HO1, NQO1 and GCLM. Significantly, shRNA-mediated knockdown of Nrf2 almost completely abolished MIND4-17-induced cytoprotection against UVR. Further studies showed that MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs. Together, MIND4-17 protects retinal cells from UVR by activating Nrf2 signaling.Nrf2 activation would efficiently protect retinal cells from UV radiation (UVR). Recent studies have developed a Nrf2-targeting thiazole-containing compound MIND4-17, which activates Nrf2 through blocking its association with Keap1. In the current study, we demonstrated that pretreatment with MIND4-17 efficiently protected retinal pigment epithelium (RPE) cells (RPEs) and retinal ganglion cells (RGCs) from UVR. UVR-induced apoptosis in the retinal cells was also largely attenuated by MIND4-17 pretreatment. MIND4-17 presumably separated Nrf2 from Keap1, allowing its stabilization and accumulation in retinal cells, which then translocated to cell nuclei and promoted transcription of ARE-dependent anti-oxidant genes, including  Retinal degenerative diseases are characterized by progressive loss of retinal cells –3. UV raNAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), γ-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC), among others  were both increased H quinone oxidoreductase 1  treatment also increased mRNA expressions of HO1, NQO1 and GCLM, but not Nrf2  method was employed to knockdown Nrf2 in retinal cells. As described , the Nrfs Figure . Since bs Figure , we stims Figure . Downstrs Figure . Remarkas Figure  and apops Figure  were ints Figure  and 4D. s Figure  and 4D. Reactive oxygen species (ROS) production and following oxidative stress are major causes of cell death by UVR , 27. Nrfi.e. RPEs and RGCs [et al., showed that Salvianolic acid A protected RPEs from oxidative stress through activation of Nrf2-HO1 signaling [In the pathology of AMD and other retinal degenerative diseases, UVR and subsequent oxidative stresses cause direct damages to resident retinal cells, and RGCs , 31, 32.and RGCs , 31, 32.ignaling . Similarignaling . SC79, aignaling , 34, proignaling .Recent studies have characterized MIND4-17 as a unique and highly selective small molecule activator of Nrf2 , 20. MINHO1, NQO1 and GCLM. Subsequently, activation of Nrf2 by MIND4-17 largely ameliorated UVR-induced ROS production, lipid peroxidation and DNA damages in RPEs and RGCs. Retinal cell death and apoptosis by UVR were also dramatically alleviated by MIND4-17. Importantly, we demonstrate that Nrf2 is required for MIND4-17's actions in retinal cells. shRNA-mediated knockdown of Nrf2 almost completely reversed MIND4-17-induced retinal cytoprotection against UVR.In the present study, we show that MIND4-17 treatment presumably separated Nrf2 from Keap1, thus allowing its stabilization and accumulation in retinal cells. Stabilized Nrf2 by MIND4-17 translocated to cell nuclei, causing transcription and expression of multiple ARE-dependent anti-oxidant genes, including Based on the structure in previous studies , 20 als, MIND4-1The established ARPE-19 cells were provided as a gift from Dr. Jiang , 35, 36.The primary-cultured human RGCs were also provided by Dr. Jiang . The pri2) to the cultured RPEs and RGCs was described previously [UV radiation   as well DNA formamide denaturation and subsequent the production of single strand DNA (ssDNA) is characteristic marker of cell apoptosis, which was tested by the ApoStrandTM ELISA apoptosis detection kit . ELISA OD at 450 nm was recorded.Following the applied treatment, retinal cells were harvested, washed and resuspended in binding buffer with 1 μL of Annexin V-FITC and 1 μL of propidium iodide (PI) . After incubation for 20 min at room temperature, cells were then tested by flow cytometry via CellQuest software . Annexin V ratio was recorded , 39.After the described treatment, retinal cells were further stained with TUNEL and DAPI florescence dyes . TUNEL ratio (vs. DAPI staining) was calculated .Cells were lysed via the described lysis buffer . DetermiHO-1, NQO1 and GLCM and GAPDH were described previously [GAPDHmRNA was tested as the reference gene. The 2–ΔΔCt method was employed to calculate relative expression of targeted mRNAs.Total RNA was extracted by TRIzol reagents  and then reversely transcribed using a PrimeScript RTreagent kit . Real-time quantitative PCR (“RT-qPCR”) assay was performed by a SYBR Premix Ex TaqTM kit on the ABI-7700 fast PCR system . mRNA primers for human eviously . GAPDHmThe Nrf2 shRNA lentiviral particles  and the scramble control shRNA lentiviral particles  were both commercial available. The shRNA lentivirus was added to ARPE-19 cells for 12 hours. Afterwards, puromycin (2.5 μg/mL) was added to select stable cells for 5–6 passages. Nrf2 knockdown was verified by both Western blotting assay and RT-qPCR assay.ROS intensity in the control and UVR-treated retinal cells was measured by the carboxy-H2DCF-DA dye  assay . FollowiThiobarbituric acid reactive substances (TBAR) level was examined to reflect the production of toxic aldehyde resulting from oxidative fatty acyl degradation, the malondialdehyde (MDA). The detailed protocol was described previously , 46.γ-H2AX assay of DNA damages. ARPE-19 cells were fixed in ice-cold ethanol, which were then incubated with a mouse monoclonal anti-γ-H2AX antibody (Santa Cruz Biotech). Afterwards, the FITC-conjugated anti-mouse secondary antibody (Santa Cruz) was then added. Cells were then subjected to FACS assay to determine the γ-H2AX percentage, reflecting DNA damage intensity .p < 0.05 were considered statistically significant.The results were presented as the mean ± standard deviation (SD). One-way ANOVA was employed to examine the significant differences between groups using SPSS 17.0 . Values of Together, we conclude that targeting Nrf2 by MIND4-17 efficiently protects RPEs and RGCs from UVR.


PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS.Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [ Many neurodegenerative diseases are associated with aggregation of specific proteins. Thus we are interested in factors that influence the aggregation and how the aggregated proteins are associated with pathology. Here, we study a protein called TDP-43 that is frequently aggregated in the neurons of patients with amyotrophic lateral sclerosis (ALS). TDP-43 aggregates and is toxic when expressed in yeast, providing a useful model for ALS. Remarkably, a protein that modified TDP-43 toxicity in yeast successfully predicted a new ALS susceptibility gene in humans. We now report a new modifier of TDP-43 toxicity, Sis1. We show that expression of TDP-43 in yeast inhibits degradation of damaged protein, while overexpression of Sis1 restores degradation. Thus suggests a link between protein degradation and TDP-43 toxicity. Furthermore we show that a mammalian protein similar to Sis1 reduces TDP-43 toxicity in primary rodent neurons. This identifies the mammalian Sis1-like gene as a new ALS therapeutic target and possible susceptibility gene. In aPIN+] dependent polyQ aggregation and toxicity  dependent toxicity of an overexpressed prion-like protein Pin4C. When overexpressed in the presence of [PIN+], Pin4C formed large hyperphosphorylated aggregates that co-localized with Sis1 and reduced degradation of a ubiquitin proteasome system (UPS) reporter protein. Aggregation, hyperphosphorylation and toxicity were suppressed by overexpression of Sis1  dependent toxicity caused by overexpression of the [PIN+] prion protein, Rnq1. Here, overexpressed Sis1 appears to enhance aggregation of toxic detergent soluble Rnq1 oligomers into apparently non-toxic larger detergent resistant aggregates in the nucleus  cells [PIN+] and [pin-] yeast [PIN+] and [pin-] cells  ,49–51 wepin-] and [PIN+] cells in three strain backgrounds. The rescue appeared to be weaker in 74D-694 [PIN+] cells where TDP-43 is more toxic PIN+], bePIN+] cells reduced degradation of a UPS reporter, CG*  and [PIN+] 74D-694 cells but was more dramatic in [PIN+] cells where TDP-43 is more toxic. Furthermore, simultaneous overexpression of Sis1 and TDP-43 resulted in rapid degradation of CG*. This is consistent with the hypothesis that TDP-43 toxicity in part results from interference with the UPS system.Previous results showed that overexpression of polyQ or Pin4C in  than [pin-] 74D-694 cells and likewise has a greater effect on UPS in [PIN+] vs. [pin-] cells. By analogy, the presence vs. absence of heterologous aggregates in mammalian cells may also influence the proteostasis of disease-causing aggregates.Aggregation of TDP-43 is frequently associated with ALS and other neurological diseases –79 and oPIN+] effects toxicity and UPS inhibition is unknown. While [PIN+] dramatically enhances aggregation and toxicity of polyQ and Pin4C [PIN+] vs. [pin-] cells are indistinguishable. Nonetheless, the fact that [PIN+] prion aggregates sequester some essential chaperones including Sis1 [PIN+] cells more vulnerable to TDP-43 overexpression, e.g. the lower level of Sis1 available for the UPS in [PIN+] cells could make cells more sensitive to the inhibitory effect of TDP-43 on the UPS. Indeed, we found that overexpression of Sis1 reduces the deleterious effects of TDP-43 on cell growth, cell shape and UPS inhibition. Several genetic screens in yeast have identified modifiers of TDP-43 toxicity; however, Sis1 was not among them  and other unknown differences between strains alter the toxicity of TDP-43, repeating screens for toxicity modifiers in additional yeast strain backgrounds may reveal new modifiers.Why and how  cells  cells . In that+] cells ,38,66,71Thus, the mechanism by which Sis1 rescues cells from TDP-43 toxicity appears to be distinct from its rescue of polyQ, Pin4C or Rnq1 toxicity. Previous studies implicated a direct effect of a different HSP40 chaperone, DNAJB6 (a member of the DNAJB7 and 8 family) on TDP-43 via interactions with the TDP-43 C-terminal domain ,83. In cDBR1 knockdown)  versions of strains were obtained on YPD plates with 5 mM GuHCl and were confirmed to be [pin-] by checking for lack of aggregates after transforming with an Rnq1-GFP plasmid  and [pin-] versions of 74D-694, W303 and BY4741 were doubly transformed with GAL1 controlled TDP-43-DsRed, GAL1 controlled modifier or control empty vector plasmids listed below. Transformants were selected on SD-Leu-Ura plates. Normalized suspensions of cells taken from SD-Leu-Ura were 10X serially diluted in water and 15 μl were spotted on SD-Leu-Ura (dextrose), and 2% Gal-Leu-Ura  plates, which were photographed after 3 (dextrose) or 5  days of incubation at 30°C. We show hUpf1 because it had a bigger effect than the yeast homologue Ecm32 found in the initial screen  and [pin-] versions of 74D-694 (A), BY4741 (B) and W303 (C) carrying pGAL1-TDP-43-DsRed (p2173) or pAG415 GAL1-ccdB-DsRed (p2302) were grown on 2% galactose plates for 4 days and then imaged at the same magnification. Shown are representative fields of many fields photographed and examined.