previous studies demonstrate that mTOR inhibition is associated with a feedback activation of AKT which may result in resistance to mTOR inhibition, no substantial increase in the phosphorylation of AKT was observed in a reaction to RAD001 in these CCC cell lines. Growth amount of RAD001 treated mice was in contrast to that of placebo treated mice and analyzed by Wilcoxon exact test. HDAC2 inhibitor Immunoreactivity was analysed using Fisher s exact test. The frequency of strong phospho mTOR immunoreactivity was significantly higher, and frequency of tumors without immunoreactivity was significantly lower in CCCs than in SACs. These results show that CCCs could be more highly influenced by mTOR for tumor progression than SACs. When analyzed by medical stage, phospho mTOR appearance was noticed in 76% of advanced stage CCCs and in 96-hours of early stage CCCs. Ergo, many patients with CCC might be candidates for treatment with a mTOR inhibitor. In contrast, in SACs, phospho mTOR expression was uncommon in early stage tumors, although it was significantly increased in advanced stage tumors. pro-protein For that reason, in SACs, mTOR inhibition might be a therapeutic alternative only in high level stage infection. Collectively, these results suggest that pharmacologic inhibition of mTOR may be a promising therapeutic strategy in the management of CCCs, both in early stage and in high level stage infection. In vitro growth inhibitory effect of RAD001 on cisplatin sensitive and painful CCC cell lines Given the frequent mTOR activation present in human CCC tumefaction specimens, we evaluated the expression of phospho mTOR in four human CCC cell lines by western blotting. As shown in Fig. 2A, under serum misery circumstances, mTOR was phosphorylated in all CCC cell lines tested, that is consistent with immunohistochemical effects observed with tumefaction samples. We next examined the efficacy of mTOR Avagacestat gamma-secretase inhibitor process inhibition by RAD001 on the expansion of CCC cells in vitro. For this purpose, we performed a MTS assay using two of these CCC cell lines with activated AKT/mTOR signaling. As shown in Fig. 2B, RAD001 inhibited the expansion of RMG1 and KOC7C cells in vitro, with 25-gauge inhibition at the highest drug concentration tested. RAD001 attenuates phosphorylation of p70S6K in vitro To find out if the anti-proliferative effects of RAD001 derive from inhibition of mTOR signaling, we examined the consequence of RAD001 on the phosphorylation of downstream p70S6K in RMG1 and KOC7C cells. AKT, mTOR and p70S6K were phosphorylated in both cell lines, indicative of the hyperactivation of the AKT/mTOR pathway. Not surprisingly, phosphorylation of the downstream effector p70S6K was considerably reduced in both cell lines by therapy with RAD001, indicating that RAD001 effectively prevents mTOR signaling in CCC cells.
The chemical and molecular determinants of raltegravir strength are actually well-understood and the nature of the interactions with its goal in the context of the integrase/vDNA complex is buy Ganetespib beginning to be elucidated owing to the contribution of molecular modeling. This knowledge plays a role in our comprehension of the reasons for the emergence of the resistance pathways, mainly on the basis of the N155, Q148 and Y143 residues. The mutation of these key residues, involved in the specific interaction of integrase with its DNA substrate, into well-defined amino acids, avoid raltegravir to bind effectively to integrase while keeping the catalytic activity of the enzyme. Modeling reports suggested that second generation inhibitors must elements depart from the type of inhibition demonstrated by raltegravir, involving concurrently metal chelation and interaction with the catalytic cycle or risk seeing the beginning of cross resistance as already demonstrated with elvitegravir. The pol secured HIV 1 integrase is really a important enzyme within the reproduction mRNA mechanism of retroviruses. . It catalyses the insertion of the viral cDNA in to the chromosomes of the infected cells. Two responses are expected for covalent integration of viral DNA. First, IN binds to a quick sequence found at either end of the long terminal repeat of the vDNA and catalyzes an endonucleotide cleavage, 3 processing reaction, resulting in the removal of two nucleotides from each of the 3 ends of LTR and the supply of hydroxy groups for nucleophilic attacks. The DNA is then used as a substrate for strand exchange reaction, ultimately causing mapk inhibitor the covalent attachment of the DNA in to the host genome. . Inhibitors of the strand transfer reaction INSTIs constitute a novel family of anti-retroviral drugs, with raltegravir in the cape, which is a first INSTI approved for AIDS therapy. Other inhibitors in high level phase of growth are GSK572 and elvitegravir. Human immunodeficiency virus type one indicates a great degree of genetic variability, that might influence the viral properties including infectivity, transmissibility, or a reaction to antiviral treatment. The most common HIV 1 group M genetic forms are circulating recombinant form CRF02 AG and subtypes A, B, C. Mentioned a largely stable distribution of HIV 1 sub-types worldwide using a notable increase in the proportion of circulating recombinant forms, a decrease in unique recombinant forms, and an overall increase in recombinants. CRF02 AG will be the main HIV strain circulating inWest and West Central Africa. Lately the recombinant CRF02 AG type was identified in the Amazon region of Brazil and in China. This progress in distribution was due mainly to a greater proportion of people from sub-saharan countries.
