The original model structure describes IKK dependent I Ba degrada

The original model structure describes IKK dependent I Ba degradation in two steps, phosphorylation of I Ba catalyzed by IKK, and degradation of phosphorylated I Ba. However, this two step description omits many intermediate LY188011 steps which occur prior to I B degradation by the 26S proteasome. We therefore extended the model to include two intermediate reac tions following I Ba phosphorylation and preceding I Ba degradation, which we posited might be sufficient to account for the missing dynamics. The reactions roughly correspond to recognition of phosphorylated I Ba by an E3 ligase intermediate, and attachment of a ubiquitin chain to the substrate.

It must be noted that each of these reactions potentially encom passes numerous intermediate steps and may not corre spond directly to the reactions as they are described here, however, the mechanistic details of this pathway obtained from the literature provide a biological basis for develop ing this model. With the new model structure in place, the parameters corresponding to the new stimulus induced I Ba degrada tion reactions were estimated using the optimization algo rithm while fixing all other parameters downstream of I Ba degradation to their previously estimated values. Remarkably, parameters were found to closely match microglial NF B activation, decreasing the data fitting error by nearly 67%, with over a 9 fold improvement dur ing the first 20 min in particular. Re estimating the other parameters with the modified model provided even better agreement with the data, further reducing the fitting error from 0. 67 to 0.

30. The consistency between simulations of the new model and the data was assessed using the a posteriori sta tistical test as before. At these parameters the test yielded a P value of 0. 038, implying that the null hypothesis could not be rejected with a high significance level. This result was corroborated by obtaining a large number of para meter estimates and finding that nearly 50% of the esti mates with this model structure had Anacetrapib P 0. 01. These results provide strong evidence that the addi tion of dynamics roughly corresponding to the steps involving phosphorylated I Ba recognition and binding by the E3 ligase, polyubiquitination, and proteasomal degradation is sufficient to account for the slightly delayed NF B activation observed in microglia. Nonlinearities in IKK activation and inactivation produce the rapid transient IKK activity in microglia We next focused our attention on the upstream signal ing pathway governing IKK activation in response to TNFa stimulation. The upstream signaling module was decoupled from the downstream model by using the concentration of free nuclear NF B produced by the downstream module as a fixed model input.

Mounting medium with the nucleus specific fluorescent marker 4,6

Mounting medium with the nucleus specific fluorescent marker 4,6 diamidino 2 phenylindole, was used to maintain fluores cence. Finally, the preparations were e amined by transmission and fluorescence microscopy or a Zeiss LSM 510Meta laser scanning confocal microscope. all PG Hitec, Lisbon, Portugal. Evaluation of dysfunction and damage of cultured selleck chemical neurons There is controversy regarding the quantification of neur onal viability, because all available methods display accuracy problems, which depend on the e perimental conditions. Therefore, we decided to use two different methods previously used by our group to assess the effect of a short e posure to glutamate on neuronal viability, dys function, and or damage, namely staining with propidium iodide and SYTO 13, and assessment of lactate dehydrogenase release.

SYTO 13 and propidium iodide assay SYTO 13 is a cell permeating nucleic acid stain that increases its fluores cence upon binding to nucleic acids, thus, the pattern of SYTO 13 staining allows the visualization of viable cells and apoptotic cells in which the plasmatic membrane is still intact. PI also binds nucleic acids, resulting in strong red fluorescent enhancement. however, because this dye cannot penetrate cytoplasmic membranes, it only stains cells with a damaged plasma membrane, that is, necrotic cells and cells undergoing secondary apop tosis. To determine neuronal damage, cultured neurons were washed three times with Krebs buffer, then incubated for 3 minutes with a mi ture of SYTO 13 and PI pre pared in Krebs buffer.

After slides were coverslipped, neu rons were visualized and counted using fluorescence microscopy. At least si fields per coverslip were analyzed, counting a total of ap pro imately 300 cells. Lactate dehydrogenase assay LDH is a cytoplasmic o idoreductase that cat alyses the interconversion of pyruvate and lactate with con comitant interconversion of NADH and NAD. Upon overt cell damage leading to a compromise of plasma membrane integrity, LDH is released into the e tracellular space. Being a fairly stable enzyme, it has been widely used to evaluate the degree of damage induced by insults to cells, especially in the conte t of cell death occurring mainly through necro sis. In this study, LDH activity was measured spectrophoto metrically by assessing the rate of conversion of NADH to NAD using optical density at 340 nm.