[(PDF)Click here for additional data file.S3 Figpin-] 74D-694 with pTETR-TDP-43-YFP (p2223) were selected on SD-Trp supplemented with doxycycline (10 μg/ml). Transformants were then grown in liquid SGal-Trp media with the indicated amount of doxycycline for 24 h and were examined and photographed at same magnification. The levels of TDP-43-YFP were determined by immunoblotting SDS-PAGE gels of normalized cell lysates probed with anti-TDP-43 antibodies, and anti-Pgk1 antibodies as an internal loading control.Transformants of [(PDF)Click here for additional data file.S4 FigPIN+] and [pin-] 74D-694 doubly transformed with pGAL1-TDP-43-DsRed (p2173) and pGAL-Sis1 (p1759) were grown in plasmid selective synthetic liquid media containing 2% galactose and 2% raffinose for 2 days. Cells were then crossed to [pin-] 64D-694 MATα (L2642) bearing plasmid p1185 (pCUP1-RNQ1-GFP). Diploids were selected on SD-Leu-Ura-Trp, expressed on SD-Leu-Ura-Trp supplemented with Cu++ (50 μg/ml), and examined for Rnq1-GFP dots or diffuse fluorescence, which was diagnostic for the presence or absence, respectively, of the [PIN+] prion.[(PDF)Click here for additional data file.S5 FigHSP104 does not prevent Sis1 overexpression from reducing TDP-43 toxicity. Isogenic [psi-] [pin-] versions of 74D-694 with (hsp104Δ) and without (WT) a deletion of HSP104 doubly transformed with p2042 (pGAL1-TDP-43-YFP) and p1767 (pGAL1-SIS1) or vector control p1752 (pGAL1-YFP) and p1768 (pGAL1) were selected on SD-Ura-Trp plates. Normalized suspensions of cells taken from SD-Ura-Trp were 10X serially diluted in water and 15 μl were spotted on SD-Ura-Trp (dextrose), and 2% SGal-Ura-Trp  plates which were photographed after 3 (dextrose) or 7  days of incubation at 30°C. B. Reduction of Ssa1 activity does not prevent Sis1 overexpression from reducing TDP-43 toxicity. Strain 1014 bearing a deletion of SSA2 and expressing a dominant negative allele of SSA1 (SSA1-21 ssa2Δ) and its isogenic parent strain, L3504 (WT) were doubly transformed with pGAL1-TDP-43-DsRed (p2173), pGAL-SIS1 (p1759), or vector controls (p2302 or p484). Normalized suspensions of cells taken from plasmid selective SD-Leu-Ura medium were 10X serially diluted in water and 15 μl were spotted on SD-Leu-Ura (dextrose), and 2% Gal-Leu-Ura  plates, which were photographed after 3 (dextrose) or 5  days of incubation at 30°C.A. Deletion of (PDF)Click here for additional data file.S6 Fig-5; # HR 3.18, p < 2x10-16, Cox proportional hazards analysis. Results were pooled from two independent experiments.*Rodent primary cortical neurons were dissected and transfected with plasmids encoding EGFP and TDP-43(WT)-mApple or mApple. In each case, neurons were also transfected with scrambled siRNA or siRNA targeting DNAJB1. (A) Knockdown was validated by immunocytochemistry using antibodies against DNAJB1. Scale bar, 50 μm. (B) Transfection with siRNA against DNAJB1 resulted in a 60% reduction in anti-DNAJB1 antibody reactivity  In longitudinal assays of neuronal survival, DNAJB1 knockdown enhanced the risk of death by 20% in control neurons expressing EGFP alone and in neurons overexpressing TDP43. * HR 1.20, p 0.004; ** HR 1.21, p 2.3x10(PDF)Click here for additional data file.


PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to  PcG mutations, genes that are normally silenced—such as oncogenes—are expressed aberrantly. Due to the activation of oncogenes and the loss of other PcG functions, PcG mutant cells often begin to display hallmarks of cancer, such as proliferating beyond control, acquiring stem-cell-like properties, and migrating to distant sites. If the transcriptional mechanisms underlying aberrant gene expression in PcG-mutant cancer cells differ from gene expression in normal cells, we may be able to selectively inhibit the growth of cancer cells without affecting their normal counterparts. Here we show that the difference between these two types of gene expression resides in their sensitivity to dBRWD3, a negative regulator of the deposition of histone H3 variant H3.3. Our results indicate that the inactivation of dBRWD3 or promotion of H3.3 deposition may selectively suppress ectopic gene expression and tumorigenesis driven by mutations in PcG.Genetic information is stored in our genomic DNA, and different cells retrieve distinct sets of information from our genome. While it is important to activate genomic regions encoding proteins that are essential for a given cell type, it is equally important to silence genomic regions encoding proteins that are potentially harmful to this type of cells. One of the gene silencing mechanisms frequently used during and after development is mediated by the Polycomb group (PcG) proteins. If this guardian function does not perform correctly due to  These phenotypes are effectively suppressed by mutations in yem or H3.3, indicating that dBRWD3 functions largely through restricting YEM-dependent H3.3 deposition . To. To44]. lls Figs . However of upd3 . This faRWD3-RFP . Therefo3 ligase . The ΔN-nt cells . TogethedBRWD3s5349 mutant cells with ΔN-dBRWD3-RFP or dBRWD3-2BC-RFP, the H3.3 levels in the dBRWD3s5349 mutant cells remained higher than those in wild-type cells  and Pc. The ChIP-qPCR analysis revealed a reduction of H3K27me3 in the distal region of Antp and at Ubx when E(z) was depleted  or Pc, the H3K27me3 or H2A118ub levels at Antp and Ubx loci remained low or became lower  depleted wings at the global level, as revealed by the equally reduced H3K27me3 immunostaining signals in the E(z) knockdown, and E(z), dBRWD3 double-knockdown wings  encodes an H3K4 monomethyltransferase [trx [trx in ectopic expression of Antp in eyes, we generated ScmD1, trxE2 double-mutant eye clones and found that the ectopic expression of Antp was suppressed -associated factors (TAF). Although TAFs are essential factors, animals with 65% reduction of  defects . Ubx-expectively . Next, wins Figs . By cont or CycH . The TrxycH Figs  and S18Anot Figs  and S18BycH Figs  and S18Ad-B Figs  and S18Antp Figs  and S18CdBRWD3 suppresses ectopic gene expression and the tissue proliferation caused by the loss of PcG function, but not orthotopic gene expression. Loss of dBRWD3 also suppresses the ectopic gene expression induced by Trx. This suppression is related to enhanced H3.3 deposition at transcription start sites and reduced H3K4me3, activated PolII, and total PolII levels around the 5' regions of the ectopically expressed genes.In this study, we provide evidence that loss of Cyp26A1 in embryonic stem cells, H3.3 is actively deposited to the enhancer before induction. Upon induction, H3.3 is depleted from the enhancer but deposited into the promoter. Knockdown of H3.3 reduces the binding of RAR and Tip60 to the enhancer region, indicating that deposition of H3.3 at enhancer regions facilitates the activation of inducible genes [A genome-wide H3.3 ChIP study revealed that H3.3 is enriched in enhancers, promoters, and gene bodies of actively transcribed genes . This cole genes . HoweverdRBWD3 is required for the ectopic gene expression observed in PcG mutants or upon over-expression of trx. Firstly, only dBRWD3 transgenes that are able to reduce H3.3 levels in dBRWD3 mutant cells are capable of restoring ectopic expression of upd3 in Scm, dBRWD3 double-mutant cells. Secondly, the loss of yem, which prevents the aberrant incorporation of H3.3, also restores the ectopic expression of upd3 in Scm, dBRWD3, yem triple-mutant clones. Conversely, the over-deposited H3.3 induced by YEM is sufficient to suppress the ectopic Antp expression and Trx-induced ectopic Abd-B expression.It is even less clear how H3.3 affects ectopic gene expression, a pathological condition frequently associated with various cancers in humans ,55. In tdBRWD3 depletion-enhanced H3.3 deposition at transcription start sites interferes with H3K4me3 and PolII enrichment at the same regions as well as the 5' ends of the gene bodies. Taken together, these observations suggest that H3.3 deposition at these regions disrupts transcription by interfering with trimethylation of H3K4 as well as PolII engagement or activation. This notion is supported by a recent finding that ectopic gene expression persists longer in Hira mutants, in which H3.3 deposition is reduced at the promoter and 5' regions [In ectopic gene expression, H3.3 deposition at the enhancers is variably regulated by dBRWD3. Nevertheless, through a not entirely clear mechanism, the H3K4me1 induced by Trx at enhancers is insensitive to dBRWD3. By contrast, the  regions . DifferedBRWD3 regulates the deposition of H3.3 more prominently at the promoters and transcription start sites in both ectopic and orthotopic gene expression. However, dBRWD3 regulates the PolII occupancy and H3K4me3 levels only in ectopic gene expression. Due to a not-yet-defined “robustness” of transcription, orthotopic gene expression is rendered insensitive to the increase of H3.3. Our data suggest that the same robustness of orthotopic gene expression closely cooperates with TFII-D and TFII-H since orthotopic gene expression remains intact under the suboptimal TFII-D or TFII-H activities. Further studies are needed to understand molecular nature of the robustness.Pc, pho, and tna but down-regulates phol, Jarid2 and the trithorax group genes ash1 and Iswi [ash1, which encodes H3K4 monomethylase, is particularly interesting because an independent transcriptome analysis of adult heads in dBRWD3 hypomorphic mutants also confirmed a lower level of ash1 mRNA. However, functional studies revealed that ash1 depletion by RNAi rescues rather than exacerbates the rough eye phenotype caused by dBRWD3 depletion. In addition, the H3K4me1 levels in dBRWD3 mutant cells are similar to those in wild-type cells. Further studies are needed to determine the significance of dBRWD3 regulation of ash1 mRNA. Finally, knockdown of dBRWD3 causes further reduction of H3K27me3 or H2AK118ub in the E(z) or Pc depleted brains, suggesting that H3.3 deposition may accelerate the removal of these repressive marks. Further investigations are warranted to understand whether the accelerated removal of repressive marks is mainly contributed by the nucleosome turnover associated with H3.3 deposition or involves activation of demethylases and deubiquitylase.In addition to their negative effects on ectopic gene expression, H3.3 deposition and dBRWD3 may also interact with PcG in different contexts. For example, it has been reported that H3.3 deposition directs PRC2 to bivalent promoters in ES cells . In addiand Iswi . The regTaf5 or Taf7 affects ectopic but not orthotopic gene expression. In other words, ectopic and orthotopic gene expression may require different levels of TFII-D activities.The H3K4me3 levels at the promoter and 5' ends of genes correlate well with active transcription. In fact, it is both a cause and a consequence of active