The current study aimed to resolve some of the inconsistencies in the literature on which mental processes affect auditory cortical activity. To this end, we studied auditory cortical firing in four monkeys with different experience while they were involved in six conditions with different arrangements of the task components sound, motor action, and water reward. Firing rates changed most strongly when a sound-only condition was compared to a condition in which sound was paired with water. Additional smaller changes occurred in more complex conditions in which the monkeys received water for motor actions before or after sounds. Our findings suggest that auditory cortex is most strongly modulated by the subjects’ level of arousal, thus by a psychological concept related to motor activity triggered by reinforcers and to readiness for operant behavior. Our findings also suggest that auditory cortex is involved in associative and emotional functions, but not in agency and cognitive effort. These conditions were complemented by three task conditions. Here, specific motor behavior was required either before, after, or both before and after the sounds in order to receive the water reward. In the MSW-condition, subjects had to exhibit motor behavior (touching a bar) to generate sounds. In the SMW-condition, subjects had to exhibit motor behavior after the presentation of the tone. In the MSMW-condition, subjects had to exhibit two motor behaviors, one to generate sounds (touching a bar) and another to the tone (releasing the bar). We note that the MSMW-condition and the MSW-condition were self-initiated conditions and the remaining conditions were externally initiated conditions. We also note that the MSMW-condition and the SMW-condition required higher efforts than the corresponding conditions in which no such motor responses were required (the MSW-condition and the SW-condition). Comparisons between pairs of conditions which differed in the presence (or absence) of one component allowed us to identify which arrangement of task components affected the firing in auditory cortex and also to make inferences on the mental processes to which auditory cortex contributes. For example, comparison between the S-condition and the SW-condition helps to clarify whether neuronal activity in auditory cortex provides a correlate of the value of sounds. This and other comparisons were also used to address the question whether auditory cortex activity is related to audio-motor associations, the sense of agency, effort, and arousal. Because the comparisons were carried out on the same neuronal populations we could also determine the relative effect size of the different mental processes on the neuronal firing.The current study aimed to resolve some of the inconsistencies in the literature on which mental processes affect auditory cortical activity. To this end, we recorded neuronal firing from the primary auditory cortex of monkeys while they were involved in six conditions differing in the arrangement of the task components sound, motor action, and water reward . ConditiWe trained two monkeys until they were able to perform all three tasks, to rapidly switch between them, and to be idle during the three simple conditions within a single experimental session. It is possible that the task-related neuronal activity identified in these instrumentally-trained monkeys differs from that of monkeys with different training experience on the same task components. To address the effects of experience on auditory cortical activity, we also tested two other monkeys on the three simple conditions only.Experiments were performed on adult female (monkey B and monkey E) and male (monkey W and monkey D) long-tailed macaques (Macaca fascicularis). More than 1 year before initiation of the current study, the two male monkeys had participated in experiments in which they performed auditory tasks different from those used here . The twoUnder deep anesthesia [Ketamine HCl (4 mg/kg) and Xylazine (5 mg/kg)], a headholder  device was implanted onto the monkeys’ head to allow atraumatic head restraint .All experiments were approved by the Animal Care and Ethics Authority of the State of Saxony-Anhalt  and conformed to the EU Directive (2010/63/EU) on the protection of animals used for scientific purposes. We support the principles of the consortium on Animal Research Reporting of Experiments were carried out in a double-walled soundproof room . For all behavioral training and testing, a monkey squatted in a primate chair. Its front compartment accommodated three light-emitting diodes for visual stimulation , a touch bar, and a spoon which could be filled with small amounts of fluids for reinforcement.A computer was used to generate visual and auditory stimuli, to monitor touching of the bar, and to control the delivery of fluids to a monkey. Auditory stimuli were either generated with a waveform generator  or with an array processor , interfaced with the computer. The output of the two devices was amplified  and presented through two loudspeakers  which were placed at a distance of ∼1 m and at an angle of 40° on the left and the right side of the monkey also see .As reinforcers, or unconditioned stimuli, we used small amounts of fluid (0.3–0.8 ml), either in form of drinking water or of a thin smoothie (made from pureed raw fruit and vegetables and diluted with water). This fluid was deposited on the spoon by means of a pump  equipped with a plastic syringe. The spoon was located in front of the monkey’s mouth such that the monkey had to actively lick the water with their tongue from the spoon.The monkeys were monitored with a digital camera . In some of the experimental sessions the video signal was recorded into a computer, using Pinnacle Studio software (25 frames per second).In most sessions, we used a multichannel drive  equipped with up to five micro-electrodes (impedance 1.5–2.5 MΩ) and a built-in pre-amplifier to record the action potentials fired by a small group of neurons from different sites of auditory cortex. The drive was attached to the monkeys’ headholder through a rigid connecting piece. In about half of the sessions of monkey W, we used a single microdrive  which was directly clamped to the recording chamber by means of a custom-made adaptor. The adaptor also allowed x-y-z positioning of the microdrive. The microdrive was equipped with a multitrode  with seven gold contacts (each with a diameter of 40μm and an impedance of 1.5–1.9 MΩ) and with a spacing of 125 μm between the contacts. An eighth contact was on the tip of the multitrode (impedance 0.5 MΩ), 700 μm from the first of the other contacts. The signals on the multitrode were pre-amplified . In sessions in which the microdrive was directly clamped to the recording chamber, the head of the monkey was not rigidly fixated but only partially restrained by surrounding the head with a custom-made box-like device made of plexiglass which was tightly fitted around the head. Based on