qPCR studies were carried out on components obtained at different time points after disease to gauge the effect on virus entry and early replication events. HIVCX05045 Bosutinib 380843-75-4 entered cells as effectively as HIVDMSO in a synchronized disease as determined by quantification of gRNA by RT qPCR evaluation at 2 hpi. . Heat inactivation of herpes or improvement of the entry inhibitor DS10000, although not the RT inhibitor efavirenz, resulted in paid down gRNA copy number, as expected. We next examined the RT phase by profiling viral DNA synthesis kinetics using qPCR research. Compared to HIVDMSO, we observed a five fold drop in the quantities of both early and late reverse transcripts in from HIVCX05045 infected cells ingredients at 12 hpi. Efavirenz blocked reverse transcription of both infections as shown by back ground level of both early and late RT services and products, displaying that HIVCX05045 carries practical RT. Of note, CX05045 checks RT neither in vitro or in vivo. In comparison to HIVDMSO infected cells, background PTM quantities of 2 LTR communities and integral copies were shown in cells infected with HIVCX05045, suggesting that the disease shows additional defects in the nuclear import step. . As expected, the integration block sustained by raltegravir all through infection was accompanied by a rise in 2 LTR circles in cells infected with HIVDMSO. Nevertheless, we observed a background amount of 2 LTR sectors in HIVCX05045 infected cells, which remained similar despite raltegravir therapy, indicating that there is minimum viral cDNA translocated into the nucleus. The reduced number of 2 LTR circles raised the question whether HIVCX05045 can also be defective for nuclear transfer of the PIC, a conference believed to be at least partially dependent Canagliflozin cell in vivo in vitro on the dynamic interaction between IN carried within the PIC and karyopherins. To deal with this matter, we performed a nuclear PIC import assay using fluorescently labeled HIV 1 particles. We created VSV. H pseudotyped particles, carrying fluorescently described IN through Vpr mediated transincorporation, in the presence of CX05045 or DMSO. HeLaP4 cells were contaminated with either HIVCX05045 or HIVDMSO after normalizing for p24 antigen. The catalytically lazy IND64E protected by the construct was successfully transcomplemented by the Vpr merged IN eGFP as established by activity at 48 hpi. In two independent experiments, the cellular distribution of the PICs was examined in cells at 7 hpi and the amount of whole and nuclear PICs was quantified by confocal microscopy. In addition, an analysis of the final distribution likelihood unmasked a statistically significant difference between HIVDMSO and HIVCX05045.