transcription. As a cause, H3K4me3 recruits the TFII-D subunit TAF3, a general transcription factor involved in PolII engagement and transcription initiation . Based oCdk7 or CycH indeed affected ectopic gene expression without a discernible effect on orthotopic gene expression, perhaps because it was more dependent on the phosphorylation of PolII CTD Ser5. A complement study in RING1A, RING1B double knockout embryonic stem cells revealed that de-repressed loci displaying higher levels of PolII phospho-CTD Ser5 are ectopically expressed at higher levels [During active transcription, H3K4me3 is established by hSet1A/B, which is recruited to actively transcribed gene regions by CTD Ser5-phosphorylated PolII . The phor levels , supportPcG mutations and reduced expression of PcG proteins contribute to tumorigenesis in several human malignancies [upd1, upd2, and upd3 also underlies tissue overgrowth in Drosophila PcG mutants, suggesting an evolutionarily conserved role for PcG in tumor suppression from insects to humans. Based on our results, we speculate that inhibition of the BRWD3, TFII-D, and TFII-H complex, for example by the CDK7 inhibitor THZ1 [PcG mutations.gnancies ,35,61–64tor THZ1 –67, mighIn summary, we found that ectopic gene expression differs from orthotopic gene expression in their sensitivities to dBRWD3. Inactivation of dBRWD3 selectively suppresses ectopic gene expression and tissue overgrowth induced by loss of PcG function.p-ENTR-dBRWD3-N1287A-3XFlag, p-ENTR-dBRWD3-N1451A-3XFlag, p-ENTR-dBRWD3-N1287A, N1451A-3XFlag (p-ENTR-dBRWD3-2BC-3XFlag) were generated with the Thermo Scientific Phusion Site-Directed Mutagenesis kit using the previously described p-ENTR-dBRWD3-3XFlag as a template. p-ENTR-HA-yem was generated by PCR from the cDNA clone RE33235, Drosophila Genetic Resource Center. p-ENTR-dBRWD3-N1287A-3XFlag, p-ENTR-dBRWD3-N1451A-3XFlag, and pENTR- dBRWD3-2BC-3XFlag were recombined into the pUWR vector (DGRC Gateway collection) to generate pUWR-dBRWD3-N1287A-3XFlag-RFP, pUWR-dBRWD3-N1451A-3XFlag-RFP, pUWR-dBRWD3-2BC-3XFlag-RFP. p-ENTR-HA-yem was recombined into the pTWF vector (DGRC Gateway collection) to generate pTWF-HA-yem.dBRWD3s5349, and yemGS21861 were described earlier [SceKO and ScmD1, trxE2 were kindly provided by Dr. Muller [hs-H3.3-GFP was a gift from Dr. Kami Ahmad [GMR-GAL4 (stock number 9146), Elav-GAL4 (stock number 458), ScmD1 (stock number 24158), Sce1 (stock number 24618), wtsx1 (stock number 44251), ex-LacZ (stock number 11067), UAS-mCD8-GFP (stock number 5146), UAS-Taf5-shRNA (stock number 35367), and UAS-Taf7-shRNA (stock number 55216) were obtained from the Bloomington stock center. UAS-trx (stock number 12194) and OK107-GAL4, were obtained from the Drosophila Genetic Resource Center, Kyoto. The ScmD1, dBRWD3s5349 double mutant, ScmD1, dBRWD3PX2 double mutant, dBRWD3s5349, Sce1 double mutant, ScmD1, dBRWD3s5349, yemGS21861 triple mutant, and wtsx1, dBRWD3s5349 double mutant were generated by recombination. UAS-Psc-dsRNA (NIG3886R-4) UAS-ph-p-dsRNA (NIG18412R-1), UAS-E(z)-dsRNA (NIG6502R-3), UAS-Pc-dsRNA (NIG32443R-1), UAS-CycH-dsRNA (NIG7405R-1), and UAS-Cdk7-dsRNA (NIG3319R-1) were obtained from the fly stocks of the National Institute of Genetics, Kyoto, Japan (NIG-FLY). UAS-dBRWD3-dsRNA (VDRC40209) was obtained from the Vienna Drosophila RNAi Center (VDRC). The transgenic flies ubi-dBRWD3-N1287A-3XFlag-RFP, ubi-dBRWD3-N1451A-3XFlag-RFP, ubi-dBRWD3-N1287A, N1451A-3XFlag-RFP (ubi-dBRWD3-2BC-3XFlag-RFP), and pTWF-HA-yem were generated by microinjection for germ-line transformation. The transgenic flies ubi-H3.3-dendra2, ubi-dBRWD3-3XFlag-RFP, ubi-delta-N-dBRWD3-3XFlag-RFP, and 10XSTAT-nlsGFP were described previously [Flies were raised in standard conditions at 25°C except as otherwise mentioned. The  earlier ,68. SceK. Muller ,47. hs-Hmi Ahmad . GMR-GALGenotypes for mosaic mutant clones in eyes:ey-flp/+; FRT82B/FRT82Bubi-nlsGFP . Following the manufacturer’s protocol, cDNA was synthesized using oligo(dT) and SuperScript reverse transcriptase (Invitrogen). OmicsGreen qPCR 5X Master Mix (Omics Bio) was used for real-time quantitative PCR on a CFX96 connect Real-Time PCR System (Bio-Rad). RPL32 was used as an endogenous loading control.rd instar larval eye imaginal discs were dissected in PBS and fixed for 17 minutes in 4% formaldehyde, followed by three 10-min washes in PBS supplemented with 0.3% Triton-X-100 (PBT) and 30-min blocking in PBT containing 5% normal donkey serum (NDS). After blocking, discs were incubated with primary antibody either overnight at 4°C or 2 hours at room temperature in PBT containing 5% NDS. After incubation with primary antibody, discs were washed three times in PBT before incubating with secondary antibody in PBT containing 5% NDS for one hour at room temperature. After three subsequent washes, discs were mounted with glycerol. Primary antibodies used in this study include mouse anti-H2AK118ub , rabbit anti-H3K27me3 , rabbit anti-H3K4me1 , rabbit anti-H3S10ph , mouse anti-Ubx , mouse anti-Antp , rabbit anti-upd3 (1:750), and mouse anti-β-Galactosidase . Secondary antibodies include goat anti-mouse Cy3 , goat anti-mouse Cy5 , and goat anti-rabbit Cy3 .3Antp, Ubx, and Abd-B. Primary antibodies used include rabbit anti-H2AK118ub , mouse anti-H3K27me3 , rabbit anti-H3K4me1 , rabbit anti-H3K4me3 , mouse anti-PolII , and rabbit anti-PolII phospho-CTD Ser5 , rabbit anti-HA  and mouse anti-Flag Chromatin immunoprecipitation was done with a ChIP-IT High Sensitivity (HS) Kit (Active Motif) following the instructions provided by the manufacturer. Briefly, 300 pairs of brain lobes  or wing imaginal discs of 3rd instar larvae were collected. The collected tissues were fixed with complete tissue fixation solution  at room temperature for 15 minutes. Fixation was stopped with the stop solution at room temperature for 5 minutes. The fixed tissues were washed with ice-old PBS wash buffer and then immersed in the tissues with the chromatin prep buffer. The fixed tissues were sonicated using the UP50H Ultrasonic Processor (Hielscher-Ultrasound Technology), with 30% amplitude and 20 pulse cycles of 30 seconds on followed by 30 seconds off. 6 μg sheared chromatin was incubated with 1μg antibodies overnight at 4°C. 30 μl Protein G agarose beads were added to each IP reaction. The mixture was rotated at 4°C for 3 hours. The ChIP reactions were loaded into columns and washed. The ChIP DNA was obtained by eluting the columns with elution buffer AM4. The elute was treated with Protease K at 55°C for 30 minutes, 80°C for two hours, followed by column clean-up. OmicsGreen qPCR 5X Master Mix (Omics Bio) was used for real-time quantitative PCR on a CFX96 connect Real-Time PCR System (Bio-Rad) to measure the amount of ChIP DNA and input DNA containing indicated sequences from enhancers, promoters, and 5' transcription regions of All confocal images were obtained by LSM 700 laser scanning confocal microscope (Carl Zeiss). For quantitative analysis of protein levels, the antibody staining conditions, laser power, and pinhole sizes were kept identical among groups. Pixel number, pixel intensity, and area were provided by the built-in software in LSM 700. The areas of clones (marked by the absence of GFP) and non-clones (marked by GFP) were calculated by the total GFP positive and GFP negative areas respectively. Antp-positive regions in ventral nerve cords were manually marked. The Antp expression areas and lengths were calculated by the built-in software according to the marked regions. H3S10ph-positive mitotic cells and Antp-positive brain cells were manually counted.S1 Text(DOCX)Click here for additional data file.S1 FigScmD1, dBRWD3PX2 double-mutant clones generated in the 3rd instar eye imaginal discs by ey-flp and marked by the absence of GFP. Scale bars indicate 50μm. (B) A schematic illustration of how protein levels are calculated from a confocal image of a mosaic imaginal disc. (C and D) Quantifications of Antp expression. The ectopic expression of Antp in ScmD1, ScmD1, dBRWD3PX2 and ScmD1, dBRWD3s5349 mutant clones (C), Sce1 and dBRWD3s5349, Sce1 mutant clones (D). (E)The orthotopic expression of Antp in wild type and dBRWD3s5349 mutant clones. a.u. indicates arbitrary unit. Data are shown as means ± S.D. *, **, *** indicate P< 0.01, 0.001, 0.0001, respectively, by Student's t-test. ns. indicates not significant.(A) Antp levels (arrows) in (TIF)Click here for additional data file.S2 FigPc-dsRNA driven by Elav-GAL4. (B-D) The mean intensity (B), expression area (C), and length of expression domain (D) of Antp in control, Pc depleted, dBRWD3 depleted, and Pc, dBRWD3 doubly depleted ventral nervous cords. (E) The knockdown efficiency of E(z)-dsRNA driven by Elav-GAL4. (F-H) The mean intensity (F), expression area (G), and length of expression domain (H) in the control, E(z) depleted, dBRWD3 depleted, and E(z), dBRWD3 doubly depleted ventral nervous cords. a.u. indicates arbitrary unit. Data are shown as means ± S.D. ns. indicates P>0.05 by Student's t-test. *, *** indicate P< 0.01, 0.0001, respectively, by Student's t-test.(A) The knockdown efficiency of (TIF)Click here for additional data file.S3 FigScmD1 and ScmD1, dBRWD3s5349 mutant clones (A) or Sce1 and dBRWD3s5349, Sce1 mutant clones (B). Data are shown as means ± S.D. *, **, *** indicate P< 0.01, 0.001, 0.0001, respectively, by Student's t-test. ns. indicates not significant. a.u. indicates arbitrary unit.(A and B) Quantification of Upd3 ectopic expression in (TIF)Click here for additional data file.S4 FigScale bar indicates 50μm.(TIF)Click here for additional data file.S5 Figexpanded-LacZ (ex-LacZ) expression (arrows) in wtsx1 mutant clones (A), and dBRWD3s5349, wtsx1 double-mutant clones (B). Scale bars indicate 50μm.(A and B) (TIF)Click here for additional data file.S6 FigScmD1, dBRWD3s5349 double-mutant clones complemented with ubiquitously expressed dBRWD3-N1287A-RFP (B) or dBRWD3-N1451A-RFP (C). Scale bars indicate 50μm.(A) A diagram illustrating the molecular structure of dBRWD3. (B and C) upd3 levels (arrows) in (TIF)Click here for additional data file.S7 FigdBRWD3 transgenes plus endogenous dBRWD3 and are shown in means ± S.D., n = 4. *, **, *** indicate P< 0.01, 0.001, 0.0001 respectively in comparison to that in Canton-S by student's t-test. a.u. indicates arbitrary unit.Data represent the mRNA levels of indicated (TIF)Click here for additional data file.S8 Figubi-promoter was expressed in dBRWD3s5349 mosaic mutant eye discs. dBRWD3s5349 mutant clones were marked by the absence of RFP (arrows), with concomitant expression of ΔN-dBRWD3-RFP (A), dBRWD3-2BC-RFP (B), dBRWD3-N1287A-RFP (C), and dBRWD3-N1451A-RFP (D). Scale bars indicate 50μm. (E) The levels of endogenous H3.3 in dBRWD3-depleted brains and ventral nerve cords.