSCC.RNA quality was evaluated using Agilent 2100 bioanalyser (Agilent Technologies) prior to microarray experiment. All the samples had rRNA Ratio 28s/18s) over 1.8 and RNA Integrity Number (RIN) over 8.0 . Reaction was performed at 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Table Total RNA from the cell lines and tissues were extracted with TRIZOL reagent (Invitrogen). For microRNA cDNA preparation, microRNA was polyadenylated by using the Poly (A) Polymerase Tailing Kit (Illumina). The cDNA product was then generated using High-Capacity cDNA reverse transcription kit (Invitrogen) following the methods described by Reichenstein et al. . For conA stem-loop shRNA with imperfect complementation was constructed according to the antisense miR-744-3p sequence (5′-GACAACGGTGATTGGAGTTGGA-3′). The miR-744-3p shRNA coding sequence was cloned into lentivector pGreenPuro (System Biosciences) to generate the miR-744-3p shRNA expressing vector (pGreenPuro-miR-744-shRNA vector). PDCD4 insert for construction of stable LSCC clone expressing PDCD4 was amplified using Roche FastStart High Fidelity PCR System (Roche Applied Science). The forward primer for PDCD4 insert amplification was: 5′- GCA TAATGGATGTAGAAAATGAGCA-3′, and the reverse primer was: 5′- CGTACTCAGTAGCTCTCTGGTTTAA −3′. The PDCD4 coding sequence was cloned into lentivector pCDH-CMV-MCS-EF1-copGFP (System Biosciences) generating the pCDH-PDCD4 vector. The pGreenPuro-miR-744-shRNA or pCDH-PDCD4 vector as well as their mock vectors was further packaged into lentiviral particles by 293-FT cells (Life technologies) using the Lenti Starter Kit (System Biosciences). Subsequently, the lentiviral particles were transduced into the LSCC cell lines SNU899 and SNU1076 by TransDux™ (System Biosciences).xCELLigence Real-Time Cell Analyzer (Roche Applied Science) was used to measure the changes in migration and invasion ability of LSCC cells in response to the changes in miR-744-3p or PDCD4 levels. CIM-Plate 16 was adopted for the migration and invasion assay. The dynamic changes of cell number (expressed as cell migration index) from upper chamber (coated with fibronectin) to lower chamber due to the migratory capacitance of LSCC cells were measured continuously over 24 hours. For the measurement of invasion propensity, the upper chamber of CIM-Plate 16 was coated with Matrigel  for 4 hour at 37°C before cell seeding. Subsequently, the dynamic invasion of LSCC cells through the Matrigel layer was recorded and expressed as cell invasion index.http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) was used to predict the candidate transcripts which are targeted by miR-744-3p [MiRWalk  on 10% SDS-PAGE. Proteins were electroblotted onto polyvinylidine difluoride (PVDF) membranes  by semi-dry transfercell (Bio-Rad). Membranes were incubated with PTEN (D4.3) XP® rabbit monoclonal antibody  or PDCD4 (D29C6) XP® rabbit monoclonal antibody  or Phospho-Akt (Ser473) antibody  or Akt (C67E7) Rabbit monoclonal antibody  or phospho-IKKα/β (Ser176/180) (16A6) rabbit monoclonal antibody  or phospho-IκBα (Ser32/36) (5A5) mouse monoclonal antibody  or NF-κB p65 (L8F6) mouse monoclonal antibody  or IKKα/β (H-470) rabbit polyclonal antibody (Santa Cruz) or IκB-α (C-21) rabbit polyclonal antibody (Santa Cruz) at 1:1,000 dilution, or phospho-NF-κB p65 (Ser536) (93H1) rabbit monoclonal antibody  or MMP-9 (D6O3H) XP® Rabbit monoclonal antibody  at 1:500 dilution, or beta-actin specific antibody (Sigma-Aldrich) at 1:10,000 dilution at 4°C overnight. Signals were developed using chemiluminescence Plus Western Blotting Detection System (Amersham) and analyzed using ImageJ software version 1.48 (http://rsbweb.nih.gov/ij/).Western blot was performed using Mini-PROTEAN® 480.Interaction between miR-744-3p and its target genes was confirmed by microRNA:mRNA immunoprecipitation using anti-Ago2 monoclonal antibody-immobilized beads (Wako) and human Argonaute 2 miRNA isolation kit . Expression levels of miR-744-3p, PTEN mRNA and PDCD4 mRNA within the Ago2-IP complex were quantitated by Real-time PCR on LightCyclerThe sense and antisense strands of the 3′-UTR of PTEN containing the binding sites of hsa-miR-744- 3p were synthesized. The oligonucleotides for wild-type 3′-UTR of PTEN were as follows: sense strand: 5′-CCACATCCTACCCCTTTGCACTGGCAACAGATA AGTTTGCAGTTGGCTAA-3′; antisense strand: 5′- AGC TTTAGCCAACTGCAAACTTATCTGTTGCCAGTGCA AAGGGGTAGGATGTGGACT-3′. The two single strands of the 3′- UTR of PTEN harboring a mutation in the binding sites of hsa-miR-744-3p were also synthesized. The oligonucleotides for mutant 3′-UTR of PTEN were as follows: sense strand: 5′-CCACATCCTACCCCTTT GCACCTATCGTCGATAAGTTTGCAGTTGGCTAA-3′; antisense strand: 5′-AGCTTTAGCCAACTGCAAACTT ATCGACGATAGGTGCAAAGGGGTAGGATGTGGAG CT-3′. The sense and antisense strands of wild-type and mutant 3′-UTR of PTEN were annealed and cloned into the SacI and HindIII sites of the pMIR-REPORT Luciferase vector (Applied Biosystems) to generate Luc-wild-type vector and Luc-mutant vector respectively. The Luciferase vectors containing the binding sites for miR-744-3p in 3′-UTR of PDCD4 with or without mutation were constructed using the above methods. The oligonucleotides for wild-type 3′-UTR of PDCD4 were as follows: sense strand: 5′-CGGTGCCATTGCTC TCGTTTGGGCAACAAGAGTGAAACTCTTGTCTCA AAA-3′; antisense strand: 5′-AGCTTTTTGAGACAA GAGTTTCACTCTTGTTGCCCAAACGAGAGCAATG GCACCGAGCT-3′. The oligonucleotides for mutant 3′-UTR of PDCD4 were as follows: sense strand: 5′-CGG TGCCATTGCTCTCGTTTGTATCGAGAGAGTGAAAC TCTTGTCTCAAAA-3′; antisense strand: 5′-AGCTTTTT GAGACAAGAGTTTCACTCTCTCGATACAAACGAG AGCAATGGCACCGAGCT-3′. MiR-744-3p silencing LSCC cells and their controls were seeded in 96 well plates for 24 hour before transfection. Cells were co- transfected with 50 ng of Luc-wild-type vector or Luc-mutant vector, along with 50 ng of pMIR-REPORT β-galactosidase control vector (Applied Biosystems) using Lipofectamine™ 2000 (Invitrogen). Firefly luciferase and β-galactosidase activities were measured 48 hour after transfection by Dual-Light luminescent reporter gene assay kit . Luciferase activity was normalized against β-galactosidase activity. The assay was performed in an LB 96V microplate luminometer (EG & G Berthold).5) were injected intravenously into the tail veins of athymic nu/nu mice . The animals were euthanized on day 60 after injection and the lung were removed for histological examination. Hematoxylin and Eosin (H&E) staining was performed on the lung sections to evaluate the number of metastatic foci. The protocol for animal study was approved by the Institutional Committee on the Use of Live Animals in Teaching and Research (Protocol number 3675–15).LSCC cells . All results were presented as mean ± SE from three or more independent experiments. The difference between primary tumors and their normal counterparts was calculated by Wilcoxon Signed Rank test. Chi-square test and Pearson correlation test was used for evaluation on the clinical significance of microRNAs or genes in LSCC. The difference between control groups and engineered cell lines were analyzed used student-


Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHELIgm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis.Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, “natural” antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that  Dynll1. While ASCIZ and DYNLL1 are also required for producing normal numbers of B-2 cells, we discovered that they regulate B-1a cells and B-2 cells by distinct genetic mechanisms. Finally, we found that ASCIZ also contributes to the early onset of B-1a B cell-derived lymphoid cancers in juvenile mice. The results provide insight into the development of an important cell population of the immune system.Antibody-producing B cells can be segregated into two major populations: The better known conventional B-2 cells typically produce high-affinity and mono-specific antibodies, but only after they encounter a particular pathogen or in response to vaccines. In contrast, the B-1a cells constitutively produce lower-affinity broad-specificity “natural” antibodies that serve as a preemptive defense against a wide range of microbes. Here we reveal that the transcription factor ASCIZ and its target DYNLL1 are essential for mice to have a normally sized pool of B-1a cells in place shortly after birth. We show that these two factors function in a single linear pathway during the development of B-1a cells. This interaction represents a rare example where the activity of a transcription factor, in this case ASCIZ, can be explained by the effects of a single target gene, in this case  B lymphoid cells are essential for the humoral immune response by producing a diverse range of antigen-specific antibodies. Antibody-mediated immunity is provided by two distinctive B cell lineages that diverge early in life. The better-known conventional, or B-2, B cells provide adaptive immunity by producing high-affinity pathogen-specific antibodies, typically in a T cell-dependent manner. The pool of B-2 cells is constantly replenished from hematopoietic stem and progenitor cells in the bone marrow . B-1a ceIgh locus through V(D)J recombination at the pro-B cell stage (Hardy fraction B); association of this unique IgH with a generic surrogate Ig light chain (λ5 and v-preB) to form the pre-B cell receptor (pre-BCR) in pre-B cells (fraction C); pre-BCR-dependent clonal expansion by ~5 cell division cycles during the large pre-B cell stage (fraction C’) [Igλ/κ light chain (IgL) loci during the small pre-B cell