Thus, to determine neuronal damage, the medium was aspirated and kept at 4 C until analysis. The plated neurons were lysed by three freeze thaw cycles with 1 ml HEPES buffer containing 0. 02% Triton 100. The lysates were also kept at 4 C for analysis. Before the assay, both intracellular and e tracellu lar fractions were separated by centrifugation for 10 Dacomitinib min utes at 14,000 rpm in a microcentrifuge at 4 C.

Consistently, hUCMSCs treatment attenuated the e pression of surv

Consistently, hUCMSCs treatment attenuated the e pression of survival genes, such as Bcl 2, Bcl L, Survivin, Mcl 1, and cIAP 1 in PC 3 cells, imply ing an inhibitory effect of hUCMSCs on antiapoptotic proteins. To confirm the selleck chemicals llc role of JNK in hUCMSCs induced apoptosis in PC 3 cells, JNK inhibitor study was carried out. Conversely, treatment of JNK inhibitor SP600125 reversed the apoptotic ability of hUCMSCs to cleave caspase 9 3 and PARP in PC 3 cells by Western blotting and immunofluorescence assay, indicating that the JNK pathway mediates hUCMSCs induced apoptosis in PC 3 cells. Consistent with our data, Aikin et al. claimed that PI3K inhibition led to increased JNK phosphoryl ation and pancreas islet cell death, which could be re versed by the specific JNK inhibitor SP600125.

Of note, the homing of hUCMSCs to PC 3 cells and TUNEL positive cells as an apoptotic feature was de tected in the tumor section of PC 3 cells, implying that hUCMSCs on the left flank can move to PC 3 cells on the right flank, as the homing of hUCMSCs to PC 3 cells, possibly for cell death. Likewise, Liang et al. reported that systemically infused hUCMSCs could home to the inflamed colon and effectively ameliorate colitis via modulation of IL 23 IL 17 by live in vivo im aging and immunofluorescent microscopy. Overall, our findings demonstrate the antitumor po tential of hUCMSCs for PC 3 prostate cancer treat ment, but further study is required for animal tumor study via direct or indirect injection of hUCMSCs in the near future.

Conclusions Based on our results, UCMSCs inhibit the tumor growth and have an antitumor potential for PC 3 prostate can cer treatment. Introduction The two main hallmarks of Alzheimers disease are e tracellular deposits composed of B amyloid peptide and intracellular filamentous aggregates composed of self assembled hyperphosphorylated Tau proteins. Histopathological studies show that these hallmarks spread, each in their own stereotyped fashion, within specific regions of the brain during disease evolution. This progression follows neuro anatomical pathways and could be the sign of ne opathy related processes. A large body of evidence indicates that neurons GSK-3 affected in AD follow a dying back pattern of degeneration, where abnormalities in synaptic function and a onal integrity long precede somatic cell death. Since neurons are highly polarized, this raises the question whether local AB and Tau protein abnormalities in the vicinity of different neuronal subparts lead to local degenerative pro cesses or could initiate distant dysfunction within neurons or even within neuronal networks, through synap tic alterations.

We also were interested in using the

We also were interested in using the the intergenic segment to gain insights to ICK regulation that in turn might sug gest functions. E pression of ICK mRNA is confined to the region in normal mouse epithelium where prolifera tion and lineage specifications occur and where B catenin TCF7L2 is most active. Loss of a tumor suppressor causes activation of B catenin TCF4 in colon cancers. We hypothesized that ICK promoter activity may be increased in colon cancer cell lines and in stomach cancer cells because of this correlation. We also studied breast cancer cell lines because B catenin TCF4 is highly active in breast cancers. The FB 9 ICK intergenic segment has bidirectional promoter activity We obtained a clone for a portion of the p12. 3 p11. 2 region of human chromosome 6 from the Sanger Institute.