(A-D) H3.3-dendra2 driven by a (TIF)Click here for additional data file.S9 Figwild type eye discs. (B-D) H3.3-dendra2 and Antp levels in ScmD1, dBRWD3s5349, double-mutant clones (B), ScmD1, dBRWD3s5349, yemGS21861 triple-mutant clones (C), and ScmD1 single-mutant clones (D) that were generated in the eye disc and marked by the absence of RFP (arrows). Scale bars indicate 50μm.(A) H3.3-dendra2 and Antp levels in (TIF)Click here for additional data file.S10 Figwild type and ScmD1dBRWD3s5349, yemGS21861 triple-mutant clones. *** indicates p<0.0001 by Student's t-test. (C) The mean intensity of Antp staining in ScmD1 and ScmD1UAS-yem. *** indicates p<0.0001 by Student's t-test.(A and B) The mean intensity of Antp (A) and Upd3 (B) staining in (TIF)Click here for additional data file.S11 Figwild-type (A) and yem-expressing (B) salivary glands. (C) The quantification of H3.3-GFP levels in (A) and (B).(A and B) Heat-shock inducible H3.3-GFP was expressed in (TIF)Click here for additional data file.S12 Figms1096-GAL4. Expression in the dorsal compartment (dark grey) is higher than in the ventral compartment (light grey). (B-F) Immunofluorescence studies of H3K27me3 levels in the E(z), dBRWD3 doubly depleted (B), E(z) depleted (C), Pc, dBRWD3 doubly depleted (D), Pc depleted (E), and control (F) wing discs. (G and H) H2AK118ub levels in SceKO (G) and dBRWD3s5349, SceKO (H) mutant clones. Scale bars indicate 50μm.(A) A schematic illustration of the expression domain of (TIF)Click here for additional data file.S13 FigAntp in ScmD1, trxE2 double-mutant clones marked by the absence of GFP (arrows). Scale bars indicate 50μm.The ectopic expression of (TIF)Click here for additional data file.S14 Figtrx-expressing (B) and trx-expressing, dBRWD3-depleted (C) peripodial epithelia. Scale bars indicate 50μm. (D-E) trx was overexpressed under the control of GMR-GAL4. The TRX-induced Antp expression (arrows) in wild-type (D), dBRWD3 depletion (E) backgrounds. (F-G) trx was overexpressed under the control of ms-1096-GAL4. The TRX-induced Ubx expression (arrows) in wild-type (F), dBRWD3 depletion (G) backgrounds. Scale bars indicate 20μm.(A) A schematic illustration of the disc proper and the peripodial epithelium of the eye disc. (B and C) Antp levels in the (TIF)Click here for additional data file.S15 Figapterous-GAL4 expression region in the 3rd wing imaginal discs. D indicates the dorsal compartment. (B-D) Immunofluorescence studies of H3K4me1 levels in the dorsal compartment of the apterous-GAL4 control (B), trx-expressing (C), and trx-expressing, dBRWD3 depleted (D) wing discs marked by GFP. Scale bars indicate 50μm. (E) A quantitative analysis of H3K4me1 levels in control, trx over-expression, and trx over-expression, dBRWD3-depleted wings. *** indicates p<0.0001 by Student's t-test. ns. indicates not significant.(A) A schematic illustration of the (TIF)Click here for additional data file.S16 FigAntp in the UAS-mCD8-GFP control, in wings over-expressing trx, or in wings with concurrent trx over-expression and dBRWD3 depletion. ns. indicates not significant. ChIP-qPCR Data are shown as means ± S.D from 4 technical replicates. *, ** indicate P<0.05, 0.01 respectively by Student's t-test.A ChIP-qPCR analysis of PolII levels at the promoters and transcription start sites of (TIF)Click here for additional data file.S17 FigTaf5 (A), Taf7 (B), Cdk7 (C), and CycH (D) RNAi. *** indicates p<0.0001 by Student's t-test.(A-D) A quantitative analysis of the knockdown efficiencies of (TIF)Click here for additional data file.S18 FigCycH knockdown, Trx and CycH-over-expressing, and CycH-over-expressing wings. (B and C) quantitative analyses of orthotopic expression of Antp in the CycH knockdown (B) and the CycH-over-expressing (C) wings. *** indicates p<0.0001 by Student's t-test. ns. indicates not significant.(A) A quantitative analysis of Abd-B expression in the control, Trx-over-expressing, Trx-over-expressing plus (TIF)Click here for additional data file.


Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling. Dexamethasone (Dex) and other glucocorticoids (GCs) are common anti-inflammatory and immuno-suppressive medicines . Yet, prAMP-activated protein kinase (AMPK) is the master regulator of energy and metabolism in eukaryotic cells ,16,17. I2O2)-induced osteoblastic cell death -2,5 diphenyltetrazolium bromide (MTT) assay described in our previous studies ,11.We applied the Histone-DNA Apoptosis ELISA Detection Kit  to quantify cell apoptosis. The detailed protocol was described in our previous studies ,11.The two lentiviral shRNAs (GV248-puromycin vector) against human AMPKα were designed, synthesized and verified by Genepharm Co. . The OB-6 cells were seeded onto 6-well plates with 50% of confluence. The lentiviral shRNA (10 μL/mL) was added directly to the OB-6 cells for 24 hours. Afterwards, cells were cultured in puromycin (1 μg/mL)-containing medium (with FBS). After 10–14 days, the resistant colonies were formed. AMPKα expression in the stable cells was tested by Western blot assay. The scramble lentiviral shRNA (Santa Cruz) was added to the control OB-6 cells.The dominant-negative mutant of AMPKα  construct was a gift from Dr. Lu’s group at Nanjing Medical University (see ). The coAssay of NADPH activity was described in previous literatures ,27. NADPROS intensity was measured via a DCFH-DA fluorescent dye . After treatment, cells were incubated with 1 μM of DCFH-DA (Invitrogen) for 30 min. Cells were then washed and analyzed for fluorescence via a Fluorescence/Multi-Detection Microplate Reader . The ROS intensity OD in the treatment group was normalized to that of control group.p < 0.05 were considered statistically significant.The data presented were mean ± standard deviation (SD). Statistical differences were analyzed by one-way ANOVA followed by multiple comparisons performed with post hoc Bonferroni test (SPSS) ,11. ValuOSU53 is a newly-developed AMPK activator , we firsIt has been shown that AMPK activation could protect osteoblasts/osteoblastic cells from Dex ,23,24, wWe compared OSU53 with other known AMPK activators in Dex-treated osteoblastic cells. Two other AMPK activators, A-769662   and CompAbove results have shown that OSU53 activated AMPK in human osteoblastic cells. Further, this novel AMPK activator protected osteoblastic cells from Dex. We next wanted to know if activation of AMPK is required for OSU53’s activity. To block AMPK activation, we utilized genetic strategies. Two non-overlapping lentiviral AMPKα shRNAs (“-a” or “-b”) were introduced to OB-6 cells, and stable cells expressing these shRNAs were established. As shown in Next, a dominant-negative AMPKα   was intrRecent studies have shown that Dex induces oxidative stress in osteoblasts/osteoblastic cells . In the in vitro studies [Osteoblasts are mesenchymal progenitor cells-derived cells, which are essential for maintain bone growth ,30,31. Y studies ,9,10,11 Dex kills osteoblasts/osteoblastic cells partly via provoking oxidative stress ,32. ROS in vivo.We conclude that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent anti-oxidant signalings. Although many AMPK specific activators have been developed and are being tested in preclinical studies, there are few specific AMPK activators entering clinical trials for various diseases . Therefo


Glycerophosphodiesterase 5 (GDE5) selectively hydrolyses glycerophosphocholine to choline and is highly expressed in type II fiber-rich skeletal muscles. We have previously generated that a truncated mutant of GDE5 (GDE5dC471) that lacks phosphodiesterase activity and shown that transgenic mice overexpressing GDE5dC471 in skeletal muscles show less skeletal muscle mass than control mice. However, the molecular mechanism and pathophysiological features underlying decreased skeletal muscle mass in GDE5dC471 mice remain unclear. In this study, we characterized the skeletal muscle disorder throughout development and investigated the primary cause of muscle atrophy. While type I fiber-rich soleus muscle mass was not altered in GDE5dC471 mice, type II fiber-rich muscle mass was reduced in 8-week-old GDE5dC471 mice. Type II fiber-rich muscle mass continued to decrease irreversibly in 1-year-old transgenic mice with an increase in apoptotic cell. Adipose tissue weight and blood triglyceride levels in 8-week-old and 1-year-old transgenic mice were higher than those in control mice. This study also demonstrated compensatory mRNA expression of neuromuscular junction (NMJ) components, including nicotinic acetylcholine receptors  and acetylcholinesterase in type II fiber-rich quadriceps muscles in GDE5dC471 mice. However, we did not observe morphological changes in NMJs associated with skeletal muscle atrophy in GDE5dC471 mice. We also found that HSP70 protein levels are significantly increased in the skeletal muscles of 2-week-old GDE5dC471 mice and in mouse myoblastic C2C12 cells overexpressing GDE5dC471. These findings suggest that GDE5dC471 mouse is a novel model of early-onset irreversible type II fiber-rich myopathy associated with cellular stress. The physiological process of aging critically affects an individual's quality of life via gradual functional, structural, and biochemical changes. Age-related decline in skeletal muscle mass and functions is defined as sarcopenia, a condition characterized by muscle weakness and fiber atrophy –3. PersoMost skeletal muscles consist of intermixed mitochondria-rich slow-twitch fiber (type I) and fast-twitch muscle fiber (type II) . It was We recently generated transgenic mice with selectively inducible skeletal muscle atrophy in type II-rich gastrocnemius and quadriceps muscles at around 12 weeks . These mThe aim of this study was to reveal the pathology and underlying cause of type II-rich skeletal muscle atrophy in GDE5dC471 mice skeletal muscles by examining cross-sections of myofibrils, muscle functions, and neuromuscular junctions through time. This study provides detailed information on the skeletal muscle pathology of GDE5dC471 mice as a useful animal model for drug development and evaluation of functional food products that protect against the onset/progression of type II-rich skeletal muscle atrophy.GDE5dC471 transgenic mouse was generated as described previously . In thisTo measure cross-sectional muscle fiber area, samples of the skeletal muscle (gastrocnemius) of GDE5dC471 mice and age-matched control mice at 4 and 8-week, and 1-year old of age were fixed in neutral buffered 10% formalin, and processed by staining paraffin-embedded transverse sections (2 μm) with hematoxylin and eosin (H&E). To detect terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) positive nuclear in gastrocnemius muscle of 4-week-old (frozen section), 8-week-old and 1-year-old (paraffin section) GDE5dC471 mice and age-matched control mice were subjected. Frozen section for TUNEL staining was prepared that the gastrocnemius muscle was harvested, fixed in 4% paraformaldehyde for 1 day, then transferred to Holt's hypertonic gum-sucrose medium (0.88M sucrose containing 1% gum acacia) for 2Days at 4°C, embedded in optimum cutting temperature compound and immediately frozen in liquid nitrogen-cooled isopentane , then prepared as 6 μm sections using a cryostat. TUNEL staining was performed using ApopTag® Peroxidase In Situ Apoptosis Detection Kit  according to the manufacturer's instructions. 