stage (fraction D); and association of IgH and IgL chains to form an IgM complex/B cell receptor (BCR) at the immature B cell stage (fraction E). The immature B cell stage represents a critical quality control phase during which cells with high-affinity self-reactive BCRs are eliminated through BIM-dependent apoptosis [Ig loci through somatic hyper-mutation of Ig variable regions and class-switch recombination of Igh constant regions.During their differentiation in the bone marrow, B-2 lineage precursors go through a series of well-defined developmental stages , 4. Critpoptosis . ImmaturWhereas adult B lymphopoiesis in the bone marrow almost exclusively produces B-2 cells, B-1a cells are derived from fetal stem cells, and B-1a precursors dominate early B cell development in the fetal liver and early post-natal spleen , 7. Thus2+-finger protein ASCIZ , whas ATMIN –16, playas ATMIN  and B ceas ATMIN . DYNLL1 as ATMIN –22, but as ATMIN , 24, incas ATMIN . We haveDynll1 largely phenocopies the B-2 cell developmental defects seen in Asciz-deleted mice, although the two genes function in part by different mechanisms. We also show that the ASCIZ-DYNLL1 axis is particularly critical for the development of B-1a cells. Surprisingly, the B-1a and B-2 cell deficiencies in Dynll1-deleted mice were differentially affected by genetic modifiers, indicating that DYNLL1 regulates the development of the two B cell lineages via distinct genetic mechanisms.Based on its established role in the B cell defects observed in ASCIZ-deficient mice, we sought to directly investigate the role of DYNLL1 during B cell development. We show here that conditional deletion of Dynll1 allele (Dynll1fl) using the Mb1-Cre knock-in allele, which is efficiently expressed from the late pre-pro-B cell stage onwards [Mb1-Creki/+Dynll1fl/fl-deleted mice compared to Mb1-Creki/+ or Dynll1fl/fl controls  ..Fig 2). tion C’) . This dicontrols . These d+ IgM-) were stained for DNA content analysis, and gated on the larger cycling pre-B cells  and via-B cells . This anse cells . Moreove animals . There w animals . This inTp53 gene in these mice. However, concomitant conditional deletion of Tp53 did not increase the numbers of cycling fraction C’ cells (and subsequent fraction D and E cells) in Mb1-Cre Dynll1-deleted mice , recirculating mature B cells (fraction F) and splenic B cells compared to the single knockout animals . This inAsciz/Dynll1 double mutant animals (which completely lack DYNLL1) compared to the Asciz single mutants  depends on the successful recombination of the Igλ/κ loci during the small pre-B cell stage (fraction D), we tested whether complementation with a pre-arranged BCR transgene could restore the numbers of immature B cells in Mb1-Cre Dynll1-deleted mice.The more severe phenotype of the f DYNLL1 , 17, 32)-deleted  and AsciSWHELIgh knock-in and Igl transgene [Mb1-Cre Dynll1-deleted mice to a level comparable to those seen in Mb1-Cre control mice (Mb1-Cre SWHEL control mice (presumably because the chicken-specific SWHEL BCR does not activate auto-reactive apoptotic programs in mice). It should be noted that the SWHEL transgene did not increase the numbers of immature B cells in Mb1-Cre Asciz-deleted mice [Mb1-Cre Dynll1-deleted mice supports the notion that DYNLL1 may play an ASCIZ-independent role in some aspect of Igl gene rearrangement or BCR surface expression.Remarkably, the anti-hen egg lysozyme-specific ransgene  restoredrol mice , althougted mice . Thus, tDynll1-deleted, Asciz-deleted and Asciz/Dynll1-double deleted mice. At this age, B-1a cell pools are established in the peritoneal cavity and thereafter expand through self-renewal [Next, we quantified B-1a cells in 4-week-old -renewal , 3, 7. R- CD93+ B220-/low CD19+)[Dynll1-deleted embryos compared to control littermates  were redtermates , while etermates . In the ecursors . Interesge cells , than liDynll1 and Asciz single mutants, presumably through accelerated self-renewal, but interestingly, they did not increase in the Asciz/Dynll1-double mutants  VH10 heavy chain knock-in, led to a ~5-fold reduction of B-1a cells compared to control mice, and thereby further compounded the B-1a lymphopenia in Dynll1-deleted mice  has a specific synthetic lethal effect on the B-2 but not the B-1a cell lineage.We recently reported that loss of DYNLL1 (or ASCIZ) leads to a dramatic synthetic lethal interaction with oncogenic MYC expression driven by the ge cells . We ther animals . Notablyent mice . Similarcontrols . In factTp53 in Eμ-Myc mice leads to the extremely early development of aggressive B cell lymphomas that cause severe morbidity requiring euthanasia by 40 days of age [Eμ-Myc Tp53+/- mice suggests that they are most likely derived from the B-1a cell lineage. To test if the ASCIZ-DYNLL1 axis contributes to the development of early postnatal lymphomas, we generated compound mutant Mb1-Cre Tp53flAscizflEμ-Myc mice. Similar to germline Tp53+/- heterozygosity, conditional B-lymphoid-specific loss of one allele of p53 caused early-onset MYC-driven lymphomas with a median survival of 37 days  in our SWHEL mice, even without the Vκ10 light chain transgene, led to a notable reduction of B-1a cells, and a corresponding compounding effect on the B-1a lymphopenia in Dynll1 mutant animals , and approved by the St. Vincent’s Hospital Melbourne Animal Ethics Committee, approval numbers 047/12, 019/13, and 002/17.Asciz , Bim [Dynll1 [tp53 [Bim knockout [Mb1-Cre , SWHEL  and Eμ-Myc [SWHEL mice used here were homozygous for the anti-HEL VH10 heavy-chain knock-in, and contained an anti-HEL Vκ10 light chain transgene. Primers used for genotyping are listed in references [Mice were housed in specific pathogen-free micro-isolators. All mouse mutations were generated on an inbred C57BL/6 background, or had been backcrossed to the C57BL/6 background for at least 10 generations. Conditional  [Dynll1  and tp53l1 [tp53  alleles,knockout , Mb1-Cred Eμ-Myc  mice havferences , 18.Mice were analysed at the ages indicated in the figure legends. Ethical endpoints of survival analyses of tumor-prone mice were determined by trained animal technician who were blinded to the genotypes. Mice with different genotypes were co-housed in the same micro-isolators.Spleen, fetal liver, bone marrow and tumour cell suspensions were prepared as described . Bone maThe following reagents were used for staining: B220-APC (eBioscience), B220-FITC (Biolegend), BP1-PE (BD), CD1d FITC (BD), CD3ε-biotin (eBioscience), CD5-FITC (BD), CD8α-biotin (Biolegend), CD11b-APC-Cy7 (BD), CD11b-biotin (Biolegend), CD19-APC eFluor780 (eBioscience), CD19-PerCP-Cy5.5 (eBioscience), CD21-PE (Biolegend), CD21/35 APC (BD), CD23-biotin (BD), CD24-FITC (BD), CD43-PE (BD), CD93-PerCP-Cy5.5 (eBioscience), GR1-biotin (eBioscience), IgD-eFluor450 (eBioscience), IgM-biotin (eBioscience), IgM-PE-Cy7 (BD), IgM(b) Biotin (BD), NK1.1-biotin (Biolegend), Pre-B Cell Receptor Biotin , Streptavidin-BV605 (Biolegend), Ter119-biotin (eBioscience) and AnnexinV-eFluor450 detection kit (eBioscience).Western blot analysis was performed as described  using anBcl2 and Hmbs were purchased from ThermoFisher Scientific. Ct values were normalized to Hmbs as the endogenous control and expressed as deltaCt (dCT).Total RNA was isolated using Isolate II RNA Micro Kit (Bioline) and reverse transcribed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems) following the manufacturer’s protocol. Real time PCR was performed using AmpliTaq Gold DNA Polymerase (Applied Biosystems) and a LightCycler 480 (Roche). TaqMan primers for Statistical analysis was performed using GraphPad Prism software (San Diego). P values were calculated by two-tailed unpaired Student’s t test, or Mantel-Cox test for survival analyses. Grouped data were analyzed using the multiple T test function  using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Error bars represent the mean ± SEM. Numbers of mice per analysis, and numbers of independent experiments are indicated in the figures or legends.S1 Fig(A) Representative FACS plots and gating strategies for the analysis of follicular and marginal zone B cells.  Quantification (mean ± S.E.) of follicular B cells (B) and marginal zone B cells (C) in the spleens of 8-10-week old mice.(TIF)Click here for additional data file.S2 Fig+CD43- bone marrow cells from one Mb1-Cre control mouse, one Dynll1fl/fl control mouse and two Dynll1-deleted mice with fractions D , E (immature) and F (recirclating) highlighted. (B) Mean fluorescence intensity (M.F.I.) of the IgM signal in fraction E cells (mean ± S.E.).(A) FACS plots of B220(TIF)Click here for additional data file.S3 Fig+B220+ bone marrow lymphocytes (mean ± S.E.). Representative FACS plots and gating strategies to determine pre-BCR (SL156) expression on bone marrow pre-B lymphocytes (mean ± S.E.) in 8-to-10-week old mice. (C) Mean fluorescence intensity (M.F.I.) of the pre-BCR (SL156) signal on CD43(TIF)Click here for additional data file.S4 FigRepresentative FACS plots indicate gating strategies to determine total and mature splenic B cell numbers.(TIF)Click here for additional data file.S5 FigRepresentative FACS plots used for the quantification of B-1a cell numbers in the peritoneal cavity of 8-week old mice in (TIF)Click here for additional data file.S6 FigRepresentative FACS plots of peritoneal cavity cells of 8-week old mice related to the quantitative analyses in (TIF)Click here for additional data file.S7 FigRepresentative FACS plots of peritoneal cavity cells of 8-week old mice, related to (TIF)Click here for additional data file.