the spatial distribution of best frequencies of the multiunits at different sites and the depth relative to cortical surface we inferred that most sites were located in the primary auditory cortex.Following preamplification, the signals from each electrode were amplified and filtered at 0.5–5 kHz with a PGMA-64 (Thomas Recordings). The action potentials of small groups of neurons (multiunits) were isolated with an A/D data acquisition system , and their time stamps and waveforms were sampled at a rate of 44.1 kHz.S-condition with sounds only and the W-condition in which only water was delivered. The M-condition was not implemented because the monkeys would not execute the same hand movement over and over again without being reinforced with water. The only two-component condition was the SW-condition, in which sounds were followed by water. Motor behavior was only required in the remaining three multi-component task conditions. In the MSW-condition, subjects had to exhibit a specific motor behavior (touching a bar) to generate sounds and to receive water. In the SMW-condition, subjects had to exhibit specific motor behavior after the presentation of sounds to receive water. In the MSMW-condition, subjects had to exhibit two motor behaviors, one to generate sounds (touching a bar) and another to the sounds to receive water (releasing the previously touched bar). We note that the MSMW-condition and the MSW-condition were self-initiated conditions and the remaining conditions were externally initiated conditions. We also note that the MSMW-condition and the SMW-condition required higher efforts than the corresponding conditions in which no such responses were required to the tones (the MSW-condition and the SW-condition). The three task conditions were also used to estimate neuronal activity related to motor behavior alone.We generated six experimental conditions which differed in the number and the order of the components sounds (S), motor behavior (M), and water (W) . We limiAs S-component we used two sounds (a pure tone and a noise burst), instead of one, because we wanted the monkeys to perform a sound discrimination task, and not a sound detection task, and thus to involve them in a task that required auditory cortex . The useAs the M-component, we considered two types of motor behavior of the monkeys. They either touched and held the bar for some time (in the MSW- and SMW-condition and at the first M-component of the MSMW-condition), or they released the bar (at the second M-component of the MSMW-condition).As the W-component, a small amount of fluid was delivered for a period of 600 ms onto a spoon which was located very close to the monkeys’ mouth and where the fluid remained until it was licked off by the monkeys.idle period of variable duration (3.5–7.0 s) before the sounds of the next trial were presented. Generally, the idle period was defined as the time between the end of the last component in the current trial and the beginning of the first component in the subsequent trial. In the S-condition and other simple conditions, every bar touch restarted the idle period to discourage such behavior. In the W-condition, only water was delivered and this was followed by an idle period, which varied randomly between 5.0 and 10.3 s. In the SW-condition, water was delivered from 1500 to 2100 ms after tone onset, both in tone-alone trials and in noise-tone trials. The idle period varied between 3.5 and 7.0 s.In the S-condition, we presented either the tone with a duration of 2200 ms, or the noise burst with a duration of 1600 ms followed by the tone. The sounds were followed by an In the MSW-condition, the monkeys received water for holding the bar for some minimal period of time, during which also the sounds were presented. In tone-alone trials, the tone started 750 ms after bar touch and the minimal hold period was 2250 ms. In noise-tone trials, the noise burst started 1050 ms after bar touch and the minimal hold period was 4350 ms. The different delays between the M- and S-component in tone-alone and noise-tone trials were accidentally implemented. In both trial types, water was delivered from 1500 to 2100 ms after tone onset to have the same temporal relationship between the sounds and the water as in the SW-condition. After tone offset, the monkeys had to release the bar and stay idle for at least 4000 ms before they could start the next trial by touching the bar. Br releases during the minimum hold period immediately stopped the sounds and the trial was scored as mistake. Br touches during the idle period reset the minimum idle time to 4000 ms, thus providing a mild form of punishment.In the SMW-condition, a tone was presented for maximally 3400 ms, either alone or 200 ms after the presentation of the noise burst. The monkeys had to discriminate the noise burst from the tone by holding the bar for a period of 1500 ms at the earliest 300 ms but not later than 1200 ms after tone onset. If they did so, the trial was scored correct and water was administered. The tone stayed on during the hold period and during water delivery and was turned off 100 ms after water delivery, as in the SW-condition. Br touches earlier than 300 ms after tone onset as well as bar releases during the 1500 ms hold period, immediately stopped the sounds and were scored as mistake . Br touches later than 1200 ms after tone onset were also scored as mistake (miss). After sound offset, the monkeys had to release the bar and stay idle for 3500–7000 ms before the next trial started with sounds.The MSMW-condition was initially identical to the MSW-condition and thus was also a self-initiated condition. Thus, the monkeys had to hold the bar which either triggered the tone after 750 ms , or triggered the noise burst after 1050 ms, which was followed by the tone . The remainder of the trial was identical to the SMW-condition, i.e., the monkeys had to release the bar from 300 to 1200 ms after the onset of the tone to receive the water reward. After sound offset, the monkeys had to stay idle for at least 4000 ms until they could start a new trial by bar touch.We defined the trial beginning as the moment in time at which the first component of a condition commenced. This was the bar touch in the MSMW- and MSW-conditions, the sound in the SMW-, SW-, and S-conditions, and the water in the W-condition.We divided the training of the monkeys on the three task conditions into five stages, in each which the monkeys remained until they performed correctly in more than 70% of the trials for more than 3 consecutive training sessions. (1) Initially the monkeys were trained for 2 months on the MSW-condition. During the subsequent training stage. (2) The monkeys were trained over a period of 1 month to perform the MSMW condition. (3) After having learnt to perform the MSW condition and the MSMW condition the monkeys were trained for 2 months to be able to switch between the two tasks within the same training session. During training stage 4, the monkeys