classes of anti-retroviral drugs that attack HIV 1 at different points in the viral replication cycle could be useful additions for the microbicide development direction. The lower genital tract of women constitutes Dabrafenib 1195768-06-9 a significant site for HIV invasion. Thus, reduction of oral illness would have been a significant landmark in curbing the global AIDS epidemic. While male condoms are very good at stopping vaginal human immunodeficiency virus transmission, women are frequently not empowered to negotiate their use by their sexual partners or may object to their use due to sociocultural norms or the want to conceive. The most effective longterm way of HIV prevention is just a vaccine, as it would confer immunological protection, but a successful HIV 1 vaccine is unlikely to arise in the forseeable future. Hence, alternative methods are urgently required to slow the spread of HIV. Endemic antiviral preexposure prophylaxis and vaginally or rectally sent topical microbicides are receiving increased attention as preventive tools. Currently, only soap, pHbuffering, and polyanion topical microbicides have completed testing in large-scale clinical trials. None of these nonspecific microbicides has shown a protective effect against vaginal HIV transmission, with one recently reported potential exception. An elevated risk of HIV transmission was indeed reported for your soap nonoxynol 9. These disappointing Plastid findings experienced two major effects to the microbicide industry. . First, efforts have increased to build up and standardize pre-clinical and animal testing types with high predictive power for clinical microbicide efficiency. The use of these types in appropriate screening algorithms needs to have the capability to screen out compounds such as cellulose sulfate before they enter stage II and III clinical trials. Next, the focus has shifted to substances with certain antiretroviral activity. Like, the reverse transcriptase inhibitors tenofovir, which can be already used to treat HIV disease, and dapivirine Celecoxib 169590-42-5 are currently entering as prophylactic vaginal gel formulations in phase III efficacy trials and phase IIb screening. There are problems, however, the use of as microbicides RTIs might increase the spread of HIV 1 resistance. They include more HIV entry inhibitors and particular fusion some of which may have already shown protection of macaques from vaginal transmission of integrase inhibitors in addition to simian human immunodeficiency virus. Recently, improved animal models using humanized and macaques rats have now been developed that may become part of a standard go/no get decision algorithm for choice microbicides under development. Nevertheless, these animal models have drawbacks.
Tissue sections were cut from blocks of formalin fixed paraffin tumor tissue from glioblastoma individuals treated with lapatinib or rapamycin. These TMAs have been used for other studies. TUNEL Staining Paraffin sections were deparaffinized Lonafarnib price and subjected to graded rehydration as with the method. Peroxidase activity was quenched with three minutes hydrogen peroxide in water.. TUNEL staining was performed utilizing digoxigenin conjugated dUTP and HRP conjugated anti digoxigenin antibodies after its protocol. Visualization for staining was performed with NovaRed substrate and cells were then counterstained with hematoxylin. For TUNEL immunofluoresence staining, tissue sections were stained for apoptosis using the In Situ Cell Death Detection Kit, TMR red and following its protocol. ChIP assays were done on U87 EGFR cells 4 hours of EGF treatment. Cells in two 15 cm plates were pooled for each 8 replicate. ChIP was done essentially as described. Quickly, cells were crosslinked for five minutes in 10 percent formaldehyde in PBS. After substantial Skin infection sonication, pre cleaning with protein G sepharose, and removal of the 50 uL fraction for normalization, soluble chromatin from each replicate was split three ways for overnight immunoprecipitations with 2 ug of the following antibodies: Mouse IgG, anti Pol II, or anti SREBP1. DNA Protein processes were drawn down by incubation for 2 hours with protein G sepharose, cleaned, and processed as previously described. gDNA was assayed by qPCR with primers increasing the FAS transcription start site, and a fragment upstream of the Transcription Start Site. Isogenic human U87 malignant glioma cells were incorporated in to immunodeficient SCID/Beige mice for subcutaneous xenograft studies. SCID/Beige rats were bred and maintained under defined flora pathogen free conditions at the AALAC permitted Animal Facility of the Division of Experimental Radiation Oncology, UCLA. natural product libraries For s. . H. implantation, dramatically growing tumefaction cells in culture were trypsinized, included by Trypan Blue exclusion, and re-suspended at 1 106 cells/ml in an answer of dPBS and Matrigel. Tumefaction growth was checked with calipers by measuring the perpendicular diameters of each s. H. Cyst. U87 and U87 EGFRvIII cell lines were inserted s. D. on opposite sides of the mouse stomach for treatment with atorvastatin, C75 alone or in combination. Mice were euthanized if tumors reached 14 mm in maximum diameter, or animals showed signs of infection. All experiments were conducted after approval by the Chancellors Animal Research Committee of UCLA. Tumefaction specimens were obtained based on a process accepted by the Institutional Review Board of UCLA. The very first pair of combined pre and post treatment tumor tissues for lapatinib trial, and 9 sets of pre and post treatment tumor tissues for the rapamycin trial, were examined.