By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments , whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals; however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0); catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1  and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function.P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson’s disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity; however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes,  P-type ATPases are an ancient family of transmembrane proteins that use the energy derived from hydrolysis of ATP to actively transport substrates across membranes . These tC. elegans genome encodes three P5B ATPases: CATP-5, CATP-6 and CATP-7. These proteins have a high degree of similarity, particularly in the M4 transmembrane domain , the National Bioresource Project , and the laboratory of Dr. Barth Grant . The following alleles were used in this study: xnIs459[yfp::ral-1a + unc-119(+)] III ) IV, catp-7(dx185[gfp::catp-7 + loxP]) IV, catp-7(dx191[mcherry::catp-7 + loxP]) IV, catp-7(dx193[gfp::catp-7 + loxP]) IV, ltIs44(Ppie-1mCherry::phPLCδ) V [catp-5(dx187[gfp::catp-5 + loxP]) X, catp-5(tm4481) X [bgIs312[pes-6::gfp] [xnIs484 [mCherry::sec-10 + unc-119(+)] [pwIS1039[psnx-1citrine::hgrs-1unc-54UTR-cb-unc-119], pwIs1175[psnx-1tgn-38::gfpunc-54UTR-cb-unc-119]; unc-119(ed3), pwIs1176[psnx-1ss-gfp-cd4-hcimprunc-54UTR-cb-unc-119]; unc-119(ed3) [All strains were maintained at 23.5°C on nematode growth medium (NGM) plates with  AMA1004  as food  AMA1004 . Some of(+)] III , catp-6(4438) IV , catp-7(m4481) X , bgIs312-119(+)] , pwIS103Alignments were performed by using CLC Main Workbench 8.0 (Qiagen Bioinformatics). Parameters were kept as default . Transmembrane domains were predicted by using TMHMM 1.6 CLC Main Workbench Plugin. Domains and motifs were identified according to the literature .Core sequences were identified according to . Alignmehttps://blast.ncbi.nlm.nih.gov/Blast.cgi or directly from http://flybase.org and http://www.wormbase.org. Alignment was performed by using the alignment tool of CLC Main Workbench 7.9.1 (Qiagen Bioinformatics). Parameters were kept as default . We tested for the appropriate model by using MEGA6.06 ) IV, by using sgRNA(catp-7#12): catgaggaggcggatgggg (underlined PAM). gfp + loxP integration was obtained as described in the previous section.We targeted exon 2 of catp-7a in the background of catp-6(ok3473) by using the following sgRNAs: sgRNA(catp-7#12): catgaggaggcggatgggg and sgRNA(catp-7#2): cgttcatccttttctaccgtggg (underlined PAM). The PAM of the first sgRNA was mutated silently from ggg to gcg. DMSC gfp-fusion repair template was constructed according to [gfp + loxP was inserted into the genome 3’ of the following sequence: cgaccgcctccaccccatcc. And 5’ of the sequence: cgtgggagttcaattggaaa. The integration led to a frameshift 3’ of gfp. Worms were propagated as heterozygotes (catp-6(ok3473) catp-7(dx189)/ nT1[qIs51] ).We targeted exon 2 of rding to  by usingWorms were dissected in M9 to release gonadal tissues. Gonads were transferred to polylysine-D-coated slides and covered with coverslips . The sliAnimals were mounted on 4% agarose pads for confocal, DIC and epifluorescence microscopy. Whole worms were immobilized with 10 mM levamisole and/or 1 mM sodium azide. Animals were imaged by using either Leica TCS SP5 II confocal microscope (Leica Application Suite LAS software) or Zeiss Axioskop 2 and MetaMorph software (Molecular Devices). Image processing was performed in Fiji/imageJ 2.0.0 , brightncatp-5(tm4481) + Ppgp-12catp-5::gfplet-858 + rol-6(su1006), bgIs312[pes-6::gfp], xnIs459[yfp::ral-1a + unc-119(+)] III and xnIs484 [mCherry::sec-10 + unc-119(+)]. Fluorescence recovery over time was measured via Fiji/imageJ 2.0.0 ) X), as native GFP::CATP-5. The expression pattern of native GFP::CATP-5 is nearly identical to that of CATP-5::GFP. However, we also detected expression on the plasma membrane of the distal tip cells (DTC)   and on tcatp-6 coding sequence, with expression driven by the putative catp-6 promoter (1135 bp 5’ of exon 1 of catp-6a). The expression pattern of this transgene was essentially identical to that of the gfp-modified version of fosmid WRM067B_F08 used in our previous study ) IV) utilized GFP, whereas the other (catp-6(dx179[catp-6::degron::mKate2::3xFlag + loxP]) IV) utilized mKate2 tag preceded by a degron motif . On. Oncatp-on motif . . To tag the native locus, we inserted the coding sequence of either gfp or mcherry into the second exon of isoform catp-7a via CRISPR/Cas9 (catp-7(dx185[gfp::catp-7 + loxP]) IV and catp-7(dx191[mcherry::catp-7 + loxP]) IV), To construct a tagged version of CATP-7 for use on extrachromosomal arrays, we inserted fp-fusion strains were not backcrossed after they were generated, but they did not show any obvious differences compared to wt N2. We estimated the average brood size for each single gfp-fusion strain in comparison with wt N2 .The CRISPR/Cas9-mediated single th wt N2 . There ifp-fusion strain had reached the L4 stage, which is comparable to wt N2.Additionally, we estimated the growth rate of the progeny (1 h layoff) from 30 worms . 48 h afCATP-5 and CATP-7 are both expressed in the intestine. Native GFP::CATP-5 and transgene CATP-5::GFP localize to the apical brush border of the intestinal cells , Fig 5A.excretory specifically expressed in the excretory cell of catp-5 mutant animals (catp-5(tm4481) + Ppgp-12catp-5::gfp + rol-6(su1006)) and cytosolic GFP (bgIs312[pes-6::gfp]) [xnIs459[yfp::ral-1a], marker for cytosolic vesicles) and mCherry::SEC-10  , plus twmembrane ). To quaexcretory displays a Mf of only 0.33%, which is highly significantly different from cytosolic GFP . Thus, it is very unlikely that the signal we observe is coming from cytosolic GFP in the excretory cell for transgene catp-5::gfpexcretory.As expected, cytosolic GFP shows a calculated Mf of 92% . Whereasp-value = 0.0003369 and adj. p-value = 0.0001015 respectively). But there is no statistical differences detectable between the Mfs of: CATP-5::GFPexcretory vs. YFP::RAL1, CATP-5::GFPexcretory vs. mCherry::SEC-10 nor YFP::RAL1 vs. mCherry::SEC-10.The cytosolic vesicle-associated control YFP::RAL1 shows a Mf of 26% and the luminal membrane control SEC10::mCherry shows a Mf of 14%. Both Mf values are significantly different from the Mf value of cytosolic GFP  in relation to RFP-labeled compartment markers expressed in the spermatheca .In living spermathecal cells, CATP-6::GFP strongly colocalizes with LMP-1::RFP expressed from a transgene . HoweverIn the case of RFP::RAB-5 we observed overlap with CATP-6::GFP in proximity to the plasma membrane of basolateral cell-cell junctions. This is consistent with the expected localization of RAB-5 to early endosomes . The loccatp-6 and catp-7 loci are very tightly linked on chromosome IV, it was not possible to obtain a homozygous double-labeled strain by meiotic recombination. Therefore, we used CRISPR/Cas9 to generate a strain that expresses both native CATP-6::mKate2 and native GFP::CATP-7. The strain was not backcrossed but did not show any obvious difference compared to wt N2. We estimated the average brood size of this strain  within the mitotic, transition, and pachytene regions  is a 705 bp deletion that removes the coding sequences for 235 aa of M3 and M4 including the putative substrate binding site, the phosphorylation site (P domain) within the large intracellular loop, plus half of the N domain and also results in a frameshift (catp-5(0) animals have an overtly wt phenotype, as expected  ; catp-5(tm4481)). Among the double mutant progeny obtained from this strain, we found that most (catp-6(0), this suggests that catp-6(+) probably provides maternal rescue . Young adult catp-6(0); catp-5(0) animals have a fully developed gonad, but the number of germ cells is smaller than in wild type (catp-6(0); catp-5(0) animals showed no obvious phenotype in terms of DTC migration (hat most . The remhat most  and appehat most . Since tild type . These aild type . Mid to igration .catp-6(0); catp-5(0) animals are obviously different from wt N2. The DTCs inappropriately continue to migrate, leading to abnormal gonad arm positions (catp-6(0); catp-5(0) animals are fertile (1%), but these have a greatly reduced brood size, ranging from 1 to 19 progeny  catp-7(0) double mutant by meiotic recombination. Therefore, we used CRISPR/Cas9 to generate a null allele of catp-7 in the background of catp-6(0). We exchanged 1492 bp between exon 2 and exon 5 of isoform catp-7a with the coding sequence of gfp. The deletion removes the coding sequences for Ma to M3, the A domain (dephosphorylation motif TGE) and results in a subsequent frameshift (catp-7(dx189), catp-7(dx189) is not able to encode a functional transporter protein. Since we found that catp-6(0) catp-7(dx189) animals are usually sterile (Ste); the double mutant chromosome was maintained over a balancer (catp-6(ok3473) catp-7(dx189)/ nT1[qIs51] ).catp-6(0) catp-7(dx189) worms that are derived from a heterozygous hermaphrodite display a high fraction of sterility (81% n = 59). These animals also typically exhibit abnormal vulva phenotypes, including Pvl, Evl (everted vulva) and Muv (Multivulva). Furthermore, 12% (n = 59) of catp-6(0) catp-7(dx189) were Vul . Homozygous catp-6(0) catp-7(dx189) worms derived from a homozygous hermaphrodite, are more severely affected: 55% (n = 78) are Vul (Ste (Vul)) and 100% (n = 78) are sterile (Ste (other), catp-6(+) and/or catp-7(+) probably confer maternal rescue of these gonadal defects .Homozygous catp-6(0) catp-7(dx189) worms exhibit a delay in DTC migration when compared to wt L4 of the same stage (catp-6(0) catp-7(dx189) animals often show a fully reflexed gonad (catp-6(0) catp-7(dx189) worms have often completed spermatogenesis, but oogenesis and ovulation are typically delayed  catp-7(dx189); catp-5(0) from hermaphrodites of genotype catp-6(ok3473) catp-7(dx189)/ nT1[qIs51] ; catp-5(tm4481). All of the catp-6(0) catp-7(dx189); catp-5(0) animals produced by this strain were sterile. Furthermore, 76% (n = 123) had an abnormal vulva phenotype—everted, protruding, or multivulva (Ste (other)) and 24% (n = 123) were Vul (Ste (Vul), We obtained animals of genotype catp-6(0) catp-7(dx189); catp-5(0) animals showed a strong delay in DTC migration when compared to wt from the same stage , catp-6(0) and catp-7(0) is viable and fertile. However, we observed distinct synthetic sterile phenotypes in the catp-6(0); catp-5(0) and catp-6(0) catp-7(dx189) double mutants. We do not observe any mutant phenotypes in the CRISPR/Cas9 tagged strains ; catp-5(0) double mutants, have nearly normal somatic gonad development, but exhibit delayed germline proliferation and have very small brood sizes. Since both CATP-5 and CATP-6 are present on the plasma membrane of pachytene-stage germ cells, the simplest interpretation is that these two proteins function redundantly within this tissue to take up a common substrate from the extracellular fluid. However, CATP-6 is also expressed on the plasma membrane of the gonadal sheath cells, which are physiologically connected to the pachytene-stage germ line via numerous gap junctions  IV.(EPS)Click here for additional data file.S3 Figcatp-6(ok3473) catp-7(dx189[delta 1492 bp Ma-M3 + gfp + loxP] IV.(EPS)Click here for additional data file.S4 Figcatp-6(ok3473) catp-7 X.(EPS)Click here for additional data file.