were trained for 1 month to perform the SMW condition. This training was followed by training stage 5, in which the monkeys learnt over period of 2 months to switch between the three tasks within the same training session.task block were followed by the simple block in which we tested, in random order, the SW-condition again together with the other two simple conditions. In the passive monkeys, only the three simple conditions were tested. The currently active condition was indicated by illuminating one or two of the three LEDs located in front of the monkeys. Although we noticed that the monkeys were clearly alerted when the illumination of the LEDs changed, it is unclear whether they used the information provided by the LEDs. The monkeys rather appeared to gain knowledge about the currently active condition by constantly monitoring the task contingencies. This monitoring likely resulted in a constantly increased level of general attention despite individual conditions not being highly demanding.During an individual experimental session the different conditions were tested in separate blocks, each consisting of 60–160 trials. In the instrumentally-trained monkeys, we initially tested the three task conditions together with the SW-condition in random order that was counterbalanced across sessions. In some of the sessions, the four conditions of this At the beginning and at the end of a session, 400 pure tones with 40 different frequencies were presented at ∼60 dB SPL to assess the best frequency and the tuning curve of each multiunit . FrequenAll data analyses were carried out with custom-written scripts in MatLab (Versions 7.5 and 9.0).We re-examined the waveforms of all online detected events using a principal component analysis with the aim to identify and further exclude artifactual waveforms from our multiunit recordings.To determine how the firing rate of a given recording site varied relative to the individual components in the different experimental conditions, we calculated peri-event time histograms (PETHs) with a bin size of 100 ms which were triggered either on sound onset, on the beginning of water delivery, or on bar touch. For each PETH a minimum of 12 trials (mean 49) were averaged. The number of trials used for averaging was lower for time bins after the minimal idle period of the condition. For the task conditions, only correct trials were included. For the simple conditions, only trials with no bar touches were included. The number of error trials was too small to compute interpretable PETHs.In this report, we used population PETHs computed from all multiunits that were used for a specific comparison across conditions. For each multiunit, we first computed PETHs for each of the conditions tested in an experimental session. We then found the two bins of all PETHs of this multiunit in which the firing rate was minimal and maximal. Subsequently, we subtracted the minimal firing rate from the firing rates in all PETHs and divided this difference through the difference between the maximal and minimal firing rate. In a last step, the normalized PETHs of all multiunits of a specific condition were combined into the median normalized PETH of that condition.We used Wilcoxon signed rank tests to compare the firing rates in the population PETHs between two conditions in specified time intervals, with a significance level of 0.01. We selected this significance level to account for the multiple comparisons that were performed between conditions. In addition to this analysis across multiunits, we also tested the multiunits individually whether their firing rates differed across trials between pairs of conditions by performing Wilcoxon signed rank tests. Results of these tests revealed the fraction of multiunits with a difference for specific conditions and allowed to draw the same conclusions as those that are made in this report based on the population analysis (see tables) and, for this reason, are not shown here.The contrast of the firing rates in a given time interval between two conditions was calculated by subtracting the firing rate of the condition with the smaller number of components from the firing rate of the condition with the larger number of components and dividing this difference by the firing rate of the first condition; contrasts are expressed in percent.All recorded videos were analyzed to detect licking activity . Using MatLab, we first extracted one frame from the video where the monkey did not make any mouth movements for several seconds. In this resting frame, the coordinates of the “mouth area” with a size of 60 by 50 pixels were determined, which covered the snout of the monkey and the end of the spoon with the deposited water. These coordinates were used to mark the “mouth area” in all other frames from the video. We calculated the averaged difference in the pixel intensities of the mouth area in the resting frame to those in every other frame of the video. The resulting curve was filtered using a rational transfer function with a window size of 400 ms to remove rapid changes. The filtered curve was squared and then, similar to the PETHs, was normalized by first finding the two bins in the curves of all conditions tested in an experimental session in which the licking activity was extreme. Subsequently we subtracted the lowest licking activity from the licking activity in all conditions and divided this difference through the difference between the largest and lowest licking activity. In a last step, the normalized curves of individual sessions were combined into the median licking activity of all available sessions in the monkeys under consideration.Two monkeys (monkey W and monkey B) were trained until they were able to differentiate the six conditions and to perform the three tasks within the same experimental session .noise-tone trials), the licking activity decreased slowly from shortly after the beginning of the first sound until the water was delivered. Subsequently, the licking activity increased and peaked 1–2 s after water delivery, after which it slowly decreased to baseline. These differences were described by determining, by linear regression, the 1000 ms interval where the licking activity increased most strongly. This revealed that the strongest increase occurred earlier after water delivery in the SW-condition  than in the W-condition (2840 ms). This indicates that the monkeys associated the (conditioned) auditory stimulus with the (unconditioned) water stimulus.Inspection of the licking movements by which the monkeys drank the water indicates that the monkeys differentiated the three conditions of the simple block. This is depicted in For the task block, analysis of the bar touches and releases obtained in 2861 trials from six sessions (three from each monkey) showed that the monkeys readily switched between the three task conditions and the simple SW-condition and that they performed correctly in 92–99% of the trials in each task. Failures resulted