3, 3'-diaminobenzidine (DAB) was used as peroxidase substrate, and for calculate the ratio of TUNEL positive nuclear to total myonuclei (%), counterstaining with hematoxylin was also performed. In order to observe morphology of nicotinic acetylcholine receptor, gastrocnemius muscles of 1-year-old GDE5dC471 mice and control mice were frozen in liquid nitrogen-cooled isopentane , and longitudinal sections (20 μm) embedded in optimum cutting temperature compound were stained with fluorescent α-bungarotoxin Alexa Fluor®594 TM . The PCR reactions were initiated with denaturation for 10 min at 95°C, followed by amplification with 40 cycles each for 15 s at 95°C, and extended for 1 min at 60°C. The primers used for semiquantitative RT-PCR or realtime PCR analysis were as follows: Acsm3, sense, 5’-CCTGATCCTATGAGAGTCTTG-3’, and antisense, 5’-GCAGTATCACCATTACTCTGTC-3’; Slc27a2, sense, 5’-GCGGCAACCATCAATCATCA-3’, and antisense, 5’-CGGTGTGTTGCACAGGTACC-3’; nAchRα1, sense, 5′-GCCGGACGTCGTTCTCTATA-3′, and antisense, 5′-GTAGAACACCCAGTGCTTCC-3′; nAchRε, sense,5′-GCGTGCTCATTTCTGGCTTG-3′, and antisense, 5′-CGCGGCAGCAGCTCTAATAA-3′; nAchRγ, sense, 5′-CAAAGGCAGCGCAATGGATT-3′, and antisense, 5′-GTAGTGGGCCATGAGGAAGA-3′; Ache, sense, 5′-GCTCCTACTTTCTGGTTTAC-3′, and antisense, 5′-AAAGATGTAGGCATAGACCC-3′; L19, sense, 5′-GGCATAGGGAAGAGGAAGG-3′, and antisense, 5′-GGATGTGCTCCATGAGGATGC-3′; β-actin, sense, 5’-TTGGGTATGGAATCCTGTGGCATC-3’, and antisense, 5’-CGGACTCATCGTACTCCTGCTTGC-3’. Realtime PCR samples were normalized according to L19 mRNA levels.Semiquantitative and quantitative PCR analyses were performed on total RNA, which was extracted from dissected quadriceps muscles of 2-, 4-, 8-week and 1-year-old GDE5dC471 and age-matched control mice, prepared with RNeasy Lipid Tissue Mini Kit  according to the instructions of the manufacturer. The reverse transcriptase reaction was carried out with 1 μg of total RNA as a template to synthesize cDNA using RevaTra Ace  reverse transcriptase. For semiquantitative PCR analysis, cDNA and specific primers were added to the GoTaq Master Mix  to give a total reaction volume of 20 μl. The reactions were sampled after 25, 28, and 30 cycles under different PCR conditions to monitor product accumulation. For quantitative PCR analysis, cDNA and specific primers were added to THUNDERBIRD™SYBR ®qPCR Mix (Toyobo) to give a total reaction volume of 15 μL. PCR reactions were performed using StepOnePlusSix hours before blood collection, food was withdrawn from mice. Serum was obtained by centrifugation of the whole blood sample at 900 × g for 10 min at 4°C and stored at -20°C for late use. Serum glucose, triglycerides and total-cholesterol were measured by Hitachi 7180 Biochemistry Automatic Analyzer .Motor coordination in 8-week and 1-year-old GDE5dC471 and control mice was assessed by KN-75 rotating rod apparatus , which consisted of a plastic rod  with a gritted surface flanked by 2 large discs. Each mouse was placed on the rod rotated 15 times per minute, and latency to fall from the rod was recorded.www.ncbi.nlm.nih.gov/geo) under accession number GSE80704.DNA microarray analysis was performed in accordance with our previous study . Total RGastrocnemius samples were homogenized in TNE buffer  using a Polytron homogenizer. The homogenates were centrifuged at 12000 rpm for 15 min at 4°C. Protein concentrations of the supernatants were determined using the BioRad Detergent Compatible (DC) assay kit (BioRad). Tissue lysates containing 16 μg protein were separated SDS–PAGE using a 10% polyacrylamide gel and stained with Coomassie Brilliant Blue (CBB) for determination of separated protein concentration or transferred to Immobilon P filters . The filters were blocked for 2 h at room temprature by soaking in 4% nonfat dried milk  in PBS; then they were incubated for 18 h at 4°C with the anti-HSP70 mouse monoclonal antibody  and after incubation in peroxidase labeled anti-mouse secondary antibody (Sigma-Adrich Japan) for 1 h the proteins were detected using ECL Western Blotting Detection Reagents . The signals were visualized by exposing the membranes to X-ray films .p values of < 0.05 (two sided) were considered statistically significant.To determine significant differences in pathophysiology, parametric comparisons between GDE5dC471 mice and age-matched control mice were carried out the two-tail unpaired t-test. Time-course data was analyzed with a two-way ANOVA followed by the two-tail unpaired t-test for evaluation the statistical significance between GDE5dC471 mice and age-matched control mice at 4 and 8-week, and 1-year old of age. Statistical analysis was executed with Prism 7 version 7.01 software . Supporting materials and methods were described in We examined morphological alterations in type II fiber-rich muscle tissues of juvenile (4-week-old), young (8-week-old), and middle-aged (1-year-old) heterozygous GDE5dC471 mice and age-matched control mice. In 8-week-old and 1-year-old GDE5dC471 mice, gastrocnemius and quadriceps muscles were smaller than those of age-matched control mice . Muscle To investigate whether GDE5dC471-related muscle atrophy affects passive motion, we performed a rotarod test using GDE5dC471 mice and age-matched control mice. GDE5dC471 mice demonstrated worse motor performance on the rotarod than control mice in all trials . Next, mIn various murine models of skeletal muscle atrophy including age-associated myopathy, several genes related to NMJs were up-regulated in skeletal muscles –24, 33, s-transferases) were up-regulated [in vivo skeletal muscle factors that sensitively respond to GDE5dC471 expression can be identified in 2-week-old GDE5dC471 mice and performed DNA microarray analysis (whole-mouse-genome microarray) using RNA samples from type II fiber-rich skeletal muscle (quadriceps) of 2-week-old GDE5dC471 and control mice to isolate genes that are differentially expressed in response to GDE5dC471 overexpression. Ninety-six genes were up-regulated and 148 genes were down-regulated in 2-week-old GDE5dC471 mice relative to age-matched control mice (p<0.05)  . We then(p<0.05) . Heat sh(p<0.05) . Sesn2 i(p<0.05) . We also(p<0.05) . Finally(p<0.05) , suggestIn this study, transgenic mice overexpressing GDE5dC471 in skeletal muscles showed reduced type II-rich skeletal muscle mass and consistently smaller cross-sectional areas of type II-rich fibers from at least 8 weeks of age. In addition, decreases in passive motor function and increased adipose tissue weight were observed in 8-week-old and 1-year-old GDE5dC471 mice. On the other hand, type I-rich soleus muscle mass of GDE5dC471 transgenic mice was similar to that of age-matched control mice from juvenile to middle-aged. The type II fiber-specific atrophy that is characteristic of GDE5dC471 mice may be because endogenous GDE5 is highly expressed in type II-rich skeletal muscle . AlternaPrevious studies have indicated a close relationship between skeletal muscle atrophy and the degeneration of NMJs that are located at pre- and post-synapses –17. In ss-transferases, were up-regulated in the quadriceps muscles of 12-week-old GDE5dC471 mice [In the present study, we found that nAchRα1, γ, and ε subunits were up-regulated in the gastrocnemius muscles of 8-week-old and 1-year-old GDE5dC471 mice, showing a similar feature as those in Duchenne muscular dystrophy model mice. However, NMJs were not morphological altered in GDE5dC471 mice even in 1-year-old mice, suggesting a distinct mechanism of up-regulation of genes related to NMJs from Duchenne muscular dystrophy model. On the other hands, various types of cellular stress  are supposed to be primary causes of skeletal muscle atrophy , 28. In 471 mice . Hence, 471 mice , 47. We 471 mice , because471 mice , 50. On 471 mice .Although NMJs were not morphological altered in GDE5dC471 mice in 1-year-old mice, the numbers of centrally located nuclei and apoptotic cells were increased in the type II fibers of 8-week-old and 1-year-old GDE5dC471 mice. Importantly, increased mRNA expression of nAchR subunits was observed even in 4-week-old GDE5dC471 mice that did not show altered skeletal muscle mass. Taken together, these observations suggest that the increased nAchR subunits expression in the skeletal muscles of GDE5dC471 mice may indicate an adaptation against cellular stress signaling and/or skeletal muscle fiber degeneration and regeneration. Thus, GDE5dC471 mice can be applied for basic research on certain human myopathies that exhibit compensatory expression of NMJs components that are a possible consequence of increased cellular stress rather than NMJ degradation. The current study characterized the pathophysiological features of GDE5dC471 mice with type II skeletal muscle atrophy without a neurotransmission disorder and further suggests the possibility that intracellular abnormal protein accumulation is associated with primary skeletal muscle pathology.S1 File(DOCX)Click here for additional data file.S1 Figp<0.05, **p<0.01.Mean ratio of centrally localized myonuclei per myofiber number in transverse sections of hematoxylin and eosin (H&E) stained gastrocnemius muscle of 4 and 8-week-old and 1-year-old GDE5dC471 mice (Tg) and age-matched control mice (Wild). 4 and 8-week-old: n = 4, and 1-year-old: n = 6. Data represent mean ± SD. *(PDF)Click here for additional data file.S2 Figp<0.01.Total RNA from gastrocnemius muscle of 1-year-old of GDE5dC471 mice (Tg) and age-matched control mice (Wild) was subjected to quantitative PCR to examine mRNA expression level of genes related to mitochondrial functions (A) or glucose metabolism (B). n = 5, Data represent mean ± SE. **(PDF)Click here for additional data file.S3 FigQuantitative PCR was performed to compare myosin heavy chain (MHC) mRNA expressions in gastrocnemius muscle of 1-week-old of GDE5dC471 mice (Tg) with age-matched control mice (Wild). n = 5, Data represent mean ± SE.(PDF)Click here for additional data file.S4 FigSpontaneous locomotive activity was examined by running wheel test. Mice were transferred to cages with a running wheel and monitored for the number of wheel revolutions made for 3 days (n = 3). There was no statistical significance between GDE5dC471 mice (Tg) and age-matched control at 1-week-old. Data represent mean ± SE.(PDF)Click here for additional data file.S5 FigGreen; GDE5, red; HSP70 and blue; DAPI.A, C2C12 cells were transiently transfected with a construct for GDE5dC471. After transfection, the C2C12 myoblasts and myotubes were fixed and stained with anti-GDE5 and anti-HSP70 antibodies. Primary antibody was visualized by fluorescein isothiocyanate- or Cy3-conjugated secondary antibody. (PDF)Click here for additional data file.