Necrosis, apoptosis and autophagic cell death are the main cell death pathways in multicellular organisms, all with distinct and overlapping cellular and biochemical features. DNA damage may trigger different types of cell death in cancer cells but the molecular events governing the mode of cell death remain elusive. Here we showed that increased BH3-only protein BIK levels promoted cisplatin- and UV-induced mitochondrial apoptosis and biphasic ROS production in HCT-116 wild-type cells. Nonetheless, early single peak of ROS formation along with lysosomal membrane permeabilization and cathepsin activation regulated cisplatin- and UV-induced necrosis in p53-null HCT-116 cells. Of note, necrotic cell death in p53-null HCT-116 cells did not depend on BIK, mitochondrial outer membrane permeabilization or caspase activation. These data demonstrate how cancer cells with different p53 background respond to DNA-damaging agents by integrating distinct cell signaling pathways dictating the mode of cell death. In the cytosol, cytochrome c, APAF-1 and caspase-9 form the apoptosome complex and activated caspase-9 triggers the activation of executioner caspase-3 and caspase-7 by cleaving them.The mitochondrial apoptotic pathway is strictly regulated by the selective protein-protein interactions between antiapoptotic and proapoptotic BCL-2 protein family members. In respBik locushave been reported in colorectal cancers, head and neck cancers, gliomas and renal cell carcinomas. AlgiMatrix dissolving buffer (ThermoFisher Scientific) was used to harvest spheroids before lysis in 1% CHAPS buffer. Proteins (600–1000 μg) were immunoprecipitated with BCL-2 , BCL-XL , MCL-1  antibodies at 4°C for 16h and coimmunoprecipitates were captured by Dynabeads Protein G at 4°C for 2 h. Beads were recovered using DynaMag spin magnet and washed twice in 1% CHAPS buffer. Total cell lysates and immunoprecipitates were separated on NuPage 10% Bis-Tris gels. After SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore) and then blocked with 5% dried milk in PBS-Tween20. Membranes were incubated with primary and secondary antibodies  in a buffer containing 10% milk diluent-blocking concentrate (KPL), detected with Luminata Crescendo Western HRP substrate (Millipore). Blots were imaged with LAS4000 image analyzer (Fujifilm) on chemiluminescence mode. The following antibodies were used for immunoblotting: BCL-2 , BCL-XL , Actin , BIK , MCL-1 , p53 .JC-1 (ThermoFisher Scientific) plate-based BH3 profiling was done as described before, 29. GenCells were transfected with BIK siRNA  and negative control (scrambled) siRNA  by using Hiperfect transfection reagent (Qiagen) according to manufacturer’s instructions. Protein knockdown efficiencies by siRNA transfection were verified by immunoblotting 24h following transfection. For 3D cell culture experiments, cells were transfected with corresponding siRNA duplex for 12h and transferred to AlgiMatrix 3D culture plate for the growth of cell spheroids in the presence of siRNA treatment for every 24h. Protein knockdown efficiencies in cell spheroids were verified by immunoblotting.The activity of caspase-3 and caspase-9 was determined by ApoAlert Caspase Profiling Plate (Clontech) according to manufacturer’s protocol. The release of fluorochrome AMC was analyzed at 380 nm excitation and 460 nm emission using Spectramax Gemini microplate fluorometer. Data shown are mean±SEM of three independent experiments and expressed in arbitrary fluorescence units per mg of protein.2.?? and subjected to treatments as indicated. Cells were fixedin 4%paraformaldehyde for 10 min at room temperature, washed once in cold PBS and incubated for 10 min in ammonium chloride solution. Cells were washed twice in cold PBS and then permeabilized using 1% BSA, 0.3% Triton X-100 in PBS containing 5% goat serum for 20 min. Following permeabilization, cells were incubated with galectin-3 antibody  overnight at +4°C, washed three times in 0.25% BSA, 0.1% Triton X-100 in PBS and incubated with Alexa Fluor-488 antibody  for 1h at room temperature. Cells were washed three times in 0.05% Tween-20 in PBS and once in cold PBS, mounted using SlowFade Diamond AntifadeMountant with DAPI (ThermoFisher Scientific) and examined by using EVOS FLoidCell Imaging Station (ThermoFisher Scientific).Galectin-3 immunofluorescence staining to evaluate lysosomal membrane permeabilization was performed as described before. Briefly2DCFDA (ex/em 490/520) and MitoSOX Red (ex/em 510/580) reagents (ThermoFisher Scientific) were used to determine Total ROS and superoxide production, respectively. Briefly, cells were grown on 96-well transparent bottom black plates and stained with CM-H2DCFDA or MitoSOX Red according to manufacturer’s protocol. Kinetic measurements of total ROS and superoxide fluorescence levels were processed for 12h every 15 min on Spectramax Gemini microplate fluorometer (Molecular Devices). Data shown are mean±SEM of three independent experiments.CM-Hg to pellet cellular debris and for 20 min at 16000g to obtain mitochondrial fraction. The resulting supernatant was centrifuged at 100000g for 45 min to obtain cytosolic extract. 10 μl cytosolic extract was mixed with 85 μl experimental buffer . 5 μl of z-FR-AMC (25 μM) was added and incubated for 30 min at 37°C. The release of fluorochrome AMC was analyzed at 380 nm excitation and 460 nm emission using Spectramax Gemini microplate fluorometer.Cathepsin B/L-specific activity was measured as described before. BrieflyStatistical significance of the results was analyzed using Student’s t-tail test using GraphPad Prism 6.2 software. *P<0.05 and **P<0.01 were considered significant.in vivo. To test the alteration of BIK levels in response to DNA damage in HCT-116 wt and p53 -/- cells, we treated the cells with (20 μM) or UV (100 mJ/cm2) for0-12h. BIK protein levels were detected by immunoblot analysis. As shown in Previous reports demonstrated the essential role of BIK in both caspase-dependent and caspase-independent cell death pathways. It has been shown that BIK was largely localized in the endoplasmic reticulum (ER) and BIK-induced release of Ca2+ from the ER in augmented mitochondrial cytochrome c release occurs through Drp-1-mediated remodeling of mitochondrial cristae , 6, 31. c release[in vivo milieu than 2D cell culture systems  for 12h and BIK mRNA levels were detected by real-time qPCR. Results are expressed as fold over control . (B) HCT-116 wt and HCT-116 p53 -/- cells were treated with 10 μg/ml cycloheximide (CHX) and BIK protein levels were detected at 0-24h by immunoblot analysis. Protein expression levels were semi-quantitatively determined by densitometry and shown as a ratio of BIK/Actin. (C) p53 transcriptional activity following treatment with cisplatin (20 μM) or UV (100 mJ/cm2) for 12h was measured in untreated or BIK siRNA-treated HCT-116 wt cells using a dual luciferase reporter assay . 100 μM BIK BH3 peptide was added to cell lysates before measurement for Firefly/Renilla dual luciferase activities where indicated. (D) BIK siRNA- or scrambled siRNA-treated HCT-116 wt cells were exposed to cisplatin (20 μM) or UV (100 mJ/cm2) for 16h. The mitochondrial release of cytochrome c was evaluated by using an ELISA-based assay .(A) p53 expression status in HCT-116 wt and HCT-116 p53 -/- cells were evaluated by immunoblot analysis. HCT-116 wt and HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm(AI)Click here for additional data file.S2 FigBIK siRNA- or scrambled siRNA-treated HCT-116 wt cells were exposed to cisplatin (20 μM) or UV (100 mJ/cm2) for 12h. Cells were stained for active conformation-specific (A) BAX (6A7) or (B) BAK (Ab-1), followed by incubation with FITC-conjugated secondary antibody. Active BAX-related immunofluorescence was analyzed by flow cytometry. Red, control cells; blue, treated cells.(AI)Click here for additional data file.S3 FigBIK siRNA- or scrambled siRNA-treated HCT-116 p53 -/- cells were treated with cisplatin (20 μM) or UV (100 mJ/cm2) for 12h. Cells were stained for active conformation-specific (A) BAX (6A7) or (B) BAK (Ab-1), followed by incubation with FITC-conjugated secondary antibody. Active BAK-related immunofluorescence was analyzed by flow cytometry. Red, control cells; blue, treated cells.(AI)Click here for additional data file.S1 Filec release, measurement of BIK protein half-life, p53 reporter assay and flow cytometry-based detection of BAX/BAK activation accompanies this paper.Supplementary materials and methods section describing additional experimental procedures including real-time qPCR, assessment of cytochrome (PDF)Click here for additional data file.