from inappropriately handling the bar: The monkeys missed to touch the bar after tone presentation in the SMW-condition or missed to release it in the MSMW-condition; or they prematurely released the bar in the MSW-condition. We also noted that in 6–15% of the trials of the conditions, the monkeys touched the bar during the idle period between trials. The different errors suggest that in some trials the monkeys confused the four conditions and that in a given condition, the monkeys actively suppressed motor behavior that was appropriate for the other conditions. The monkeys likely suppressed such motor behavior also during the simple block as indicated by occasional bar touches.p-value was < 0.01. To increase readability of the text, we report p-values exclusively in To more easily understand how combining task components affects auditory cortical activity, we first describe the results obtained from the simple block. We compared the S- and the W-condition with the SW-condition of 65 multiunits recorded during 17 sessions from monkey W and of 32 multiunits recorded during 8 sessions from monkey B. Best frequencies (BF) of these multiunits ranged from 0.12 to 20 kHz (median 1.6 kHz) and bandwidths of the tuning curves (BW) ranged from 0.46 to 7.95 octaves. The firing rate of each multiunit was normalized and then all 97 multiunits were combined into a median population firing rate for different conditions of tone-alone and noise-tone trials . ContrasAs expected, the population firing rate in the S-condition was increased in several time intervals relative to baseline , which was significant for all onset responses but for no offset response. Differences were observed both in multiunits that were tested with tones close to their BF and in multiunits that were tested with tones more than two octaves from BF. This suggests that pairing sounds with water changed the responses to all tones within the entire tuning curve of a neuron in the same direction . ReductiThe steady-state firing rates both during the sounds and during a corresponding time window of the W-condition differed between the three simple conditions. Similar to the baseline, the steady-state firing rate of the SW-condition laid between those of the two single-component conditions. It tended to be closer to that of the S-condition and, in tone-alone trials, was significantly smaller (20% contrast) than that of the W-condition.After water delivery the population firing rate of the SW-condition varied in a similar way as that of the W-condition and hence differed from that of the S-condition. From 2500 to 3500 ms after start of water delivery, the firing rate of the SW-condition was larger than that of the S-condition, both in tone-alone trials and in noise-tone trials (16 and 20% contrast).To find out how the neuronal activity was related to the monkeys’ behavior, we computed normalized cross covariance functions between the firing rate and the licking activity from 1.5 before to 10 s after water delivery for six sessions and 18 multiunits of monkey B (phasic sound responses were disregarded and replaced by the average firing rates in the two adjacent time bins in the PETH). Covariance was smallest in the S-condition  and larger both in the W-condition (0.92) and in the SW-condition . All covariance functions peaked at time shifts different from zero, indicating that the firing rates led the licking activity by several hundreds of milliseconds.After having established that pairing sounds with water resulted in large changes of auditory cortical firing compared to the unpaired conditions, we examined the task block and analyzed 108 multiunits recorded during 21 sessions from monkey W and 72 multiunits recorded during 17 sessions from monkey B. BF ranged from 0.14 to 20.47 kHz (median 2.84 kHz) and BW from 0.96 to 7.95 octaves. In almost half of the multiunits, the task block was tested before the simple block. We noticed that the population firing rates observed in the SW-condition (which was tested twice) were very similar in the two blocks (not shown).p = 0.008) and the MSMW-condition (p = 2∗10–5) only during the last 300 ms before bar touch but not from 0 to 1000 ms after bar touch (p > 0.052). Hence bar touch-related firing appears to consist of a sharp increase in rate shortly after bar touch which is followed by a slow and monotonic rate decrease. Whether this decrease continued beyond 1 s and whether there is also bar touch-related firing before bar touch cannot be clarified with our approach.We performed two comparisons between externally-initiated and self-initiated conditions on the same population of neurons, one between the SW-condition and the MSW-condition and another between the SMW-condition and the MSMW-condition. The two pairs of conditions differed in the level of effort that was required to receive water in a trial: it was low in the first pair where the monkeys automatically received water; it was high in the second pair where the monkeys had to execute a motor response to the sounds to receive water.The population firing rate of the SW-condition differed in several intervals from that of the MSW-condition . The firThe second interval with significantly different firing rates corresponded to the phasic response to the onset of the first sound in a trial, which was the tone in tone-alone trials and the noise burst in noise-tone trials (10 and 7% contrast). No significant differences were found in the phasic responses to all other sound transients, including the onset of the tone in noise-tone trials. The differences at the first sound were partially resulted from the decreased firing following bar touch in the MSW-condition: Indeed, the relative phasic sound response to the first sound was not significantly different in tone-alone trials.The third interval with significantly different firing rates overlapped the steady-state part of the first sound when firing rates were significantly lower in the MSW-condition than in the SW-condition . Since the steady-state part of the first sound largely overlapped with bar holding, the different firing rates were likely due to bar holding in the MSW-condition .We obtained similar results when we compared the two high-effort conditions SMW and MSMW . Firing We also performed two comparisons to find neuronal activity that was related to whether or not the monkeys exhibited a motor response to the sounds. We compared two externally-initiated conditions (SMW and SW) and two self-initiated conditions (MSMW and MSW).The population firing rates of the SW-condition and the SMW-condition differed significantly during baseline  discriminated the three simple conditions. Analysis of video recordings from 14 of a total of 18 sessions (9 in monkey D and 5 in monkey E) revealed that the monkeys’ baseline licking activity differed between the three simple conditions . It tendTo find out how pairing sounds with water altered neuronal firing in the passive monkeys, we analyzed multiunit activity recorded