Cre-Apaf1f/f mice) and examined the antigen-specific delayed-type hypersensitivity (DTH). Lck-Cre-Apaf1f/f mice exhibited exacerbation of DTH responses as compared with Apaf1-sufficient control mice. In Lck-Cre-Apaf1f/f mice, antigen-specific T cells proliferated more, and produced more inflammatory cytokines than control T cells. Apaf1-deficient T cells from antigen-immunized mice showed higher percentages of activation phenotypes upon restimulation in vitro. Apaf1-deficient T cells from naive (non-immunized) mice also showed higher proliferation activity and cytokine production over control cells. The impact of Apaf1-deficiency in T cells, however, was not restored by a pan-caspase inhibitor, suggesting that the role of Apaf1 in T cell responses was caspase-independent/non-apoptotic. These data collectively demonstrated that Apaf1 is a negative regulator of T cell responses and implicated Apaf1 as a potential target for immunosuppressive drug discovery.Apaf1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of Apaf1 in embryonic development was shown, the role of Apaf1 in immune responses, especially T cell responses, has yet to be elucidated. We generated T cell-specific Apaf1-deficient mice (Lck- Once the antigens are removed, circulating activated lymphocytes, especially T cells, should be eliminated to avoid unnecessary inflammation and to maintain immune homeostasis, while only a few of the activated lymphocytes are fated to develop into memory lymphocytes. In this process, apoptosis plays critical roles. Apoptosand Apaf1. In bothand Apaf1, 6.C. elegans CED-4, participates in the formation and activation of the apoptosome[c[Apaf1, the mammalian homolog of poptosome, 8. Absepoptosome, 10, Caspoptosome, 12 or Aptosome[c, leads tptosome[c. Roles optosome[c. AlthougCre-Apaf1f/f, in which Apaf1 gene was disrupted with Lck promoter-driven Cre expression, to investigate the biological function of Apaf1 in T cells. Apaf1-deficient T cells showed resistance to mitochondria-dependent apoptosis but showed susceptibility to Fas-mediated apoptosis. We then performed the delayed-type hypersensitivity (DTH) assay, using ovalbumin (OVA)-specific T cell receptors (TCR)-expressing mice (OTII mice), and found that antigen-specific T cell activation leads to enhanced proliferation and Th1-type immune responses in Lck-Cre-Apaf1f/f-OTII mice, as compared with control Apaf1-sufficient OTII mice. Apaf1-deficient T cells showed higher percentages of cells expressing early activation markers after stimulation than control T cells. Surprisingly, Apaf1-sufficient T cells treated with a pan-caspase inhibitor, z-VAD-fmk , did not reproduce the activation-related phenotypes observed in Apaf1-deficient T cells, indicating caspase-independent roles of Apaf1 during T cell activation. Our data suggested that Apaf1 in T cells is a negative regulator of immune responses.In this study, we took advantage of T cell-specific Apaf1-deficient mice, Lck-Apaf1 is shown in loxP sites. The linearized targeting vector was electroporated into E14K ES cells and homologous recombinants were selected. The heterozygous Apaf1 mutant (Apaf1f-Neo/+) ES clones were injected into C57BL/6 blastocysts to generate chimeric mice. Apaf1f-Neo/+ mice were selected and crossed into C57BL/6 mice more than 10 times; the PGK-neo cassette was removed by mating the heterozygotes with CAG-Flpe transgenic (Tg) mice . Mice heterozygous for Apaf1 mutation (Apaf1f/+) were then crossed with Lck-Cre Tg mice and transgene-positive Apaf1f/+ mice were intercrossed to generate Lck-Cre-Apaf1f/f mice and Lck-Cre-Apaf1f/+ mice. Lck-Cre-Apaf1f/f -OTII mice were similarly generated by crossing Lck-Cre-Apaf1f/f mice with OTII-Tg mice expressing OVA-specific TCR. Lck-Cre Tg mice and OTII mice were kindly provided by Dr. A. Yoshimura, Keio University, Japan. Successful disruption of Apaf1 gene was confirmed with genomic Southern blot analysis and absence of Apaf1 protein in Lck-Cre-Apaf1f/f T cells was confirmed with Western blot analysis using anti-Apaf1 antibody (PharMingen)The design of the conditional targeting vector for This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Saga University . All efforts were made to minimize suffering.5 cells/well) were cultured in 96-well plates with or without the following agents or stimulation; dexamethasone , staurosporine , γ-irradiation , anti-Fas antibody  plus cycloheximide . After 20 hours, apoptotic cells were detected by staining with Annexin V- FITC (PharMingen) and propidium iodide .For induction of apoptosis in thymocytes, thymocytes (5 × 106 cells/well) were activated with plate-bound anti-CD3ε  plus anti-CD28 antibodies  for 48 hours in 6-well plates. After removal of apoptotic cells by staining the cells with Annexin V-APC followed by anti-APC magnetic beads (MACS) procedure, remaining cells (Annexin V-negative cells >97.6%) were cultured (2 × 105 cells/200 μl/well) either in fresh medium alone, or conditioned medium prepared from the supernatants of the primary stimulation culture (anti-CD3ε plus anti-CD28 antibodies), or with anti-CD3ε antibody in fresh medium. Twenty-four hours later, apoptotic cells were detected as above.For activation-induced apoptosis of peripheral lymph node (LN) T cells, LN cells were stained with FITC-labeled anti-Thy1.2 antibody (eBioscience) followed by positive selection procedure using anti-FITC magnetic beads  and LS columns (Miltenyi Biotec). Purified T cells  were cultured in the presence of OVA protein  or anti-CD3ε antibody (1 μg/ml) in RPMI1640 medium supplemented with 10% fetal bovine serum. For proliferation assay, cells were pulsed with 1 μCi/well of 3H-thymidine (PerkinElmer) during the last 7 hours of 48 hours of culture and incorporated radioactivity was measured. Culture supernatants were removed before 3H-thymidine pulse and IL-2, IFN-γ or IL-17 levels in the supernatants were measured using ELISA Ready-SET-Go (eBioscience). In some experiments, a pan-caspase inhibitor z-VAD-fmk  was added into the culture.Mice were subcutaneously (s.c.) immunized with 200 μg of OVA emulsified in complete Freund’s adjuvant  at the tail base. Inguinal LNs were collected and single cell suspensions were prepared. Total LN cells , anti-CD11b-FITC (eBioscience), anti-Ly6G-PE (BD PharMingen), anti-CD3ε-APC (eBioscience) antibodies, and analyzed by flow cytometry for expression of respective molecules.Seven days after immunization with OVA as above, mice were challenged s.c. at right footpad with 200 μg of OVA in 20 μl PBS. As a control, the same volume of PBS was injected into left footpad. Footpad thickness was measured with a dial vernier caliper (Teclock) on day 1 and 2. The magnitude of the DTH response was calculated as follows; footpad swelling (μm) = thickness of OVA-injected footpad − thickness of PBS-injected footpad. For histological analysis of the DTH lesions, paws were removed on day 2 and fixed with 10%-formaldehyde neutral buffer solution . After decalcification by a standard protocol, specimens were embedded in paraffin and were stained with hematoxylin-eosin (H&E). For analysis of the tissue-infiltrating cells, paws were thoroughly minced with scissors and then were incubated at 37°C for 1 hour in Hank's solution containing 1.0 mg/ml collagenase II (Worthington), 1.0 mg/ml dispase (Sigma-Aldrich) and 40 μg/ml Dnase I (Roche). After removing debris with 70 μm cell-strainers, cells were re-suspended into 33.7% Percoll  and pelleted by centrifugation at 1,000 7 cells/ml and incubated with CFSE (DOJINDO) at a final concentration of 5μM CFSE plus 0.1%BSA for 10 min at 37°C, followed by washing 3 times with RPMI1640 medium. Stained cells (2 × 105 cells/well) were cultured with anti-CD3ε or OVA Peptide  for 4 days, and evaluated for CFSE intensity.Naïve LN cells were suspended in PBS at 2 × 10TM (BD Biosciences) for detection of respective surface molecules.For detection of apoptotic cells, cells were stained as above. For detection of CD69, CD44, and CD62L, LN cells isolated from naïve mice or mice on day 8 after immunization were stimulated with OVA protein  or anti-CD3ε antibody (1 μg/ml) and stained with anti-CD69 (PharMingen), anti-CD44 (TONBO Biosciences), and anti-CD62L (BD Biosciences) antibodies on day 0 or 2. For detection of OTII-TCR-Tg-positive cells, cells were stained with anti-TCR Vα2-FITC antibody . Stained cells were analyzed with BD FACSVersefmk (10 and 100 μM). Cell lysates were prepared, electrophoresed, and blotted. Tubulin, caspases 3, 7, and 9 were detected with respective antibodies  and visualized using an enhanced chemiluminescence procedure .For detection of capase cleavage during T cell activation, inguinal LN cells were isolated from mice on day 8 after immunization and were stimulated with OVA protein  or anti-CD3ε antibody (1 μg/ml) as above with or without pan-caspase inhibitor z-VAD-t-tests. Differences between two samples among multiple samples with various experimental conditions were analyzed using unpaired t-tests with Bonferroni correction (See figure legends). Alpha value was set at 0.05 and p values below the alpha value (0.05) were considered to be statistically significant.Experiments were repeated at least three times. Values were expressed as means + SD. Differences between control (Apaf1-sufficient) and Apaf1-deficient samples were analyzed using unpaired Cre-Apaf1f/f mice) were generated as described in the Materials and methods. Genotypes of the mice were confirmed by Southern blot analysis of the genomic DNA using two external probes (not shown) and a probe at exon 4 . In addition, total numbers of thymocytes and cellularity of CD4+ and CD8+ thymocytes were comparable between Lck-Cre-Apaf1f/f and control Apaf1f/f mice (not shown). In our previous report, Apaf1-deficient thymocytes showed resistant to drug-induced and irradiation-induced apoptosis but showed susceptibility to Fas-induced apoptosis over WT thymocytes[Cre-Apaf1f/f mice showed similar resistance to dexamethasone-, staurosporine-, and irradiation-induced apoptosis over control thymocytes from Apaf1f/f mice but showed susceptibility to Fas-induced apoptosis just like Apaf1-sufficient thymocytes  phenotype in Lck-Cre-Apaf1f/f-OTII mice were comparable to those in Apaf1f/f-OTII mice  or lower