One hoI restriction fragment contains the intergenic region and the start sites for transcription of both genes. This 4. 5 kilobase fragment and portions thereof were placed into the promoterless pGL3 luciferase plasmid so as to gener ate constructs, shown schematically in. We refer to constructs as ICK 1 to 12 and as FB 9 1 to 5. We used these constructs to study the promoter in five human cancer cell lines as well as in HEK293T. The full intergenic segment was active in both orientations in all si of the lines, suggesting that ICK and FB 9 share a bidirectional promoter. Analyses in the different lines show elements in the common SspIb to PstIb fragment are important for bidirectional activity, and may account for the correlated e pression of FB 9 and ICK in microarray data that motivated this study.

Our analyses show that the intergenic segment is not a constitu tive, bidirectional promoter because the FB 9 activity relative to ICK activity is variable. Promoter activity in HER2 overe pressing breast cancer cells ICK promoter activity was 10 20 fold higher than FB 9 promoter activity in AU565 and SKBR3 cells, using con structs ICK 1 and FB 9 1 which contain the full inter genic segment. Moreover, AU565 and SKBR3 gave similar patterns of relative activity between the different constructs derived from ICK 1 and FB 9 1. This may relate to the fact that AU565 and SKBR3 were obtained from pleural effusions from the same patient. The results obtained with the truncation constructs reveal enhancer elements within the SspIb EcoRVa seg ment and a suppressor element within the unique EcoRV EcoRV fragment.

The internal deletions indicate another enhancer element for ICK lies in EcoRVb PstIb close to the ICK start site. Removal of this segment reduces ICK promoter activity 40% in both AU565 and SKBR3 cells. E tending the internal deletion from Pst1b back to SspIb, or further back to SspIa, had modest and opposite effects. The region from SspIb to PstIb is particularly comple , and appears Cilengitide likely to have several important elements. This conclu sion is borne out by data obtained from the other lines.

http:/

Deltarasin? The 118 PS26 BC8 contigs were further analyzed by aligning the corre sponding PS26 and BC8 contigs with each other, result ing in 61 inter genotype contigs with no mismatches that were aligned. The average overlapping regions of the 61 inter genotype contigs was 241 bp with an average number of 28 sequence reads. The remaining PS26 BC8 contigs, while initially identified by BlastN as having 100% identity over a region 100 bp, did not continue to share sequence similarity outside this region and therefore did not align over the whole contig. Mapping and predicted function of putative ASGR carrier chromosome transcripts Up to four primer pairs per contig were used to test for linkage of the 61 contigs to the ASGR carrier chromo some. Sequence characterized amplified region primer pairs were designed based on the PS26 contig sequence.

After screening by PCR against PS26, IA4X, N37 and a small number of progeny from apomictic BC8 segregating for mode of reproduction, 45 contigs showed specific amplification from PS26 and apomictic BC8 but no amplification from IA4X or sexual BC8 individuals establishing linkage of 45 contigs to the ASGR carrier chromosome. Single strand conformation polymorphism analysis and a CAPS screen using two to four restriction enzymes was applied to the 14 primer pairs which amplified pro ducts in both PS26 and IA4X DNA. Four additional contigs could be linked to the ASGR carrier chromo some using SSCP analysis. The CAPS screen identified a HaeIII polymorphism for PS26 c2552, a transcript also mapped by SSCP.

The markers from the 49 ASGR carrier chromosome linked contigs were initially screened on a limited num ber of apomictic and sexual F1s for mapping to the ASGR. This resulted in one contig, PS26 c9369, showing tight linkage to the ASGR as the primers amplified DNAs from only apomictic Entinostat F1s but not sexual F1s. The remaining primer sets did not show amplification specificity in the F1 population, both apomictic and sexual progeny amplified. A larger F1 population of 22 individuals was used to map the PS26 c9369 and PS26 c2552 transcripts. PS26 c2552 was mapped based on the HaeIII polymorphism found in the CAPS screen between PS26 and IA4X and also seen in the F1 population. PS26 c2552 is unlinked to the ASGR as the CAPS polymorphism segregated 1,1 in the population but with 7 sexual and 5 apomictic individuals containing the marker. In comparison, the PS26 c9369 primers remained specific to the 10 apomictic plants and did not amplify the 12 sexual plants.

A clus tering of these expression data is shown in Figure 2, with

A clus tering of these expression data is shown in Figure 2, with cultivars arranged according to their chilling requirements. In a previous work under our experi mental Pacritinib solubility conditions, early cultivars Red Candem, Flor Red, May Glo, 86 6, Precocinho and Sunraycer required less than 412 chilling hours for dormancy release, intermediate cultivars Carolina and Crimson Baby needed 412 511 chilling hours, whereas Rose Diamond and Big Top showed requirements longer than 631 chilling hours. As expected in genes up regulated after dormancy release, the overall gene expression was higher in early cultivars with low chilling requirements than in late cultivars with higher requirements.