Receptor proteins were precipitated from cell lysates with a commercial antibody against HER2 or with a non commercial antibody against HER1/EGFR. Lapatinib blocks EGFR and HER2 activation We have enzalutamide shown previously that both lapatinib and erlotinib, an EGFR selective tyrosine kinase inhibitor, block the growth agar soft of several pancreatic cancer cell lines1. . Because EGFR 5 inhibition is demonstrated to radiosensitize other cancers, including head and neck squamous cell carcinomas and breast cancer, we sought to find out whether these compounds may also radiosensitize pancreatic cancer cells and whether this radiosensitization correlated with EGFR and HER2 expression. We first evaluated by qRT PCR the relative expression degrees of all four members of the family of receptors among a panel of four pancreatic cancer cell lines. EGFR levels were 10 17 fold greater in the PANC 1 and T3M4 cells in accordance with that observed in the Capan 2 and MIA PaCa 2 cells, while HER2 levels were similar among all four lines. Phrase of HER3, a member of family that lacks kinase activity, was approximately 10 fold greater within the Capan 2 and T3M4 cells. HER4, the ultimate relative, had very-low mRNA expression levels across all cell lines. An anti proliferative effect was shown by all cell lines in response to increasing concentrations of both erlotinib and lapatinib. The dual EGFR/HER2 phytomorphology inhibitor lapatinib demonstrated improved growth inhibitory activity compared to erlotinib in 2 and MIA PaCa 2 cell lines, a finding consistent with low levels of EGFR mRNA in these cell lines. PANC 1 and T3M4 cells had higher levels of EGFR than HER2 expression, and demonstrated comparable growth inhibition by lapatinib and erlotinib. To show that lapatinib blocks ligand triggered EGFR and HER2 activation in our pancreatic cells activation of receptors was analyzed by immunoprecipitation followed by western blot analysis. In line with what we pifithrin a and others have previously reported using in vitro, in vivo, and individual samples and reviewed in, lapatinib blocked activation of both EGFR and HER2 in every four pancreatic cell lines. . Pancreatic cancer cell lines harboring E ras mutations are resistant to lapatinib mediated radiosensitization As a result of improved anti-proliferative and ligand triggered receptor inhibition of lapatinib in the tested cell lines, we chose to investigate whether lapatinib can radiosensitize pancreatic cancer cells. Clonogenic success assays were performed on our section of cells that were both treated with lapatinib or vehicle alone for your 2 hours preceding and 2 hours after irradiation. We chose this short duration of drug treatment since the clonogenic survival and cell cycle distribution of non irradiated cell lines that were pretreated in this style with either lapatinib or DMSO control weren’t statistically different, suggesting that the 4 hour exposure to lapatinib did not radiosensitize cells simply by inhibiting proliferation or by redistributing cells into a more radiosensitive cycle of the cell cycle.
The decrease in overall retinal thickness was largely due to a thinning of the inner retina layers. Retinas were incubated in Extravidin natural product library solution at room temperature for 2 h in the dark. Following PBS cleaning, each retina was incubated using a PharMingen DAB substrate Kit before the desired color intensity produced. microscopic pictures were taken, and cell counts were analyzed, just like the DTMR described retina flatmounts. Scotopic ERG was used to assess possible injury to the outer retinal layer by the elevated IOP. Shortly, animals were dark adapted over night and anesthetized. The pupils were dilated with Mydfrin and corneas were anaesthetized with Alcain. White light flashes were created by a photostimulator placed 25 cm before the rats eye. The answers were recorded and analyzed by data trend electroretinogram collection pc software. Before IOP was elevated baselines of The and Bwave amplitudes were gathered. They were used as a contrast from the individual ERG values collected Organism in the indicated time position after IOP elevation. As previously noted, the suture lever method produces rat ocular hypertension, the degree of which depends on the weights attached to the ends of the suture. These photographs show a duration dependent decrease in GCL cell density and loss of the inner retinal layer after 7 h of IOP elevation. Quantification of the improvements demonstrated that overall retinal thickness did not change significantly, except in the 7 h IOP height group. Ocular hypertension for approximately 7 h didn’t influence the thicknesses of the ONL, OPL, or INL. mapk inhibitor Significant cell loss in the GCL was seen in all three experimental groups in comparison with the control group. These changes within the retina confirm the length dependent ON damages induced by elevated IOP. Loss in DTMR Labeled RGCs Induced by IOP Elevation: To corroborate the ocular hypertension induced loss of cells in the GCL, DTMR labeled RGC counts were performed on retina flatmounts produced from eyes when the IOP was elevated to 45 mmHg for 7 h. Figure 4A shows representative pictures of retinas at different time points, from 3 days to 28 days, after a 7 h, 45 mmHg IOP elevation. It is clear from these images that gradual RGC damage was clear after the insult. Quantitative analysis of the data is presented in Figure 4B. Hence, the density of DTMR labeled RGC in the get a grip on Figure 1. Intraocular pressure elevation utilizing the suture lever approach. These findings suggest the outer retina was not functionally damaged by the morphological findings are confirmed by this procedure, which shown in Figure 3. Time dependent histological modifications of rat optic nerves induced by ocular hypertension. Analysis was performed four weeks following the injury.