from 75 sites in auditory cortex . BF ranged from 0.14 to 13.36 kHz (median 1.6 kHz) and BW from 1.58 to 7.95 octaves.Replicating the findings of the instrumentally-trained monkeys, the baseline firing of the passive monkeys increased significantly from the S-condition to the SW-condition (58% contrast). Opposite to the instrumentally-trained monkeys, the baseline firing of the passive monkeys was largest in the SW-condition and not in the W-condition, although none of the comparisons was significant in either group of monkeys. The two groups also differed in the phasic sound responses, which were larger in the SW-condition than in the S-condition in the passive monkeys, even though this was significant only for the weak response to noise burst offset (36% contrast). The reason for the opposite results in the two groups could be that the baseline differed quite strongly between the conditions in the passive monkeys. When we accounted for baseline changes by computing the relative phasic response, we confirmed that these responses were significantly reduced in the SW-condition relative to those in the S-condition in the passive monkeys (contrasts 21–30%). The baseline changes also explain why we found significantly different firing rates during the steady-state part of the tone and the noise burst in the three conditions tested on the passive monkeys. In conclusion, despite some quantitative differences, pairing sound with water had similar effects on the neuronal firing in instrumentally-trained and classically conditioned monkeys.The present study confirms and extends the knowledge that neuronal firing in primary auditory cortex can be related to the delivery of water , 2015. IWe previously proposed that water-related firing reflected physical properties of the water stimulus, or its reward value . ObservaOur study also extends previous accounts that auditory cortex can respond to unconditioned stimuli or can be active during the execution of unconditioned responses not only when aversive stimuli are present but also when appetitive stimuli are present. In the present study we describe that neuronal firing in auditory cortex can be related to two types of motor-activity of subjects. Firstly, there was firing that was related to bar touching. It was characterized by a brief interval with an increased firing rate shortly after bar touch which is followed by a decreased firing rate for at least one second. Secondly, there was firing that was related to the licking activity of the monkeys. These observations extend previous observations of motor-related firing in auditory cortex . We noteResults of the present study support and extend previous observations that the baseline firing rate in auditory cortex is increased relative to an S-condition when sounds are paired with water or other reinforcing stimuli. This was previously observed for conditions equivalent to those used here, including pairing sound with water  and requOur study also indicates that the baseline firing rate in auditory cortex can depend on non-auditory task components, such as in the W-condition in which baseline firing rates were increased relative to the S-condition. The question how the baseline firing rate in the W-condition is related to that in other conditions was not resolved by the present study and thus requires further experiments. These may also clarify why we obtained different results in the instrumentally-trained and the passive monkeys and why other studies found effects of self-initiation in conditions without reinforcement .In the present study, combining sounds with water or requiring motor behavior relative to sounds resulted in changes of both the phasic and the tonic sound responses in auditory cortex. This is compatible with earlier studies which also analyzed equivalent conditions; an S-condition with either an SW-condition , an MS-cIn the instrumentally-trained monkeys, the phasic sound responses were clearly different when we compared the S-condition to the SW-condition (up to 25%). By contrast, the phasic sound responses did not change, or changed very little and then changed inconsistently when we compared the SW-condition to the task conditions: If present, changes were limited to the first sound transient in a trial and disappeared if baseline changes were taken into account . These observations, thus, suggest that if an S-condition is compared to a task condition, the phasic sound responses are most strongly affected by the presence of water or some other reinforcing stimulus in the task conditions rather than by the subjects’ execution of goal directed movements relative to sounds.In the present study, tonic sound responses were, at most, slightly changed across conditions. One reason for this could be that already during the S-condition there was only weak evidence for tonic sound responses. Hence, it is possible that the changes in the steady-state firing rates simply reflect differences in baseline firing which continued throughout the presentation of the noise burst, or reflect that the firing rate was decreased after bar touch and increased after bar release.It may be counterintuitive that phasic responses to sounds are suppressed when sounds are paired with water or when motor responses have to be executed to the sounds because the sounds are behaviorally more relevant in the task conditions and the SW-condition than in the S-condition. Indeed, responses to sounds with a positive value were found to be enhanced . HoweverOur approach of comparing conditions with different number of task components allows us to conjecture which mental processes are reflected in auditory cortex. We also note that such discussion depends on the concepts the researchers have of a given mental process and on the cognitive strategies utilized by individual animals during task performance.The present study found that the sound-evoked responses changed from the S-condition to the SW-condition and the other task conditions. Because this was found between the S-condition and the SW-condition our study supports the view that auditory cortex is involved in the formation of associations between sounds and water see also . By contOur finding that the sound-evoked responses changed from the S-condition to the SW-condition and the task conditions is also compatible with the proposal that activity in auditory cortex reflects the value of sounds . Our finWe consider that our observations support the view that auditory cortical activity is related to the general activity level, or arousal, of animals. In our study the level of arousal was estimated from the monkeys’ motor activity. It likely changed from the S-condition, where the monkeys most of the time sat quietly but awake in the primate chair, to the SW-condition, where the monkeys had increased licking activity because of the water, to the three task conditions, where the monkeys also had to touch and release a bar at time points determined by the experimenters. The changes of