viability of control Apaf1-sufficient cells over Apaf1-deficient cells from immunized mice after antigenic stimulation , which should be shown by reduction of pro-caspase 9, was barely detectable in both Apaf1-sufficient or -deficient OTII cells; note that cleaved form of caspase 9 was not detectable with antibody used. The western blot analysis clearly demonstrated that antigen-specific activation of T cells induced caspase 3 and 7 cleavage, which was not mediated by Apaf1 (and presumably not by caspase 9) and also was not inhibited by z-VAD-fmk. As the caspase cleavage occurred similarly in both Apaf1-sufficient and Apaf1-deficient T cells, caspase activation did not account for the difference in proliferation and cytokine production between Apaf1-sufficient and Apaf1-deficient T cells. These data indicated that the negative impact of Apaf1 on antigen-stimulated T cells was largely independent of caspase activities, although the possible involvement of caspase/apoptosis was not formally excluded.To address whether or not caspase activation occurs during T cell activation, either in Apaf1-dependent or -independent way, western blot analysis was performed to detect caspase activation during antigen-induced T cell activation . StimulaTaken together, our data clearly demonstrated that Apaf1 is a negative regulator of T cell responses by attenuating proliferation, activation, and cytokine production. This negative regulatory role of Apaf1 plays a role during antigen-specific T cell response but not in the maintenance of T cell homeostasis and the role is caspase-independent.Apaf1 gene in a T cell-specific manner. Data shown in Previous reports including ours demonstrated the role of Apaf1 in the functional shaping of some organs and adjusting the number of cells during development, 14–16. Cre-Apaf1f/f-OTII mice showed remarkable exacerbation of DTH responses including footpad swelling, cell infiltration, and inflammatory cytokine production (Cre-Apaf1f/f-OTII and Apaf1f/f-OTII mice (data not shown). In addition, time course of the footpad swelling between the two groups of mice were almost comparable (data not shown). It was thus unlikely that Apaf1 attenuated T cells responses by controlling apoptosis, although the possibility was not formally excluded.To examine the role of Apaf1 in T cells during antigen-induced immune responses, we exploited OVA-specific delayed-type hypersensitivity assay using OTII mice expressing OVA-specific T cell receptors on T cells. Lck-oduction , clearlyex vivo and in vitro analyses of antigen-specific cell activation supported another possibility that Apaf1 regulates T cell activation, independently of cell survival. Apaf1-deficient OTII T cells upon stimulation proliferated more efficiently and showed higher percentages of cells with activation phenotypes . It is possible that the difference in individual cells was subtle and/or only a small fraction of antigen-specifically activated cells showed difference. Alternatively, it is possible that Apaf1 exerts its negative regulatory effect at the priming phase where naive T cells are primarily stimulated in the draining lymph nodes, before the effector phase. Higher percentages of cells in activation status in Apaf1-deficient T cells at the primary stimulation phase in vitro  may suppCollectively, our data clearly demonstrated Apaf1 as a negative regulator of T cell responses. Further analysis of the underlying mechanisms may reveal Apaf1 as a potential target for immunosuppressive drug discovery.S1 FigApaf1 allele, structure of the targeting vector, and the resultant mutated allele with (Apaf1f-Neo) or without (Apaf1flox) neomycin resistant gene are shown. DTA; diphtheria toxin fragment A gene, Neo; neomycin resistance gene, Flpe; FLPe recombinase (treatment or expression). Two probes used for confirmation of homologous recombination (ex probe 1 and 2) and a probe used for the genomic Southern blot (A part of wild-type ern blot  are show(TIFF)Click here for additional data file.S2 FigCells were prepared as in (TIFF)Click here for additional data file.S3 FigApaf1f/f-OTII or Lck-Cre-Apaf1f/f -OTII mice were assessed as in + cells (A) and of CD44highCD62Llow cells  are shown.LN cells from OVA-immunized (TIFF)Click here for additional data file.S4 Figfmk (100). Apoptotic cells were evaluated by Annexin V and PI staining. (B) Cell lysates were prepared from thymocytes in A , electrophoresed, and blotted. Caspase 3 (pro- and cleaved form) were detected with anti-Casp3 antibody. Tubulin was detected as a control.(A) Thymocytes were prepared from wild-type C57BL/6 mice and treated with indicated doses of dexamethasone  in the absence (-) or presence of DMSO (D) or 100 μM of z-VAD-(TIFF)Click here for additional data file.


Moreover, by q-summable double sequences corresponding to the space q-summable single sequences was defined by Başar and Sever 11], we iequation  and the L ifL is called the x. Throughout the paper, i.e.,K such thatLorentz  introduc Čunjalo , if for hiuddine  proved tbp-convergent double sequence with the same limit. Almost conservative and almost regular matrices for single sequences were characterized by King [et al. [Móricz and Rhoades  consider by King  and almor et al. . Mursaler et al.  introducϑ-convergent double sequence A four-dimensional matrix A four-dimensional matrix B in the spaces of all almost convergent and almost null double sequences In this present section, we define new almost convergent double sequence spaces Now we may define the spaces The sequence spacesandare Banach spaces and linearly norm isomorphic to the spacesandrespectively, with the norm defined byK such thatp-limit as Since in other cases it can be similarly proved, we prove the theorem only for the space ∥Cf (see ), it is fined by .T from T is clear. Let us take any y by equation  A four-dimensional matrixis almostregular, i.e., iff the conditions  nditions -4.5) ho hoA fourThe following statements hold: A four-dimensional matrixis almostconservative, i.e., iff iff -4.3) an anA fourA four-dimensional matrixis almostregular, i.e., iff the conditions  ho hoA fourThe following statements hold: A four-dimensional matrixis almostconservative, i.e., iff , iff -4.3) ho hoA fourA four-dimensional matrixis almostregular, i.e., iff the conditions  ho hoA fourThe following statements hold: A four-dimensional matrixiff the condition iff (hold withinstead of:Cf)iff -4.5) ho ho\docum(ii)iff (and (hold withinstead of:Cf)iff -4.3) an an\documiff (and -4.7) ho ho\docum(iii)iff (hold withinstead of:Cf)iff -4.3) ho ho\docum(iv)iff (and (hold withinstead of:Cf)iff  and (4.1iff (and -4.16) h h\docume(v)iff (and (hold withinstead ofCbp)iff  and (4.8iff (and  hold witThe following statements hold. (i)iff (hold withinstead ofCf))iff -4.3) ho ho\docum(ii)iff (hold withinstead ofCf))iff -4.5) ho ho\docum(iii)iff (and (hold withinstead ofCf))iff -4.3) an an\documiff (and -4.7) ho ho\docum(iv)iff (and (hold withinstead ofCf))iff  and (4.1iff (and -4.16) h h\docume(v)iff (and (hold withinstead ofbp))iff  and (4.8iff (and  hold witThe following statements hold. (i)iff (hold withinstead ofCf))iff -4.3) ho ho\docum(ii)iff (hold withinstead ofCf))iff -4.5) ho ho\docum(iii)iffand (hold withinstead ofiffand -4.7) ho ho\docum(iv)iff (and (hold withinstead ofCf))iff  and (4.1iff (and -4.16) h h\docume(v)iff (and (hold withinstead ofbp))iff  and (4.8iff (and  hold wit(vi)iff (hold withinstead ofMu))iff  hold witThe following statements hold. (i)iff (and (hold withinstead ofSbp)iff  and (4.8iff (and  hold wit(ii)iff (hold withinstead of:BS)iff  hold witThe following statements hold. (i)iff iff , 4.17)--\documenf;p)iff  hold wit(ii)iff (hold withinstead of);p)iff , 4.17)--\documen);p)iff  hold wit(iii)iff (hold withinstead of);p)iff , 4.17)--\documen);p)iff  hold witf. Then many mathematicians have studied the matrix domain on almost null and almost convergent sequences spaces ..f. Then The almost convergence for double sequence was introduced by Moricz and Rhoades  and studα-, γ-duals of the space In this work, we studied the domain of the four-dimensional generalized difference matrix


HER-2 positive tumors are among the most aggressive subtypes of breast cancer and are frequently associated with metastasis and poor outcome. As with other aggressive subtypes of breast cancer, these tumors are associated with abnormally high expression of galectin-7 , which confers metastatic breast tumor cells with increased invasive behavior. Although previous studies in the rat model of breast tumorigenesis have shown that gal-7 is also increased in primary breast tumor, its contribution to the development of the primary breast tumors remains unclear. In the present work, we have used genetically-engineered gal-7-deficient mice to examine the role of gal-7 in the development of the mammary gland and of breast cancer. Using histological and immunohistological analysis of whole mammary glands at different stages of development, we detected no significant changes between normal and gal-7-deficient mice. To test the involvement of gal-7 in breast cancer, we next examined the effects of loss of gal-7 on mammary tumor development by crossing gal-7-deficient mice with the mammary tumor transgenic mouse strain FVB-Tg(MMTV-Erbb2)NK1Mul/J. Finally, assessment of mice survival and tumor volume showed a delay of mammary tumor growth in the absence of systemic gal-7. These data suggest that gal-7 could potentiate the phenotype of HER-2 positive primary breast cancer. Breast cancer is among the most commonly diagnosed cancer among women and is a major cause of cancer-associated mortality . HistoriN-acetylglucosamine branched glycans leads to alterations in the clustering of membrane glycoreceptors, resulting in functional advantages for tumor cells [Galectins belongs to a family of widely expressed proteins that contains 15 members, named according to the order of their discovery , 7. All or cells . Not suror cells –14.et al. have also shown that gal-7 expression is rapidly induced in chemically-induced rat mammary tumors [Most of our knowledge on the role of galectin in breast cancer derives from studies that focused on gal-1 and gal-3, the most studied members of the galectin family. There is increasing