Total neurite length in each issue was normalized to total neurite length in get a handle on wells containing NGF. For explant tests, deborah 5 embryos Mouse types DLK knock-out mice were produced by homologous recombination using a phosphoglycerate kinase neomycin cassette flanked by homology arms of 5. 1 and 2. 8 kb. The 5 arm contained a LoxP site 1. 5 kb from the neomycin cassette. Embryonic stem cells were processed via PCR using the following primers, which zoomed over equally order Cyclopamine homology arms: blotting. In DRG explant tests 24 h after plating, media were replaced with media containing no NGF and 25 ug/ml anti NGF antibody for various schedules and were then fixed for staining. For dissociated cultures, DRGs were digested in 0. As described above 05% trypsin for 30 min at 37 C and were plated. 24 h after plating, mitotic chemical was included with the tradition and then eliminated 24 h later. NGF was Skin infection withdrawn from the culture 4 5 d after plating as described above. . In experiments using JNK inhibitor AS601245, 10 mM stock solution was manufactured in DMSO and diluted to 10 uM performing concentration in media. Compartmentalized chamber assays were done essentially as previously described. In temporary, 35 mm tissue culture dishes were coated with poly d lysine and laminin and scratched with a green rake to create tracks for axonal growth. 50 ml of culture media containing 4 mg/ml methylcellulose was added to the area to ensure that axons could grow inside the tracks. A Teflon divider that creates a central cell body chamber flanked by two axon chambers was then seated on silicon grease and put on the culture dish as such that the cell body chamber was in the center of the scratched area. Dissociated DRGs from E13. 5 mouse embryos were filled in the cell human body compartment and suspended in methylcellulose thickened medium, and both axon compartments were stuffed with culture Lapatinib Tykerb media with 4 mg/ml methylcellulose. 1 d after plating, press containing 7 mM AraC were included with the cell body compartment for a period of 24 h. 3 5 d after plating, NGF was taken from different spaces by replacing media containing 4 mg/ml methylcellulose and 25 mg/ml anti NGF antibody. Biotechnology, Inc. and two siRNAs targeted to different elements of JIP3 were purchased. Degrees of knock-down were tested by quantitative PCR at 5 d after plating utilising the Syber green qPCR set and approved primer sets for DLK, JIP3, and JIP1. The control siRNA employed was an siRNA directed against luciferase. Glyceraldehyde 3 phosphate dehydrogenase expression level with an increase of than three explants scored per embryo. For compartmentalized step experiments, more than four chambers were quantified in two independent experiments. Axon destruction quantification in dissociated DRG neurons was performed using MetaMorph pc software. A journal that quantifies whole axons only was written and used to measure all photographs, like a final read-out for every image giving an overall total neurite period.