Interestingly, the peach putative orthologs of Arabidopsis genes involved in pollen development programs were mostly grouped in two clusters, which argues for the existence of evolutionary conserved regulatory circuits orches trating the coordinated expression of these genes. Quantitative real time RT PCR confirm ation of microarray hybridization results allowed a more accurate determination of groups of similar expression. Eight genes from the cluster I of Figure 2 were analyzed by qRT PCR. All of them showed a common pattern, with higher and similar expression values in the cultivars Red Candem, 86 6 and Sunraycer, almost undetectable expression in Rose Diamond and Big Top, and intermediate values in the remaining five cultivars. On the other hand, ten genes analyzed from the cluster II showed a similar expression profile by qRT PCR, due to their higher transcriptional activity in Red Candem and Sunraycer.

The gene ppa011974m from cluster I and other five genes not included in clusters I and II in Figure 2 had a more gradual decline in expression from early to late culti vars, without drastic differences between cultivars with similar chilling requirements. We employed these qRT PCR data, based on their improved accuracy over microarray signals, to redefine two clus ters of coordinated expression in flower bud late genes, cluster A including IB153, PpB89, ppa020886m, ppa008548m, ppa018509m, ppa009789m , ppa021109m and ppa008777m, and cluster B containing ppa003797m, ppa006852m, ppa006506m, PpB71, ppa022178m, ppa019432m, ppa016810m , ppa011965m, PpB87 and ppa021373m. The predominant expression in cultivars Red Candem, Sunraycer and to a lesser extent 86 6, indicates an earlier activation of genes involved in microsporogenesis and tapetum development in these cultivars.

Flower bud late genes are transiently expressed in anthers The tissue specificity of genes belonging to clusters A and B was studied in the cultivar Big Top by qRT PCR. The transcript accumulation of these genes in vegetative buds was negligible when compared with their expres sion in flower buds, which precludes a general function of them in dormancy or growth resumption Anacetrapib processes common to both vegetative and reproductive buds.

After 21 days, 1 l of culture was ex tracted twice with an equal

After 21 days, 1 l of culture was ex tracted twice with an equal volume selleck chem of methylene chloride. The obtained crude extract was subjected to thin layer chromatographic analysis using solvent system, chloroform methanol. After chromatography, the area with silica gel on the plates containing putative taxol and bacca tin III was scraped at the appropriate relative front and exhaustively eluted with methanol and further separated by high performance liquid chromatography using Kromasil C18 column at 227 nm. Authentic taxol and baccatin III standards were used as reference. Cell lines and culture conditions HeLa, HepG2, Jurkat JR4, Ovcar 3, T47D, Jurkat JR16 and caspase 8 deficient Jurkat cells were used for the experiments. The Jurkat cell lines were grown in RPMI 1640 medium.

HepG2, HeLa, Ovcar 3 and T47D cells were cultured in DMEM. All cul ture media were supplemented with 10% fetal bovine serum, 100 iu ml 1 penicillin and 100 ug ml 1 streptomycin. Cell lines were grown in a humidified 5% CO2 environment at 37 C and were passaged every 3 4 days. Stock solutions of paclitaxel, baccatin III, fungal taxol and fungal baccatin III dissolved in DMSO were stored at ?80 C. Stocks were diluted in culture medium at the required concentration at the time of treatment. Analysis and quantification of apoptosis Analysis of hypodiploid cells were performed using Propi dium Iodide staining. Flow cytometric analysis of the cell lines was performed after treatment with taxol and baccatin III. Cells treated with different concentrations of standards or fungal taxol and baccatin III in 500 ul of medium for various time intervals were harvested and washed once with 0.