Radiation-induced genomic instability plays a vital role in carcinogenesis. Bod1 is required for proper chromosome biorientation, and Bod1 depletion increases premature chromatid separation. MiR-142-3p influences cell cycle progression and inhibits proliferation and invasion in cervical carcinoma cells. We found that radiation induced premature chromatid separation and altered miR-142-3p and Bod1 expression in 786-O and A549 cells. Overexpression of miR-142-3p increased premature chromatid separation and G2/M cell cycle arrest in 786-O cells by suppressing Bod1 expression. We also found that either overexpression of miR-142-3p or knockdown of Bod1 sensitized 786-O and A549 cells to X-ray radiation. Overexpression of Bod1 inhibited radiation- and miR-142-3p-induced premature chromatid separation and increased resistance to radiation in 786-O and A549 cells. Taken together, these results suggest that radiation alters miR-142-3p and Bod1 expression in carcinoma cells, and thus contributes to early stages of radiation-induced genomic instability. Combining ionizing radiation with epigenetic regulation may help improve cancer therapies. Radiation-induced genomic instability (RIGI), a common long-term complication resulting from exposure to ionizing radiation , is charBiorientation of chromosomes in cell division 1 (Bod1), a novel protein found in vertebrate centrosomes and outer kinetochores, locates and corrects aberrant syntelic attachments in mitotic spindles and is required for proper chromosome biorientation . Bod1 deet al. recently found that miR-142-3p expression was lower in cervical carcinoma cells than in normal cervical epithelium cells [et al. reported that miR-142-3p inhibits cervical cancer cell proliferation and invasion by targeting frizzled class receptor 7 (FZD7) [MicroRNAs (miRNAs) are an important class of conserved, small noncoding RNAs that target the 3′-UTRs of target mRNAs to repress mRNA expression post-transcriptionally or promote mRNA degradation –14. miRNum cells , and Den7 (FZD7) . HoweverIn this study, we found that radiation induced premature chromatid separation in 786-O and A549 cells. In addition, irradiation altered the expression of both miR-142-3p and Bod1. MiR-142-3p targeted the Bod1 3′-UTR sequence and inhibited its expression, and overexpression of miR-142-3p induced premature chromatid separation and G2/M arrest in 786-O cells by inhibiting Bod1. Furthermore, either overexpression of miR-142-3p or knockdown of Bod1 sensitized 786-O and A549 cells to X-ray radiation.RIGI promotes the acquisition of genetic alterations, including karyotypic abnormalities , 4, of whttp://www.targetscan.org/) and microRNA.org (http://www.microrna.org/) predicted that Bod1 is a potential target of miR-142-3p. To identify whether both miR-142-3p and Bod1 were involved in the biological effects of irradiation, we measured mature miR-142-3p and Bod1 expression in 786-O cells exposed to X-rays using quantitative RT-PCR (qRT-PCR). As shown in Figure Because Bod1 depletion causes premature chromatid separation , we inveUsing the Target Scan and microRNA.org databases, we identified two predicted, highly-conserved putative binding sites for miR-142-3p in the 3′-UTR of Bod1 Figure . To direCells were co-transfected with miR-142-3p mimics and dual-luciferase reporter vector to confirm the target prediction results. Bod1 wild-type 3′-UTR luciferase activity, but not Bod1 3′-UTR-mut activity, decreased in 786-O cells after miR-142-3p transfection Figure . qRT-PCRNext, we investigated whether Bod1 and miR-142-3p act together to influence chromatid separation in 786-O and A549 cells. First, we knocked down Bod1 expression in 786-O and A549 cells using siRNA. As shown in Figure et al. reported that miR-142-3p also inhibits cell cycle progression and induces G2/M arrest [Bod1, which corrects aberrant syntelic attachments in mitotic spindles via activation of the protein phosphatase 2A (PP2A)/Polo-like Kinase 1 (Plk1) pathway , 10, is M arrest . As showWe next investigated whether the sensitivity of 786-O and A549 cells to X-rays was affected by siRNA-induced Bod1 knockdown. As shown in Figure Because miR-142-3p targeted Bod1, we investigated whether transfection of miR142-3p mimics altered responses to radiation similarly to Bod1 knockdown in 786-O and A549 cells. As shown in Figure To determine whether Bod1 overexpression inhibited miR-142-3p-induced premature chromatid separation, we transfected a Bod1 overexpression vector into 786-O and A549 cells. As shown in Figure Because Bod1 knockdown induced premature chromatid separation and sensitized carcinoma cells to X-rays, we investigated whether Bod1 overexpression also affected irradiation-induced premature chromatid separation and radiosensitivity. 786-O and A549 cells were transfected with Bod1 overexpression vector to increase Bod1 levels. Bod1 overexpression inhibited X-ray-induced premature chromatid separation both in 786-O and A549 cells Figure . ProlifeProper timing of separation is crucial for balanced chromosome segregation. A cohesin complex maintains connections between sister chromatids until anaphase begins –27. Impaet al. reported that Bod1 is necessary for the regulation of PP2A activity and Plk1 at the kinetochore and for establishing and maintaining chromatid cohesion [et al. reported that inhibition of PP2A increases intracellular p-Plk1, TCTP, and Cdk1 levels and decreases p53 levels, ultimately increasing cell cycle arrest, aberrant mitosis, and radiation-induced inhibition of cell proliferation [We found that radiation increased premature chromatid separation in 786-O and A549 cells compared to un-irradiated cells, and western blots and chromosome spread scores confirmed that Bod1 was involved in this effect Figure . Porter cohesion . Inhibitcohesion , 34 and cohesion . PP2A incohesion –38. Lv eferation . These set al. reported that miR-142-3p suppresses a group of cytoskeletal regulatory genes, including Bod1, during megakaryopoiesis [Epigenetic modifications are important in cancer development and radiation response, and continued investigations of epigenetic mechanisms will likely identify new potential targets for clinical cancer therapies . MiRNAs opoiesis , and miRopoiesis . We founRenal cell carcinoma, a lethal urologic malignancy that originates in renal tubular epithelial cells, comprises approximately 90% of all kidney cancers and accounts for 2-3% of all cancers worldwide , 47; lunmiRNAs increase the sensitivity of tumors to radiotherapy by increasing cell cycle arrest , 54, andIn summary, we found that radiation induces premature chromatid separation via the miR-142-3p/Bod1 signaling pathway in 786-O and A549 cells; this mechanism, which involves interactions between epigenetic regulation and radiation response, may be an early contributor to the development of RIGI. Furthermore, we found that irradiation altered miR-142-3p/Bod1 signaling pathway activity, and either knockdown of Bod1 or overexpression of miR-142-3p sensitized 786-O and A549 cells to irradiation. Although the molecular mechanisms underlying premature chromatid separation are not fully understood, our results suggest that epigenetic modifications contribute to early RIGI processes.2.786-O cells  and A549 cells (human lung carcinoma cells) were obtained from the American Type Culture Collection . 786-O and A549 cells were maintained in RPMI-1640 medium  supplemented with 10% (v/v) fetal bovine serum  and 1% penicillin/streptomycin . Cells were cultured at 37°C in a humidified atmosphere containing 5% COX-ray irradiation was conducted at a Faxitron RX-650 facility  at 100 kVp and 5 mA at room temperature. The target of this instrument is wolframium (W), and the dose was 0.97 Gy/min.Chromosome spreads were performed as previously described . BrieflyThe 3′-UTR of Bod1 containing the putative miR-142-3p binding site was PCR-amplified from normal human cDNA, and the mutant construct was generated by mutating the miR-142-3p seed sequence (from ACACUACA to CATGACAA and CGATCGAC). The wild-type and mutant Bod1 3′-UTRs were cloned into the psiCHECK2 luciferase vector . The constructs were verified by DNA sequencing.MiRNA-142-3p mimics and negative controls were purchased from RiboBio . SiRNA targeting Bod1 and negative controls were purchased from GenePharma . Bod1 siRNA (5′-GCCACAAAUAGAACGAGCAAUUCAU-3′) was constructed as described . The BodThe Bod1 3′-UTR segments containing the predicted miR-142-3p binding site or a mutated miR-142-3p target site were chemically synthesized by Sangon Biotech . These segments were annealed and inserted into the pmirGLO Vector, a dual-luciferase miRNA target expression reporter vector (Promega). 786-O cells were then co-transfected with 150 ng reporter vector and 50 nM miR-142-3p mimics using Lipofectamine 2000 in a 96-well white plate (Corning). Firefly and renilla luciferase activity in cell lysates were assayed using the Dual-Glo Luciferase Assay System (Promega) after transfection, and firefly luciferase activity was normalized to renilla luciferase activity.ΔΔCt method with the following equation: RQ (Relative Quantitation) = 2−ΔΔCt.Total RNA was extracted from cells with TRIzol Reagent (Invitrogen). Reverse transcription was conducted with the Transcriptor First Strand cDNA Synthesis Kit  and qRT-PCR was conducted using SYBR Green PCR Master (Roche) to measure mRNA expression. Primers for Bod1 and the GAPDH internal control were purchased from GeneCopoeia. For miRNA detection, reverse transcription and qRT-PCR was performed using an ALL-in-one™ miRNA qRT-PCR Detection Kit (GeneCopoeia). Primers for miR-142-3p and the U6 internal control were also purchased from GeneCopoeia. qRT-PCR was performed using a Bio-Rad Chromo4 System RealTime PCR detector . All procedures were conducted according to the manufacturers' protocols. Relative fold-change in mRNA expression was calculated using the 2−Cells were lysed in RIPA buffer  with Protease Inhibitor Cocktail Tablets . Lysate total protein concentrations were determined using a protein assay kit . Equal amounts of protein were denatured with loading buffer (Beyotime) at 100°C for 10 min, then loaded onto a 12% SDS-PAGE gel for electrophoresis and transferred to a methanol-activated polyvinylidene fluoride membrane . The membrane was blocked in tris-buffered saline (TBS) containing 5% bovine serum albumin  for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. Bod1  and GAPDH  primary antibodies were used. After washing with TBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. Secondary antibodies conjugated with HRP included Goat-Anti Rabbit IgG  and Rabbit-Anti Goat IgG . Relative protein levels were quantified using Image J software.6 cells/mL, incubated with 100 μg/mL DNase-free RNase A and 0.2% Triton X-100, and stained with 50 μg/mL PI  at 4°C for 30 min. A total of 104 nuclei were examined with a BD LSRFortessa™ cell analyzer (BD Biosciences) and DNA histograms were analyzed using Flowjo software. The experiment was repeated at least three times.For cell cycle analysis, transfected cells in the logarithmic growth phase were harvested by trypsinization 48 h post-transfection, washed with PBS twice, fixed in 70% ethanol overnight at 4°C, pelleted, resuspended in PBS at 1 × 103 cells/well 24 h after transfection. 