The character of protrusion and retraction establish changes in cell shape and directionality. By mapping the spatiotemporal dynamics of mobile protrusion/retraction and buy Bortezomib PI3K signaling administered by total internal reflection fluorescence microscopy, we show that randomly migrating fibroblasts reorient polarity through PI3K dependent pivoting and branching of protrusions. PI3K inhibition did not influence the initiation of newly branched humps, nor did it prevent protrusion induced by photoactivation of Rac. Rather, PI3K signaling increased after, not before, the beginning of local protrusion and was necessary for the stabilization and lateral spreading of nascent branches. All through chemotaxis, the division experiencing the higher chemoattractant concentration was favored, and, therefore, the cell reoriented to be able to align with the external gradient. Raises after, not before, the initiation of outcropping induced spontaneously or by freedom of photoactivatable Rac. Finally, it’s shown Posttranslational modification (PTM) that biasing the part and rocker reorientation process allows chemotactic fibroblasts to align migration directionality with the external gradient. . lamellipodial branching in fibroblasts is not a normal mechanism of motility but rather a stochastic process that resets migration polarity. The critical function of PI3K signaling in this process is not in the generation of new protrusions but instead to promote lateral spreading and distribution of the branched state. Re-orientation of cell migration by control of motility character across disparate time scales We previously showed that PI3K signaling, monitored by total internal reflection fluorescence microscopy in migrating fibroblasts expressing the GFP AktPH biosensor, is localized in protrusive buildings during both random migration and chemotaxis, and, therefore, the pattern of PI3K signaling correlates with overall direction of cell migration. More over, PI3K signaling is temporary, with local regions emerging and dying out, with a characteristic time scale of 15 min in randomly migrating cells, the dynamics are globally coupled, in the perception that the emergence of a hotspot k48 ubiquitin is commonly quickly followed or preceded by the death of another. Here, for the same cohort of randomly migrating cells, we mapped the radial protrusion/retraction velocity alongside the places of PI3K signaling hotspots and regions of fingerlike morphological extension as a function of angular position and time. These spatiotemporal maps reveal specific character on short and long time scales. They are almost exclusively confined to long lived morphological extensions of the cell, while specific outcropping and signaling events tend to be relatively short lived, consistent with the previous analysis. Therefore, protrusion and retraction occur along well defined tracks inside the spatiotemporal chart. Consequently, over the cell citizenry, protrusion and PI3K signaling are definitely related, however the correlation of morphological extension with either protrusion or signaling is sustained.
The cells were incubated at 37 C in a CO2 incubator for 24 h after transfection. IKK recombinant protein was pull-down through the use of Bicalutamide 90357-06-5 Flag tagged protein immunoprecipitation Kit in line with the information. . In temporary, after transfection with Flag IKK wt for 24 h, HEK293T cells were collected and cleaned by PBS for twice. The mobile lysates were prepared by incubation with lysis buffer for 15min on ice and then centrifuged for 10 min at 12,000 gary. Theresin was prepared in line with the information, and the cell lysates were included with the glue and agitated for overnight at 4 C. The resin was obtained by centrifuging for 30 sec at 8200 h and then washed by wash buffer for three times. Eventually, the Flag IKK wt was eluted by opposition with 3 Flag peptide and stored in 80 C for completing IKK kinase assay. Cellular differentiation To determine the direct result of shikonin on IKK exercise, the IKK kinase assay was performed. . In temporary, equally GST IB substrate, FLAG IKK wt recombinant protein, and ATP were incubated with or without shikonin at 30 C for 30 min. The mixture was analyzed by 10 % SDS polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose filters.. Thenitrocellulosemembraneswere blocked by 50-acre driedmilk for 60min and then incubated with G IB for overnight at 4 C.. Themembranes were cleaned with TBS T again and further incubated with HRP conjugated secondary antibodies for 60min, following day. One way ANOVA or unpaired Students test was used to determine the significance of big difference, a value of 0. 05 was considered statistically significant. 3. 3. 1. Shikonin Checks Human T Lymphocyte Proliferation. Optimum T lymphocyte proliferation requires two signals, one is provided by the antigen specific T cell receptor complex and another could be the costimulatory receptor Fingolimod distributor CD28. In the current research, the immobilized OKT3 plus CD28 antibodies in 96 well plates or PMA plus ionomycin were employed to activate T-cells, and the hallmarks of the cell activation could be observed, particularly, cell proliferation and secretion of IL 2 and IFN. Therefore, we firstly examined the effect of shikonin on human T cell proliferation, and the showed that shikonin could suppress the T cell proliferation induced by OKT 3/CD28 or PMA/ionomycin in a dose dependent fashion and 1. To ascertain whether the suppressive influence of shikonin on human T lymphocyte proliferation is resulted from the cytotoxicity of the compound, MTT method was used to gauge the possibility of T cell in the experiment. IFN secretion and to evaluate whether the inhibitory effect of shikonin on human T cell growth was mediated by inhibition of IL 2 and IFN secretion, we examined the effect of shikonin on IL 2. IL 2 and IFN were significantly released in the cells evoked by PMA/ionomycin, while this increased secretion might be eliminated by treatment of shikonin in a dose dependent fashion, as shown in Figure 2.