2% BSA containing PBS and fixed in 70% ethanol for 1 h at ?20 C. The cells were then centrifuged at 1000 g and suspended in stain ing solution containing 50 ug/ml PI, 50 ug/ml RNase A and 100 uM EDTA in PBS for 1 h at 42 C. Analysis was carried out using a flow cytometer. Cell cycle distribution is presented as the number of cells versus the amount of DNA, and Entinostat the extent of apoptosis was determined by counting cells of DNA content within the subG1 peak. Effect of caspases on fungal taxol and baccatin III induced apoptosis In order to find out the involvement of caspases in the fungal taxol and baccatin III induced apoptotic pathway, caspase in hibitors were employed. Jurkat cells in 250 ul of RPMI supplemented with 10% FBS were first pretreated with 25, 50 and 100 uM of cell permeable Z VAD FMK or Z LEHD FMK or Z DEVD FMK or Z AEVD FMK or Z VDVAD FMK for 1 h. The cells were then cultured for 24 and 48 h with 6 nM of fungal taxol or 3. 5 uM of fungal baccatin III. The cells were processed for PI staining and sub jected to FACScan analysis as described above.

Western blot analysis Whole cell lysates were collected by adding

Western blot analysis Whole cell lysates were collected by adding SDS sample buffer. After extensive sonication, the samples were boiled for 10 min and subjected to SDS PAGE. The proteins were then transferred to nitrocellulose membranes and analysed truly by immunoblotting. Immunoprecipitation and MALDI TOF mass spectroscopy For immunoprecipitation assays, approximately 2. 5 107 MDA MB 231 cells were lysed in 500 ul of lysis buf fer supplemented with protease and phos phatase inhibitor cocktails, 2. 5 uM trichostatin, and 50 uM 2PCPA. The cell extracts were cleared by centrifuga tion and then diluted with 500 ul of dilution buffer supplemented with protease and phosphatase inhibitor cocktails, DNase I, 2. 5 uM trichostatin and 50 uM 2PCPA.

The extracts were pre cleared by 30 min incubations with 20 ul of PureProteome Protein G Magnetic Beads at 4 C with rotation. The E2F1 antibody was then cova lently coupled to Dynabeads and added to the pre cleared extracts. After immunoprecipita tion and elution, the bound proteins were digested with trypsin according to standard procedures. The data were recorded and processed with Agilent MassHunter Workstation Software to obtain the Peptide Mass Finger print. The resulting PMF mass spectra were searched against the E2F1 protein sequence with car bamidomethylation of cysteine as a fixed modification and phosphorylation of serine residues as variable modifi cation. The peptide mass tolerance was set to 50 ppm, and a maximum of three missed cleavages was allowed. Microscopy Confocal microscopy was carried out using a Leica TCS 4D confocal microscope.

For indir ect immunofluorescence studies, preparations of the cells on glass slides were fixed with cold acetone for 5 min and then washed with PBS. The cells were incu bated with 3% bovine serum albumin for 20 min and then 2 h at room temperature with specific primary antibodies. The cells were washed three times in PBS and incubated for 1 h at room temperature with Alexa Fluor Dyes as sec ondary antibodies. After 3 washes with PBS, the cells were incubated with 0. 01% 4 6 diamidino 2 phenylidene in water for 5 min. To ensure antibody specificity, primary antibodies were replaced with specific IgGs in negative control reactions. Statistical analysis In all experiments, the mean standard deviation values from three to five determinations in triplicate were calculated. Statistically significant differences were evaluated using a Students t test. Differences were con sidered to be statistically AV-951 significant at P 0. 05. Results and discussion 4OHT up regulates ER expression in ER negative breast cancer cells Breast cancer cells are classified as either ER positive or ER negative, depending on the presence or absence of ER, particularly ER.

The requirement for high O2 appeared to be se lective for

The requirement for high O2 appeared to be se lective for http://www.selleckchem.com/products/Imatinib(STI571).html induction of culmination, because terminal cell differentiation occurred normally even within the fruiting bodies formed after only 1 h of exposure to nor moxia. The effect of O2 appears to be mediated at least in part by prolyl 4 hydroxylation of Skp1, because elevated O2 levels are required by phyA and Skp1 overexpression strains, and lower O2 is required by PhyA overexpression and Skp1B cells. To further explore the role of Skp1 modification in O2 sensing and the importance of culmination as the target of regulation, we turned to a previously described submerged development model, in which pro gress beyond the loose aggregate stage is strictly dependent on elevated atmospheric O2, and terminal dif ferentiation bypasses the morphogenetic movements of culmination.