24 h later, cells were exposed to X-rays at a dose of 4 Gy. MTT solution (0.5 mg/mL) was added to each well at different time points  after radiation. After incubation at 37°C for 4 h, cell-free supernatant was removed and the resulting formazan crystals were dissolved in 200 μL of DMSO. Absorbance was measured at 490 nm with a microplate reader. All experimental treatments were repeated in five wells each. The experiment was repeated at least three times.Cell proliferation was measured using a 3--2, 5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 35-mm dishes 24 h before transfection with miRNA-142-3p, siBod1, or both Bod1 vector and miRNA-142-3p. The transfected cells were trypsinized and plated in a 96-well microplate at a density of 2×10After irradiation, cells were washed with PBS buffer, trypsinized, and counted using a cell counter (Coulter). An appropriate number of cells were plated into 60-mm dishes to produce colonies. After incubating for 10 d, the cells were fixed with 10 mL fresh Carnoy's fluid and stained with 0.5% crystal violet for 20 min. Colonies with more than 50 remaining cells were counted as survivors. Plating efficiencies (PE) were calculated as follows: number of colonies formed/number of cells plated. Survival ratios were calculated as follows: PE (irradiated)/PE (un-irradiated). All experiments were performed in triplicate and repeated at least three times.48 h after radiation, cells were fixed with Carnoy's fluid for 20 min at room temperature and stained with 20 μL of Acridine Orange in an aqueous solution (10 μg/mL). Analyses were performed with a fluorescence microscope (Axio Imager Z2) at 20× magnification. At least 500 cells were scored for each sample. The experiment was repeated at least three times.P values) of differences in means between two samples were evaluated using Student's t-tests. A P value < 0.05 (*) was considered statistically significant (**P < 0.01). Graphs show the means ± standard error of at least three independent experiments.Statistical significance (


Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of  Drosophila eye serves as an excellent model to study patterning, growth, gene expression, function and disease [Drosophila eye develops from an eye imaginal disc housed inside the larva [ disease –3. The she larva –7. It ishe larva , 9, whiche larva . The larhe larva , 12. Eache larva , 12. Durhe larva , 12. Thihe larva , 13.decapentaplegic (dpp), which encodes a homologue of secreted Bone Morphogenetic Proteins (BMPs), belongs to the TGFβ superfamily [dpp expression is restricted to a stripe of cells moving along with the MF [dpp first expresses at the ventral margin [dpp expresses along the posterior and lateral margins of the eye disc [hedgehog (hh), a ligand for Hh signaling, and other transcription factors [hh expresses in the peripodial membrane [hh expresses in the center of the posterior margin, where it triggers MF formation and its progression [yorkie (yki), can negatively regulate MF progression by activating Wg signaling in the developing eye [Several signaling pathways are involved in MF formation and progression. The MF initiation and progression depends on the expression of erfamily . In the h the MF , 15. In l margin –18. In teye disc , 20. Dpp factors , 21–24. membrane , whereasgression , 26. Dppgression –30. Inteping eye . In ordeDrosophila model is the availability of large repository of tools like Gal4/UAS system [dpp-Gal4 (BL-1553 and others) [dpp-lacZ line, lacZ reporter expression moves with the MF in the developing eye along the temporal axis.One of the strengths of S system –34, whic others) , can mar others) . Howeverdpp, which will be a great tool for studying regulation of MF formation and progression during patterning and development of the Drosophila eye.A collection of transgenic lines were generated at the Janelia farm by taking overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes of interest, which were cloned upstream of GAL4, and then inserted into a defined genomic location by site-specific recombination . The ratdpp, viz., GMR17E04-Gal4 and GMR18D08-Gal4. GMR17E04-Gal4 drives the transgene expression along the MF and GMR18D08-Gal4 drives expression in the entire eye imaginal disc but more robustly on dorso-ventral margins. However, to our surprise, the GMR17E04-Gal4 drives expression in the entire pupal retina whereas the GMR18D08-Gal4 drives expression only in the ventral half of the pupal retina. GMR18D08-Gal4 can also serve as a ventral pupal retinal specific marker. These two CRMs are different from the CRM of dpp-lacZ reporter, which exhibits eye specific dpp expression in Drosophila.Here we present characterization of two eye-specific enhancer lines of http://flybase.bio.indiana.edu). The stocks used are UAS-GFP-NLS [Sp/CyO; dpp-Gal4/TM6B Hu(BL-1553) [dpp-lacZ/CyO [wg-GFP[wgRNAi [hpo [yki3SA [GMR17E04-Gal4 (BL-48770), GMR17G08-Gal4 (BL-48784), GMR19B04-Gal4 (BL-48839), GMR19D09-Gal4 (BL-45833), GMR16G02-Gal4 (BL-47472), GMR18B08-Gal4 (BL-45437), GMR18D08-Gal4 (BL-45442), and GMR19C03-Gal4 (BL-49283) for dpp gene [dpp-CRM lines were generated with the aim to analyze their ability to drive expression of GFP as well as RFP reporter genes. We used an enhancer trap [dpp[The stocks used in this study are described in flybase    and were fixed in 4% para-formaldehyde for 20 minutes. Imaginal discs were washed in PBS after fixation and stained following the standard protocol –49. AntiDrosophila eye images were taken using a Zeiss Apotome Imager.Z1 microscope. The flies were prepared by freezing them at -20°C for around 2 hours. The legs and wings of the flies were removed and flies were mounted on a dissection needle, and the fly was positioned on a glass slide using mounting putty [Adult ng putty –54. Imagdpp-lacZ reporter under the dpp enhancer [dpp-Gal4 (dpp>GFP) driver in the larval brain, eye-, leg- and wing- imaginal disc  eye-antennal imaginal disc  eye-antennal disc  is similar to dpp-lacZ expression in the late first instar eye-antennal disc  eye-antennal disc. However, dpp>GFP expression remains restricted to the posterior margin of the eye disc and does not move along with the MF  compartmental boundary. Both dpp–lacZ  eye imaginal disc. Therefore, it is not an optimal driver for the MF specific expression.We wanted to study the expression of enhancer , 40 withnal disc . The dppnal disc , which fnal disc . At thisnal disc . The dppnal disc  and latenal disc . Unlike h the MF . In the dpp–lacZ  and dpp> lines from GMR collections   , only tw8D08>GFP  and GMR17E04>GFP  exhibit B08-Gal4 , GMR19D0D09-Gal4 , GMR16G0G02-Gal4 , GMR17G0G08-Gal4 , GMR19B0B04-Gal4  and GMR1C03-Gal4  did not al brain . GMR18B0al brain . None ofnal disc , leg imanal disc  or haltenal disc . We haveGMR17E04-Gal4 and GMR18D08-Gal4 in the third larval instar stage. During late second or early third instar, the MF is initiated at the posterior margin of the developing eye-antennal imaginal disc [GMR18D08>GFP expresses strongly on the dorso-ventral (DV) margin of the eye imaginal disc and in the entire eye disc ; haploinsufficiency (hin); and imaginal disk specific-disk (disk). These two newly identified dpp CRMs in the eye, viz.,—GMR17E04-Gal4  can drive the dpp-lacZ expression specifically along the MF in the eye-antennal disc. We found that the sequence of the two newly identified dpp CRMs are located upstream and is different from the known CRM of dpp-lacZ in the disk region.The ase pairs) and GMRase pairs) . t 3’ end . This dpwg tagged with GFP in the eye using dpp-Gal4 line (dpp>wg-GFP) suppresses the eye fate and results in “no- eye” as seen in the eye imaginal disc , which marks the ventral half of pupal retina, results in near wild type eye imaginal disc . The other dpp enhancer, GMR17E04-Gal4 driver (GMR17E04>wg-GFP), which marks MF  results in enlargement of the eye imaginal disc  exhibits wild-type phenotype both in the eye imaginal disc , which will trigger cell death and yorkie (yki), which is known to suppress the MF progression [dpp CRM lines. In comparison to the wild-type eye imaginal disc  suppresses the eye fate and results in reduced eye as seen in the eye imaginal disc , which marks the ventral domain of pupal retina, strongly suppress the eye fate in the eye imaginal disc  exhibits normal eye disc , which is expressed along the antero-lateral region of the eye disc [wg and hh do not express in the MF exclusively. However, dpp exhibits expression along MF. Thus, a MF specific enhancer of dpp may serve as an ideal candidate to drive expression along MF. However, the available dpp-Gal4 reagent does not drive expression along with the MF  construct[dpp-lacZ to dpp-Gal4 is difficult [The rap line  with a Prap line . We initconstruct, 60. Theifficult , 60.dpp CRMs in the developing eye imaginal disc. One of them GMR18D08-Gal4 drives expression in the developing eye imaginal disc. Interestingly, in the pupal retina its expression gets restricted to only the ventral half  significantly reduced the adult eye due to loss of ventral half of the eye –68. The apoptosis causing genes grim, rpr and hid are involved in tissue homeostasis. Activation of rpr triggers cell death. We found that misexpression of rpr by GMR17E04-Gal4 (MF specific marker) exhibits a normal adult eye . This dpp-Gal4 drives expression of a transgene only on the posterior margin of the developing eye imaginal disc. In case of wg, dpp>wg results in “no-eye” phenotype as seen in the eye imaginal disc and the adult eye Click here for additional data file.S2 FigExpression of (A) GMR18B08>GFP, (B) GMR18D08>GFP, (C) GMR19D09>GFP, (D) GMR16G02>GFP, (E) GMR17E04>GFP, (F) GMR17G08>GFP, (G) GMR19B04>GFP, (H) GMR19C03>GFP in (A-H) wing imaginal disc. None of these lines exhibit GFP reporter expression in (A-H) wing imaginal disc.(TIF)Click here for additional data file.S3 FigExpression of (A) GMR18B08>GFP, (B) GMR18D08>GFP, (C) GMR19D09>GFP, (D) GMR16G02>GFP, (E) GMR17E04>GFP, (F) GMR17G08>GFP, (G) GMR19B04>GFP, (H) GMR19C03>GFP in (A-H) leg imaginal disc. None of these lines exhibit GFP reporter expression in (A-H) leg imaginal disc.(TIF)Click here for additional data file.S4 FigExpression of (A) GMR18B08>GFP, (B) GMR18D08>GFP, (C) GMR19D09>GFP, (D) GMR16G02>GFP, (E) GMR17E04>GFP, (F) GMR17G08>GFP, (G) GMR19B04>GFP, (H) GMR19C03>GFP in (A-H) haltere imaginal disc. None of these lines exhibit GFP reporter expression in (A-H) haltere imaginal disc.(TIF)Click here for additional data file.S5 FigExpression of (A) GMR18B08>GFP, (B) GMR18D08>GFP, (C) GMR19D09>GFP, (D) GMR16G02>GFP, (E) GMR17E04>GFP, (F) GMR17G08>GFP, (G) GMR19B04>GFP, (H) GMR19C03>GFP in (A-H) larval brain. Only GMR18B08 and GMR17E04 exhibits robust expression in the larval brain.(TIF)Click here for additional data file.S6 Figdpp>rpr,  GMR18D08>rpr,  GMR17E04>rpr. Note that dpp>rpr results in highly reduced eye as seen in (A) the eye imaginal disc and (B) the adult eye.  GMR18D08>rpr results in the reduced (C) eye imaginal disc and (D) the adult eye with preferential loss of ventral eye. In GMR17E04>rpr results in near normal (E) eye disc as well as the (F) adult eye.(TIF)Click here for additional data file.