evidence, however, that other members of the galectin family are also important in tumor progression. We have recently shown that multiple galectins are expressed in highly aggressive breast cancer tissues . In the y tumors . In the C57BL/6-lgals7-deficient (KOG7) mice have been described [ad libitum. The MMTV-ErbB2 mice were crossed with KOG7 mice. F1 mice were bred with lgals7+/− female mice to obtain MMTV-ErbB2+/−/lgals7pos, and MMTV-ErbB2+/−/lgals7neg littermates. All experiments were conducted using virgin females. and approved by the Institutional Animal Care and Use Committee (CISAU) of INRS-Institut Armand-Frappier (Protocols #0702–08 and #0706–01). Mice were monitored twice a week until tumors developed after which they were monitored daily. Body weight was measured weekly. Tumor development was monitored by measuring length and width with calipers. The following humane endpoints were used and approved by the institutional animal care committee: signs of lethargy, weakness, hunched posture, rough hair coat, decreased mobility, rapid or labored breathing, or if there was weight loss of more than 20% of body weight. Mice also euthanized if tumors reached 2 cm3. All animals were euthanized using CO2 narcosis prior to collection and fixation of tissues. Statistical analyses of tumor growth curves were conducted on the natural log of tumor volumes in the two groups and compared with controls using unpaired t test.C57BL/6 mice and FVB-Tg(MMTV-ErbB2)NK1Mul/J transgenic mice  were obtescribed . Mice weErbB2 transgene (sense primer: 5’- CCC CGG GAG TAT GTG AGT GA -3’ and antisense primer: 5’- ACA GTC GGA AGT TTT GTC GAG T -3’), galectin-7 (sense primer: 5’- GGG CTT TGT GGG AAT ATT GAT AAC C -3’ and antisense primer: 5’- GGT ACA TTT GGA CGA TAC GCC ACT C -3’), Neo-A3 (sense primer: 5’- TCA TTA TTT GAC CCT CCG TTA CTG G -3’ and antisense primer: 5’- GCA CTG TTT ACC TTC ATC GTG CAG A -3’) and GAPDH (sense primer: 5’- CGT CGT GGA TCT GAC GTG CCG -3’ and antisense primer: 5’- GGG GTC GTT CCT GTG ACT CGT T -3’) were amplified using the following conditions: 94°C for 3 min, followed by 39 cycles of the following: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. PCR was performed in a thermal cycler . The amplified products were analyzed by electrophoresis using 1% agarose gels and SYBR Safe (Life Technologies) staining and UV illumination.DNA was extracted from the end of the tail. The MCF-7 neo (clone 1) and MCF-7 HER-2 (clone 18) cell lines were a generous gift from Dr. Mien-Chie Hung . These cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose / F12 (1:1) supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 10 mM HEPES buffer. All cell culture products were purchased from Life Technologies . Immunohistochemical reactions on smear cells were realized as described below.galectin-7  and GAPDH  cDNAs were amplified using the following conditions: 94°C for 3 min, followed by 35 cycles of the following: 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. PCR was performed in a thermal cycler . The amplified products were analyzed by electrophoresis using 1% agarose gels and SYBR Safe (Life Technologies) staining and UV illumination.Total cellular RNA was isolated from cells using the TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. First-strand cDNA was prepared from 2 μg of cellular RNA in a total reaction volume of 20 μL using the reverse transcriptase Omniscript . After reverse transcription, human Whole mammary glands collected at different stages of development were formalin-fixed and paraffin-embedded. Then, sections of 4 μm were stained with hematoxylin and eosin and scanned at a high resolution using the Nanozoomer Digital Pathology .Formalin-fixed paraffin-embedded material from each primary tumor sample and normal mammary gland was used to construct tissue microarrays with an automated arrayer designed to construct high-density tissue microarray blocks . To that end, triplicate 1 mm cores from each tumor and control tissues were punched out and arrayed into two recipient blocks. For immunohistochemical analysis, three-micrometer thick sections were prepared from each TMA. Immunostaining reactions were carried out using the Discovery XT automated immunostainer . Deparaffinated sections were incubated in cell conditioning buffer pH 8 for antigen retrieval and then with primary antibodies for 1 to 3 hrs: goat polyclonal anti-galectin-7 , rabbit monoclonal anti-estrogen receptor α , rabbit polyclonal anti-keratin 5 , rabbit polyclonal anti-HER-2 , rabbit monoclonal anti-Ki-67 . The slides were counterstained with hematoxylin and bicarbonate. Each section was scanned at a high resolution using the Nanozoomer whole-slide cell scanner .t test and Kaplan-Meier curves were assessed using GraphPad Prism 5.00 . A p value of 0.10 or less was considered statistically significant.Fisher’s exact test, lgals7−/− and age-matched wild-type (WT) female mice. No differences could be detected in mice at 15 days of pregnancy, during lactation or post-lactational involution stage . Furthermore, the absence of gal-7 did not prevent the epithelial expression of the estrogen receptor   . Similarloricrin . They alloricrin , 25–27. loricrin . Whetherloricrin , 28.neg) or gal-7-negative (ErbB2-G7pos) mammary tumors . All mammary tumors were then collected and used to construct tissue microarrays (TMAs) in order to compare the expression of gal-7, HER-2, ER, and Ki-67 in each tumor by immunohistochemistry staining . Our analysis showed that As expected for MMTV-ErbB2 transgenic mice, ER status was negative for all tumors (data not shown) [p = 0.0076) lower frequency of mammary tumors expressing high levels of Her-2 (Table 1). This association between a low frequency of Her-2-positive tumors and low gal-7 expression was also observed when we compared gal-7-positive tumors and gale-7-negative tumors in mammary tumors generated in mice that contained at least one gal-7 wild-type allele (p = 0.0037). Kaplan-Meier survival curves also showed that mice with gal-7-positive mammary tumors had a significant (p = 0.086) lower median survival time (289 days) as compared to gal-7-negative tumors derived from ErbB2-KOG7 mice (358 days) . Tumors that were gal-7-positive were also significantly (p < 0.05) larger than tumors of KOG7 mice .Previous studies using gal-3-deficient mice have revealed a role for this galectin in breast cancer progression . Thus, et shown) . Our resgal-7 mRNA expression . Such cell model will thus be useful to clarify a potential functional link between gal-7 and HER-2. Future studies will be necessary to establish the mechanism by which gal-7 accelerates the development of primary breast cancer in mice.In the present work, we have shown that: 1) the development of mammary epithelium appears normal in galectin-7-deficient mice; 2) absence of galectin-7 delayed the development of MMTV-ErbB2 mammary tumors, and 3) expression of gal-7 correlates with a higher frequency of mammary tumors with Her-2-positive tumors. Taken together, these results support the view that gal-7 plays an important role in primary breast cancer by accelerating tumor progression. They also confirm previous studies that linked gal-7 with the HER-2-positive molecular subtype. Whether expression of gal-7 and HER-2 are linked at the molecular level is an interesting possibility supported by preliminary findings using MCF-7 with or without HER-2 breast cancer cell lines. There again, we found that the presence of HER-2 in MCF-7 cells correlated with increased S1 Figneg) or gal-7-negative (ErbB2-G7pos) mammary tumors. PCR genotyping and immunohistochemistry were both used to distinguish between experimental and controls groups. Tumors were detected in mice with the ErbB2 transgene only .MMTV-ErbB2 transgenic mice were backcrossed with gal-7-deficient (KOG7) mice to generate gal-7-deficient MMTV-ErbB2 (ErbB2-KOG7) transgenic mice. Controls included mice that had at least one allele of wild-type gal-7 that were able to generate either gal-7-positive (ErbB2-G7(TIFF)Click here for additional data file.S2 Figgal-7 and GAPDH (as a loading control) were assayed by semi-quantitative RT-PCR in MCF-7 neo and MCF-7 HER-2 cells. B) Immunohistochemical staining on smear cells to confirm HER2 gene expression.MCF-7 transfectants were a generous gift from Dr. Mien-Chie Hung . Scale bars, 100 μm.A) mRNA levels of (TIFF)Click here for additional data file.


Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary “to live” or “not to live” mechanism). These data refine our knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous system.In this study, we identify the means by which segmentally homologous neurons acquire different neuropeptide fates in  Drosophila melanogaster continue to provide fundamental insights into this process . To . To Ubx mselves)  and elavineages) . Using tons Figs  and S2A.Ubx was misexpressed, however, we found posterior expansion of Va-Capa neurons in abdominal segment A5, which appears to abort the PCD program Fig 1A). Antp mutants. The posterior prevalence rule predicts that Va-neuron neuropeptidergic identities would be unaffected, as Ubx would not be affected and posterior expansion of Sex combs reduced (Scr) would not confer neuropeptide identity on T1-T3 Va-neurons. Nonetheless, T3 Va-neurons in Antp mutants expressed DH31 and Dimm   ; nonethelav-Gal4 . The datpression . AbdA leUbx activity, and Va-A2-A4 neurons terminally differentiate into Capa-expressing neurons (neuropeptidergic fate II) under the influence of abdA. In contrast, Va neurons born in abdominal segments A5-A8 are eliminated by PCD under the control of Abd-B (fate III) . Genotypes: (A) elav-Gal4/UAS-Ubx, (B) elav-Gal4/UAS-abdA, (C) elav-Gal4/UAS-Antp, (D) OregonR and as in (A-C).Expression of Dimm (green) and DH31 (red) in (PDF)Click here for additional data file.S3 FigAntennapedia mutants. Genotypes: (A) AntpNS-rvc12/Antp14 and (B) OregonR.Expression of Dimm (green) and Scr (red) in wild type and (PDF)Click here for additional data file.S4 Figcas>Antp (B). (C) Quantitation of genetic studies [n ≥8 VNC]. Genotypes: (A) OregonR, (B) cas-Gal4/UAS-Antp Expression of Capa (green) and AbdA (red) in controls (A) and (PDF)Click here for additional data file.