arousal between these conditions were paralleled by changes in the baseline firing in auditory cortex. A correspondence between auditory cortical activity and arousal has been proposed in earlier studies  and, e.gBy contrast, our findings are not consistent with the view that activity in auditory cortex is related to the efforts subjects deploy on sound processing . This isThe datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by the Animal Care and Ethics Authority of the State of Saxony-Anhalt .SK designed the study, acquired, analyzed, and interpreted the data, and wrote the manuscript. MB designed the study, interpreted the data, and wrote the manuscript. ES and AG acquired and interpreted the data, and wrote the manuscript. FO interpreted the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pre-treatment on banana slices is usually carried out to stop discolouration of the fruit by oxidative reactions, in raw presentation of the fruit especially in fruit salads. Drying is an old long preservation method for fruits and vegetables. In drying banana fruit, discolorations do occur and an attempt to stop the discoloration while maintaining quality (shelf life) is studied in this work. Dataset presented here, is on the effect of different pretreatment on the proximate analysis, microbial and sensory evaluation of dried banana during its storage. The pre-treatment methods considered in this work, were lemon juice and carbonated lemon drink while the control had no pre-treatment on the slices before drying. Proximate analysis and water activity of raw and dried samples were carried out while the microbial and sensory evaluation changes were observed in the samples over one month period. Specifications table•These data are useful for insight into the effect of pre-treatment in the shelf life of dried banana samples.•Food scientist, nutritionist and equipment fabricators can benefit from this data. Also, this work is beneficial to the Small, Medium Enterprises.•The data can provide useful information on the level of preservation needed for the dried slices for any specific period of time.0C for 18 hours. The results of the proximate analysis, rate at which moisture is reabsorbed by the dried banana slices, changes in pH during storage and changes in the Titrable Acids (TTA) during storage are presented in Tables 1 – 4 respectively.The shared data is about the effect of different pretreatment on the proximate analysis, microbial and sensory evaluation changes of tray dried banana slices during its storage. Drying of the banana slices was effected in a tray dryer at a temperature of 65citrus limon ‘Eureka’) was used in this work and the lemon juice was prepared in a 1:4 ratio (being volume of lemon extract to volume of water) and samples labeled, L while the carbonated lemon drink was purchased Sprite brand with samples labeled, S. The non-treated (raw) banana  served as the control.Raw banana fruits were peeled and sliced into cylindrical shapes of about 5 mm thickness, using a banana slicer according to the modified method of Alagbe et al. 200g each of sliced banana were pre-treated by dunking for 10 minutes in 400ml of lemon juice  and carbonated lemon drink  and after 10 minutes, the treated slices were sieved and mopped with filter paper to remove the excess water on the surface of the slices.0C, for 18 hours according to works of Alagbe et al. (unpublished).Drying of samples was accomplished by loading batches of the treated and untreated banana slices into an oven at a temperature of 65 0C and 55% respectively. Common handling procedures of the dried banana slices were mimicked for analysis . 2 – 5 g of samples were taken out of the pack every week for analysis according to standard methods After drying, 50g of each sample were packed in 12 cm x 8.85 cm resealable plastic packs and refrigerated at a temperature and relative humidity of 4 ± 2 Water activity, ash content, crude fibre, protein content, total titrable acids (TTA) and sensory evaluation changes of the fresh and dried samples were determined every week and data obtained are presented in Tables 1 – 5 below. The total duration of the packed dried banana slices was 4 weeks.


This was associated with an induction of a 48-kDa active β-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa β-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa β-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active β-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active β-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical β-catenin-dependent mechanism.Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active β-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3β at Ser Chronic liver injury due to hepatitis B/C viral infections (HBV and HCV), metabolic disorders, and alcohol are the major risk factors for HCC2. Recent reports using genetic lineage tracing models have demonstrated that mature hepatocytes are the cells of origin in injury-induced HCC3. Tumorigenesis in the liver is attributable to hepatocyte plasticity, defined as the ability of the mature hepatocytes to undergo phenotype change usually by dedifferentiation or transdifferentiation into a preneoplastic precursor cell-type6. The existing paradigm that hepatic facultative stem/progenitor cells are activated to produce different cell types during liver injury has been challenged in favor of hepatocyte plasticity8. This intuitive concept rests upon observations that mature hepatocytes retain the capacity to undergo rapid division and phenotypic alteration during regeneration of the adult liver after injury. Furthermore, hepatocyte-derived proliferative ducts retain the capacity of differentiating back into hepatocytes upon cessation of injury9. These observations are supported mostly by the lineage tracing method in genetic mouse models10. The molecular drivers of hepatocyte plasticity that results in clonogenicity and oncogenicity during chronic injury in the adult liver remain elusive.Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality and accounts for approximately 745,000 deaths per year worldwide11. Among these tumor-related proteins, DCLK113 is extensively expressed in the livers of patients with cirrhosis and HCC16. Of note, DCLK1 mRNA and protein is nearly undetectable in normal human or mouse livers18. DCLK1 can also be experimentally induced during liver injury in the DEN/CCl4 mouse model of HCC19 and in primary human hepatocytes cultured under clonogenic conditions e.g. matrigel. However, DCLK1 expression is not detected in normal human or mouse livers18. Downregulation of DCLK1 inhibits HCV replication, hepatoma cell migration, tumor growth in an HCC xenograft model, EMT, and expression of myeloid-derived S100A9 protein, which suppresses anti-tumor immunity24. DCLK1 polymerizes tubulins into microtubules and is involved in cargo transport in neuronal cells26. Thus, DCLK1 plays important roles in the regulation of cellular phenotypes.We previously reported that chronic HCV infection, one of the most potent risk factors for HCC development, induces dedifferentiated phenotypes in liver cells with enhanced expression of cellular stemness-related proteins, such as DCLK1, CD133, AFP, CK19, Lgr5, Lin28, and c-Myc27). Typically, β-catenin is ubiquitinated and subjected to proteasomal degradation by a protein scaffold of Axin, APC, GSK-3β and CK1, and the E3-ubiquitin ligase b-TrCP in the absence of Wnt signaling. During this process, β-catenin is phosphorylated first at Ser45 by CK1, followed by phosphorylation at Ser33, Ser37, and Thr41 by GSK3β. However, Wnt binding to its cell surface receptor frizzled (FZ) and co-receptor LRP5/6 inactivates the β-catenin degradation complex. The active, hypophosphorylated β-catenin translocates into the nucleus where it acts as a co-factor for the TCF/LEF family of transcription factors and activates genes involved in cell proliferation, survival, stemness, invasion, and cell cycle regulation. β-catenin also forms a bridge between the cytoplasmic domain of E-cadherin and the cytoskeleton, and is a constituent protein of adherens junctions critical to the establishment and maintenance of epithelial polarity27. The microtubule-associated protein PRC1 regulates Wnt signaling by promoting cytoskeletal sequestration of the destruction complex, which results in increased stabilization of cytoplasmic β-catenin28.β-catenin is a critical component of signaling pathways regulating cellular stemness, epithelial polarity and adherens junctions, cytoskeleton remodeling, and tumorigenesis. Aberrant activation of Wnt/β-catenin signaling has been reported in many cancers including HCC  or GFP-tagged human DCLK1 (Lenti-GFP-DCLK1). FACS-based analysis suggested that 12–15% of hepatocytes were transduced after the lentiviral infections and expressed the GFP marker within 48 h (not shown). Similar transduced and subsequently trypsinized cultures formed spheroids in a magnetic levitation assay in which newly formed spheroids grow in suspension culture29. As shown in Fig. 11 that also overexpress DCLK1 was used as a positive control  cells were infected with lentiviruses expressing either RFP or N-terminus RFP-tagged human DCLK1 as described previously16. We previously demonstrated that the expressed RFP-DCLK1 protein extensively colocalizes with microtubules, stimulates inflammatory signals, and promotes Huh7 cell migration19. In this study, live cell imaging revealed the dynamic distribution of RFP-DCLK1 with GFP-labeled microtubules and centrosomes during cell division  and RFP-DCLK1 (Huh7-RFP-DCLK1). The cells were immunostained with anti-active β-catenin [ABC(8E7)]. This monoclonal antibody is highly specific and recognizes the N-terminus unphosphorylated HSGATTTAP (aa 36–44) motif of the protein. The β-catenin form detected by this antibody has been shown to be transcriptionally activells Fig. b. Quantills Fig. c. Paralllls Fig. b.Figure To confirm β-catenin status in these cells, we performed Western blot analysis of total lysates derived from Huh7-RFP and Huh7-RFP-DCLK1 cells Fig. d. While 32 in the lysates of these cells  and size of the spheroids Fig. a and b cTaken together, these data Figs.  and 3 su-catenin was observed in DCLK1 + cells, we determined its interaction with the transcription factor TCF-4 by immunoprecipitation (IP) using nuclear lysates of Huh7-RFP-DCLK1 cells. IP with anti-TCF-4 antibody followed by probing blots with anti-ABC(8E7) showed binding of the 48-kDa form of active β-catenin with nuclear TCF-4  or FOPFlash (mutant promoter-Luc) luciferase (Luc) reporter plasmids. The results showed that TCF-4/LEF-responsive wild type promoter activities were significantly enhanced (fourfold) compared with the mutant TCF/LEF promoter in Huh7-RFP-DCLK1 cells , leading to stabilization of β-catenin34. To understand this dynamic in our cell lines, we next performed Western blot analysis of total cell lysates derived from the three cell-types described above. Huh7-RFP-DCLK1 cells demonstrated an increased Ser9 phosphorylation of GSK-3β  signaling. In addition, GSK-3β activities that are required for N-terminus hyperphosphorylation-mediated degradation of β-catenin are downregulated due to increased GSK-3β-Ser35. To determine whether the 48-kDa form of active β-catenin is generated by proteasomal activities, Huh7-RFP-DCLK1 cells were treated with increasing amounts (5 µM to 125 µM) of bortezomib or equivalent amounts of solvent (DMSO) for 48 h. Bortezomib treatment of Huh7-RFP-DCLK1 cells caused increased accumulation of 48-kDa active β-catenin  compared with normal samples  and cholangiocarcinoma  showed variable expression compared with levels found in normal tissues. The survival patients with HCC was significantly reduced with high expression  of patients with cirrhosis and HCC express DCLK1 in their livers and this expression correlates with a worse prognosis0%-70% ofTo determine whether active β-catenin and DCLK1 are expressed in the same liver cells, we performed co-immunohistochemical staining of liver tissues from patients with cirrhosis and HCC (n = 20). The immunohistochemistry results revealed specific co-staining of active β-catenin (red) and DCLK1 (brown) in the same epithelial cells Fig. . Next, w38. We determined soluble E-cadherin levels in the cell culture supernatant of DCLK1-overexpressing hepatoma cells to determine whether DCLK1 overexpression in the liver diseases is related to change in epithelial phenotypes of the cells. Within 24 h of cell passage, we observed a modest increase in the cleaved/soluble E-cadherin level in the media supernatant of Huh7-RFP-DCLK1 cells compared with the media of Huh7-RFP cultures  by a number of proteinases at the cell surface produces soluble 80-kDa ectodomain, which reduces epithelial features and induces migratory phenotypes due to disruption of cell–cell interactions39. Using this model, we cyclically withdrew NTBC from their drinking water and intrasplenically transplanted primary human hepatocytes anticipating that these cells would expand and repopulate mouse livers with the human hepatocytes  deficiency in the immunodeficient FRG mouse results in liver damage due to accumulation of toxic tyrosine catabolites. However, NTBC (nitisinone) prevents liver damage in these mice by blocking 4-hydroxyphenylpyruvate (4-HPD) two steps upstream of FAH in the tyrosine catabolic pathwayRFP or RFP-DCLK1 expressing primary human hepatocytes were transplanted into FRG mice (5 mice per group). The RFP fluorescence was clearly detected mostly in the abdominal region 10 days post-transplantation for both RFP and RFP-DCLK1 groups Fig. e. The coTo further determine whether the human hepatocytes exhibit the potential to adopt more immature or dedifferentiated phenotypes, we examined active β-catenin expression in the DCLK1-expressing cells by co-immunostaining. A number of DCLK1 + hepatocytes exhibited cytoplasmic and nuclear accumulation of active β-catenin in RFP-DCLK1 chimeric livers