Terminal differentiation in submerged cultures When normal strain Ax3 cells were incubated at a simi lar density under a height of several mm of PDF buffer under room light illumination, rather than on a surface wetted with the same buffer, development proceeded only to the loose aggregate stage. However, when the at mosphere above the culture was maintained at 70 or 100% O2, the majority of cells formed tight spherical aggregates with diameters of 100 250 um and optically dense cores. These cell aggregates were uniformly bounded by Calcofluor positive stalk cells, distinguished by their polygonal shapes due to cell expansion during terminal differenti ation.

Confocal microscopy revealed that the stalk cells comprised a cortex surrounding an interior region of spore like cells, based on their characteristic ellipsoid profiles, with an uneven boundary at the inter face. Note that Figures 3 and 4 also include comparative data on phyA cells, which will be described below. The interior cells could be liberated under pressure and consisted of a mixture of spores and undifferentiated cells. In contrast, the stalk cells remained associated with the deflated cyst like struc tures. Maximal spore number was achieved by 2 d, and ranged from 6 to 33% of the input cell number. These spores tended to be less elongated than their counterparts formed in fruiting body sori, suggesting imperfect synchronization of spore coat assembly processes. To test their au thenticity, spores were released by probe sonication in a non ionic detergent, which ruptured the cyst like struc tures and lysed non spore cells.

Spores from cysts were on average slightly more brightly labeled than authentic spores isolated from fruiting bodies by immunofluores cence probing with mAb 83. 5, which binds to the fucose epitope associated with the Drug_discovery spore coat proteins SP96 and SP75. Surface labeling was retained even after boiling the spores in urea, indicating tight associ ation of residual coat proteins with spore coat.

Increased expression of RhoA or RhoC GTPAses and or their ROCK1 2

Increased expression of RhoA or RhoC GTPAses and or their ROCK1 2 effectors has been reported in several metastatic cancers, and they play important roles in tumour progression selleck catalog and invasion. These mole cules also partake in mechanotransduction of signals in response to external tensional stimuli. This work investigates the role of dense collagen matri ces that resemble the matrix densities of mammary breast cancer tissues in regulating the migration of tumour cells. MTLn3 rat mammary carcinoma cells were observed to maneuver between collagen fibrils during migration into dense matrices utilizing cell contractility. These cells also have significantly higher ROCK1 activity levels in high density compared to low density matrices, indicating matrix dependent regulation.

ROCK1 levels and activity were sensitive to HDAC inhibition by MS 275, which was abrogated when Notch1 was blocked. Inhibition of ROCK1 and metallo proteases by themselves had no effect on cell migration indicating alternation of invasion strategies. However, in the presence of both inhibitors, cell migration was sig nificantly blocked. Results Preparing in vitro collagen matrices with similar collagen content and organization to high mammographically dense tissues Regions of low or high mammographic density in prophylactic invasive ductal carcinoma tissues were macrodissected and processed for imaging and quantitative analyses. Massons Trichrome staining showed that HMT regions contained mostly collagen with isolated clusters of glandular cells whereas low mammographically dense tissues consisted mainly of adipose cells with little presence of collagen.

Collagen concentrations were esti mated using picrosirius red to stain collagen and meas uring dye uptake at 531 nm absorbance wavelength. Compared against known standards, the collagen content in LMT and HMT were measured to be 2. 65 1. 60 and 19. 59 2. 91 mg cm3, respectively. High density collagen matrix was prepared by centrifugation to increase collagen concen trations and polymerisation using vaporised NH4OH. A centrifugation time of 60 min was found to be suitable for preparing HD matrices at 19. 16 0. 74 mg cm3, similar to that for HMT extracts. The fibril densities were comparable between HMT tissue and HD matrix measuring 0. 63 0. 08 and 0. 61 0. 07 mm of fibril mm2, respectively. Similarly, in LMT tissue, the density 0.

19 0. 09, was similar to that of LD matrix which measured 0. 19 0. 07 mm of fibril mm2. Pore sizes between tumour tissue and in vitro matrices are also comparable with HMT and HD pore sizes measuring 0. 025 0. 014 and 0. 017 0. 011, res pectively, while those of LMT and LD measure 0. 678 0. 458 and 0. 799 0. 695, respectively. The colla gen fibril size of AV-951 the HD matrix was very similar to HMT tissue, with 62% of the fibrils lying within 125 225 nm for both matrices.