Forces within the surgical milieu or circulation activate cancer cell adhesion and potentiate metastasis through signaling requiring FAK-Akt1 interaction. Impeding FAK-Akt1 interaction might inhibit perioperative tumor dissemination, facilitating curative cancer surgery without global FAK or AKT inhibitor toxicity. Serial truncation and structurally designed mutants of FAK identified a seven amino acid, short helical structure within FAK that effectively competes with Akt1-FAK interaction. Adenoviral overexpression of this FAK-derived peptide inhibited pressure-induced FAK phosphorylation and AKT-FAK coimmunoprecipitation in human SW620 colon cancer cells briefly exposed to 15mmHg increased pressure, consistent with laparoscopic or post-surgical pressures. Adenoviral FAK-derived peptide expression prevented pressure-activation of SW620 adhesion not only to collagen-I-coated plates but also to murine surgical wounds. A scrambled peptide did not. Finally, we modeled operative shedding of tumor cells before irrigation and closure by transient cancer cell adhesion to murine surgical wounds before irrigation and closure. Thirty minute preincubation of SW620 cells at 15mmHg increased pressure impaired subsequent tumor free survival in mice exposed to cells expressing the scrambled peptide. The FAK-derived sequence prevented this. These results suggest that blocking FAK-Akt1 interaction may prevent perioperative tumor dissemination and that analogs or mimics of this 7 amino acid FAK-derived peptide could impair metastasis. Disseminated tumor cells are pivotal in cancer metastasis. A one centimeter tumor sheds one million cells into the circulation daily . SurgeryExposing suspended colon cancer cells to 15 mmHg increased pressure promotes cell adhesion to collagen , 8, endoDisseminated tumor cells encounter such forces in various milieus. Circulatory pressures range from 5-10 mmHg in the portal vein to 90-120 mmHg in systemic arteries.  Tumors growing against constrictive stroma exhibit average 15-38 mmHg interstitial fluid pressures  while irPressure and shear activate dual mechanosensitive signaling pathways involving the cytoskeleton and paxillin or Src and phosphatidylinositol 3-kinase (PI3K) respectively , 24. TheThe FAK molecule is divided into the N-terminal erythrocyte band four.1-ezrin-radixin-moesin (FERM) (residues 35-362), the kinase (residues 416-676), and the C-terminal focal adhesion targeting (FAT) (residues 677-1025) domains . The FER7 SW620 cells previously used in similar pull-down assays. GST-FAK-NT1-conjugated Sepharose beads pulled down Akt1 after overnight incubation with either cell lysate or purified Akt1 , we used 0.3 micrograms of purified Akt1 in pull-down assays to approximate the Akt1 in 1,500 μg of whole cell lysate, the lysate from 1×101 Figure , suggestWe examined the role of a short helical secondary structure (LAHPPEE) using mutated variants of FAK-NT1 Figure . FAK-NT1To investigate the importance of individual subdomain structures within NT1-2-2 in FAK-Akt1 binding, we generated four variants that successively excluded secondary structures in the N- and C-terminal of the short helical segment while preserving the short helix itself Figure . We chalTo minimize the effect of non-specific binding to GST, we next used the larger FAK-NT1 (relative to GST) as bait and assessed the effects of the NT1-2-2 FAK truncations as interfering peptides. The wild-type 33 amino acid peptide (Pep-FAK-NT1-2-2) reduced binding between GST-FAK-NT1 and Akt1. A scrambled 33 amino acid control peptide containing the same amino acids in a different order. (Pep-FAK-NT1-2-2Scr) did not Figure . We subsWe created adenoviral vectors to deliver the wild type sequence LAHPPEE (Ad-FAK-Helix) or the scrambled HPELAPE (Ad-FAK-HelixScr) into intact human SW620 colon cancer cells and assessed pressure-induced signaling. 15mmHg increased pressure stimulated FAK Tyr397 phosphorylation (145±10%) in cells infected with the scrambled Ad-FAK-HelixScr control vs. cells at ambient pressure Figure . In contBecause pressure stimulates Akt1 Ser473 phosphorylation and Akt1 activation before Akt1 phosphorylates FAK , we predWe have previously demonstrated that pressure stimulates FAK-Akt1 interaction in intact SW620 cells, transfected with HA-FAK before immunoprecipitation with anti-HA to amplify the signal produced by basal FAK-Akt1 interaction , 25. We We hypothesized that peptide overexpression would similarly inhibit pressure-stimulated adhesion, the downstream consequence of pressure-activated FAK phosphorylation. Equal numbers of virus-treated cells were seeded onto collagen-I-coated plates under ambient or increased pressure for 30 minutes. The plates were washed in blinded fashion to remove nonadherent cells. The remaining adherent cells were quantified by MTS assay. Pressure-induced adhesion was inhibited by Ad-FAK-Helix infection but not by Ad-FAK-HelixScr Figure .Increased pressure stimulates cancer cell proliferation by a different mechanism , 19, 26.Since physiologic tissues are more complex than purified matrix proteins, we investigated SW620 cell adhesion to surgical wounds in BALB/c mice. SW620 cells infected with either Ad-FAK-HelixScr or Ad-FAK-Helix were labeled with Tag-it Violet dye, exposed to ambient or 15 mmHg increased pressure for 30 minutes in suspension and seeded into standardized murine surgical wounds. After 30 minutes, copious irrigation removed non-adherent cells as in surgical settings. After sacrifice and wound excision, adherent SW620 cells were distinguished from mouse tissue by fluorescence-activated cell sorting (FACS) for the Tag-it Violet dye. The dye was non-toxic and did not alter proliferation (not shown). Pressure activation increased Ad-FAK-HelixScr-infected cell implantation into wound tissue. Ad-FAK-Helix infection blocked this effect  were relabeled to blind the surgical investigator and seeded into surgical wounds in mice. After 30 minutes, the wounds were washed six times with warm PBS and closed. The mice were observed for 90 days during which tumors were assessed as palpable or non-palpable Figure  and palpPhysical forces evoke signaling responses across diverse cells by different mechanisms. The potentiation of adhesion in suspended cancer cells by a force-activated pathway represents a target for inhibiting metastasis, and the uncommon FAK-Akt1 interaction essential for this pathway seems an attractive target because blocking it may not affect other FAK signaling. Our results demonstrate that Akt1 interacts with FAK directly without an intermediary protein, likely via a short helix on the surface of the FAK F1 lobe, and that this FAK-Akt1 interaction can be blocked by peptides derived from said F1 lobe. Indeed, adenoviral delivery of this peptide into intact cancer cells blocks both pressure-activated signaling and consequent increases in cell adhesion without interfering with other aspects of FAK or Akt1 signaling. Finally, while adenoviral peptide delivery may not be a practical therapeutic modality, our findings suggest that interventions using or mimicking the FAK-derived peptide may translate to in vivo models and increase tumor-free survival by mitigating pressure-stimulated tumor adhesion.FAK-Akt1 signaling has recently been described in several contexts with respect to malignancy. FAK  and Akt1following cell adhesion [before adhesion [Pressure requires FAK-Akt1 interaction to stimulate FAK tyrosine-397 autophosphorylation and activation . The FAKadhesion , while padhesion . Similaradhesion . Changesadhesion . Mutatioadhesion . These dConsistent with the effects of pharmacologic Akt blockade , 25, adeAs for the molecular data, the inhibition of pressure-induced cell adhesion by the short helix peptide was consistent with previous work using less specific pharmacologic agents , 9, 25. Viral toxicity seems unlikely to explain our findings. Control cells were similarly infected, and Ad-FAK-Helix-infected adherent SW620 cells continued basal proliferation and responded to increased pressure with increased proliferation, similar to uninfected cancer cells , 26, 42.To power our study, we sought a tumor prevalence of 50% in the ambient control group, which came at the expense of replicating the low baseline incidence of clinically significant tumor recurrence in surgical wounds. However, physical forces activate tumor cell adhesiveness not only in the context of wound implantation but also peritoneal implantation  and distThese results suggest that perturbing FAK-Akt1 interaction, by mimicking the structure of a small segment of the F1 FAK lobe, can abate the sensitivity of suspended malignant cells to mechanical signals, potentially mitigating both the biochemical and the clinical consequences of this force-activated pathway. Such a resultant decrease in FAK and Akt1 activation and inhibition of cell adhesion could attenuate the metastatic potential of shed tumor cells during surgery and increase tumor-free survival. Potential toxicity and off-target effects limit the clinical utility of other methods to inhibit this force-activated adhesion pathway, such as high dose colchicine, disruption of the cytoarchitecture, or non-specific FAK and Akt inhibitors. Preventing FAK-Akt1 interaction without interfering with other aspects of FAK or Akt1 signaling might eventually achieve the desired effect with less compromise of other cell function and less toxicity.The crystal structure of human FAK (PDB 2AL6) and preliminary data showing Akt1 pulldown by a truncated 33 amino acid segment of the F1 lobe of FAK designated NT1-2-2 , 32 suggL113A: AAHPPEE - Enhanced α helical preferenceP116N: LAHNPEE - Similar α helical, β turn preference in the PPE region, increased polarityP116C: LAHCPEE - Structurally labile, greater hydrophobicityP116G: LAHGPEE - Stronger turn preference, greater flexibility, less hydrophobicityP117K: LAHPKEE - Structurally labile, enhanced polarityP117S: LAHPSEE - More structurally labile and polarTriple mutant L113A, P116N, P117K: AAHNKEE - Enhanced helicity and polarityTriple mutant L113A, P116C, P117G: AAHCGEE - More structurally labile and hydrophobicTriple mutant: L113A, P116A, P117A: AAHAAEE - More helical and hydrophobicBacterial expression vector pGEX-4T1  was used as a template to generate mutated and truncated human FAK as GST (Glutathione S-transferase) fusion proteins. Point mutations  and triple mutants  were generated using the Quick Change II XL Site-Directed Mutagenesis kit . Truncations were generated through PCR using forward and reverse primers to direct truncation (Table 7 Caco-2 or SW620 cells (1500 μg protein) or purified Akt1 (0.35 μg)  overnight at 4°C. Similar incubation for two hours at 4°C was performed with FAK-derived peptides (95% purity by HPLC)  reconstituted in sterile water and mixed with conjugated beads and cell lysate for a final concentration of 160mM before overnight Akt1 incubation. Bound protein was eluted for western analysis [Glutathione-Sepharose-4B beads (30 μl)  were conjugated with GST  or recombinant GST-tagged  [cDNA coding a seven amino acid segment from the F1 lobe of FAK  and a scrambled version of this sequence  were cloned in-frame into the MCS region of separate pShuttle-CMV vectors  using forward primers that added 5′-NotI and reverse primers that added 3′-HindIII restriction sites  . The PCR did not use template DNA as forward and reverse primers collectively spanned the entire product. Recombinants were generated per manufacturer's protocols , selected using kanamycin resistance, confirmed by sequencing, amplified in XL10-gold ultracompetent cells, purified, PacI-linearized, and transfected into HEK293 cells to produce adenoviral vectors coding for FAK-Helix (Ad-FAK-Helix) and the FAK-HelixScr (Ad-FAK-HelixScr). Viral particles were expanded and collected per manufacturer's protocols, and passed through a Fast-Trap Adenovirus Purification and Concentration kit  before reading OD at 260nm. Viral titer was calculated as one A260 unit to 1012 viral particles with a 50:1 ratio of particles to infectious particles via agarose overlay per manufacturer's protocols (not shown).Pressure was controlled using an airtight apparatus previously described, pressurized with filtered 5% CO2/95%, and maintaining temperature, pressure, pO2, pCO2, and pH .4 cells/well, and allowed to adhere to collagen-I-coated plates for 30 minutes at 37°C under ambient or 15mmHg increased pressure [3 vp/cell) for 1 hour before replacing infection media with growth media. After 24 hours, infected SW620s were replated in new T25 flasks at a 1:4 ratio. At 72 hours, adhesion was assayed as above. Cells were used within ten passages and authenticated by ATCC.SW620 colorectal adenocarcinoma cells  at 90% confluence were split 1:4 two days previously to achieve 50-60% confluence on the day of adhesion assay. SW620 cells were trypsinized, plated randomly at 5×10SW620 cells were transfected with Ad-FAK-Helix or Ad-FAK-HelixScr for 72 hours, trypsinized, and exposed to ambient or 15mmHg increased pressure for 30 minutes at 37°C in growth media in 48 well plates pacificated with 1% heat-inactivated BSA in PBS (to prevent adhesion). SW620 cells allowed to adhere to collagen-I-coated plates for 30 minutes at 37°C were positive controls. Cells were lysed for western analysis.4 cells/well on 96 well plates, recovered at 37°C for 12 hours, allowed 24 hours for proliferation, and counted using CellTiter 96 Aqueous One Solution Reagent as above. In adenoviral studies, SW620 cells were transfected with Ad-FAK-Helix or Ad-FAK-HelixScr 60 hours before plating on 96 well plates. For nonsurvival wound adhesion studies, cells were dyed with 10μM Tag-it Violet proliferation and cell tracking dye , or equivalent amounts of DMSO vehicle.SW620 colorectal adenocarcinoma cells were seeded at 10Coimmunoprecipitations were performed as previously  using moSW620 cells were transfected using Lipofectamine 2000 reagent  and HA-FAK(WT) plasmid (from Dr. JL Guan). Cells were grown in T75 flasks until 80% confluent, replated into 6 well plates at 80% confluence, and transfected 12 hours later per manufacturer's protocols.Protein concentrations were determined by bicinchoninic acid (BCA) protein assay . Eluate from GST pull-downs or collected cell lysates was resolved by SDS-PAGE and transferred to Hybond P 0.45 PVDF blotting membrane . Membranes were blocked for 1 hour at room temperature with Odyssey TBS Blocking Buffer  and blotted overnight at 4°C with antibodies against FAK (#3285), Phospho-FAK Tyr397 (#3283), Akt1 (#2967), Phospho-Akt1 Ser473 (#9271), GSK-3β (#9315), Phospho-GSK-3β Ser9 (#9315), or the GST tag (#2624) . Membranes were visualized by the infrared fluorescent IRDye system  and analyzed on an Odyssey scanner  within the linear range. The doublet produced by the Akt1 antibody is consistent with results with this , 51 and Akt1 frequently appears in published Western blots (34-37) as a doublet. The reason for this is not entirely clear, but may represent altered phosphorylation states or other post-translational modifications of the molecule yet to be clarified. The cell line studied and its phenotype in the experiment in question, the amount of protein loaded and concentration of primary and secondary antibody, the percentage of the SDS-PAGE used to resolve the proteins, and numerous other factors may influence this. The densitometric data presented here considers the heavier Akt1 band in each of the doublets.5 cells was randomly applied to the wounds. After 30 minutes, the fluid was aspirated and the wounds were washed with warm phosphate buffered saline six times to remove nonadherent cells. In nonsurvival studies, the mice were euthanized following wound irrigation, and wounds were excised to quantify tumor adhesion by fluorescence-activated cell sorting (FACS). The excised wound tissue was mechanically (paired scissors) and then enzymatically (3ml/sample collagenase incubation for 1hour at 37°C with agitation) disaggregated before passage through a cell strainer and 20 minute room temperature incubation in BioLegend fixation buffer . Fixed cells were resuspended in PBS with 5%FBS and Tag-IT dye. Fluorescence and cellular auto-fluorescence were detected using a LSR flow cytometer  with a filter for Pacific Blue (ex/em 410/455). In survival studies, the wounds were instead closed and followed as described [SW620 colorectal adenocarcinoma cells were transfected with Ad-FAK-Helix or Ad-FAK-HelixScr. After 72 hours, cells for nonsurvival studies were trypsinized and dyed with 10μM Tag-it Violet  per manufacturer's protocols; survival studies used undyed cells. Cells were incubated for 30 minutes at 37°C under ambient or 15mmHg increased pressure in a 48 well plate pacificated with 1% heat inactivated BSA in PBS to prevent adhesion to the plate. These cells were then collected and washed in warm PBS. In nonsurvival studies, 1 cm groin incisions were made bilaterally in 6-7 week old 22.9-24.2 gram male BALB/cAnNHsd mice  anesthetized i.p. with ketamine (100mg/kg), xylazine (10mg/kg), and acepromazine (3mg/kg). In survival studies, a single 1 cm groin incision was made in 6-7 week old 22.9-24.2 gram male athymic nude- Foxn1nu mice  anesthetized with continuous inspired 1-2% IsoFlo  in oxygen. A 50 μl suspension ambient or increased pressure of 5×10Data are expressed as mean±SEM. Results were compared by Student's unpaired t-test and log-rank test as appropriate seeking 95% confidence. In vivo studies were analyzed by Mantel-Haenszel testing. Assays were within linear ranges.