but not in RFP control livers  that is required to bind E-cadherin. This short β-catenin form accumulates in the nucleus and activates TCF-responsive genes, such as cyclin D1. Increased cyclin D1 expression leads to hepatocyte transformation in a transgenic mouse model of HCC43. It has also been reported to correlate with an aggressive form of HCC in patients44. The Huh7 hepatoma cells used in this study have been shown to have little or no expression of Wnt family members. The tumor-promoting mutations in CTNNB1 (β-catenin), APC, or AXIN genes are also absent in this cell line. Interestingly, β-catenin activation is still required for optimal growth of Huh7 cells. However, the mechanism that regulates this activation is unknown45. Our results provide a novel non-canonical/atypical mechanism for activation of β-catenin by DCLK1  predominantly originates from hepatocytes in murine models46. Recently, Goretsky et al.32 demonstrated the existence of a low molecular weight (52–56 kDa) β-catenin that was phosphorylated at Ser552 in colon cancers. This form, generated by a proteasome-dependent mechanism, lacks the significant parts of both N- and C-termini but retains armadillo repeats that interacts with TCF4. In DCLK1-overexpressing hepatoma cells and livers derived from patients with cirrhosis and HCC, we detected a distinct low molecular weight (48-kDa) β-catenin with an intact N-terminus that was not phosphorylated at Ser33 and Ser37. This form was repeatedly detected in the lysates of cultured cells and liver tissues containing protease/phosphatase inhibitor cocktails (Sigma and Abcam). Phosphorylation of Ser33 and Ser37 residues by GSK-3β is a critical early event during proteasomal degradation of β-catenin. Overexpression of DCLK1 in Huh7 cells leads to phosphorylation of GSK-3β at the Ser9 residue . Indeed, we observed accumulation of 48-kDa β-catenin and no change in total full-length β-catenin in the bortezomib-treated Huh7-RFP-DCLK1 cells  and 10-kDa 551–671 aa) fragments71 aa fra47. Further analysis showed that these spheroids contain AFP/CK19-positive undifferentiated and albumin-positive mature hepatocytes16. A similar observation was made for AFP expression in the Huh7-RFP-DCLK1 cells-derived spheroids  and cathepsins, among others, generates a soluble 80-kDa E-cadherin fragment in the extracellular space, disassembles the cadherin–catenin complex, and releases β-catenin into the cytoplasm (reviewed in Ref.36). We previously reported that DCLK1 expression in HCC strongly correlates with MMP2 and MMP1450, which are involved in E-cadherin cleavage. Unlike normal livers, which lack both DCLK1 and 80-kDa E-cadherin, livers from patients with cirrhosis and HCC clearly showed both proteins  mice with C57BL/6 background were purchased from Yecuris (Oregon). The mouse studies were conducted in compliance with the protocol approved by OUHSC Institutional Animal Care and Use Committee .The cell lines  and cryopreserved primary human hepatocytes (obtained from Corning) were tested negative for mycoplasma and viruses using h-IMPACT panel. All methods were carried out in accordance with the guidelines and regulations of the University of Oklahoma Health Sciences Center (OUHSC) Institutional Biosafety Committee . The normal and diseased human liver tissues were obtained from the Liver Tissue Cell Distribution System . Informed consent of donors (age > 18 years) was obtained by LTCDS. The use of human cells and tissues was approved by OUHSC Institutional Review Board . Fah16. The lentiviruses expressing RFP, GFP, RFP-DCLK1, and DCLK1-GFP were produced and purified as described by Tiscornia et al.51. The human hepatocytes were cultured on collagen I-coated plates in Hepato-STIM defined medium supplemented with EGF (10 ng/mL), 2 mM l-glutamine, and 1X antibiotic–antimycotic, and were infected with lentiviruses (~ MOI 20–30). The RFP or GFP expression was monitored in live cells by fluorescence microscopy. The GFP- or RFP-expressing cells were enriched or analyzed using the FACS method. Huh7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1 × Pen/Strep, 1 × non-essential amino acids, and 10% fetal bovine serum , and were maintained at 37 °C and 5% CO2. Huh7 cells expressing RFP (Huh7-RFP) and RFP-DCLK1 (Huh7-RFP-DCLK1) were prepared by infection with the lentiviruses, followed by enrichment using the FACS method. Knockdown of DCLK1 in cells using a highly effective shDCLK1-291 shRNA and its scrambled negative control have been previously described16. The media supernatants of the cells were collected 24 h post-passage and clarified for cell debris by centrifugation. Soluble E-cadherin levels in the culture supernatants were estimated by Quantikine Human E-cadherin ELISA protocol (R&D Systems). The cell lines (Huh7 and its derivatives) and primary human hepatocytes (obtained from vendors) were tested negative for mycoplasma, lymphotropic choriomeningitis virus and hantavirus using h-IMPACT panel.Lentiviruses expressing RFP- or GFP-tagged human DCLK1 (isoform 1) were constructed using Addgene vectors, as described previously+ primary hepatocytes were measured by transiently transfected with the TOPFlash or FOPFlash plasmids as described in the Promega’s protocol.Cells  grown on glass cover-slips were rinsed briefly in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde/PBS pH 7.4 for 20 min at room temperature, washed twice with ice-cold PBS, and permeabilized in ice-cold acetone. Cells were incubated with blocking buffer  for 1 h, washed with PBS, and treated with anti-active β-catenin  or total anti-β-catenin  antibodies in PBS-T containing 1% BSA for 2 h at room temperature. After thorough washing with PBS-T, cover slips were incubated in the appropriate AlexaFluor-conjugated secondary antibodies. The nuclei were counterstained with DAPI (0.1–1 μg/mL PBS). The cover slips were mounted on microscope slides in ProLong Gold and subjected to confocal microscopy. The RFP- and RFP-DCLK1-expressing cultured cells were subjected to FACS analysis using standard method. Immunohistochemistry was performed according to manufacturer’s protocol using the Leica Bond-III™ Polymer Refine Detection system . The β-catenin activity assay in RFP-DCLK1The nuclear and cytoplasmic fractions of cultured cells were prepared using NE-PER reagents . Whole cell lysates were prepared using RIPA buffer (Thermo Fisher). The lysates were prepared using Bullet Blender’s protocol . All lysates were treated with protease/phosphatase inhibitor cocktail (Abcam or Sigma). Western blots were carried out with chemiluminescence or fluorescence reagents using LI-COR imaging system. The band intensities were calculated using Image Studio Digits. The images were produced in compliance with the digital image and integrity policies.2 incubator. Nanoshuttle beads (n3D Biosciences) were then mixed with suspended cells. The plates were covered with a magnet-cover top, incubated at 5% CO2 at 37 °C, and were photographed under phase-contrast microscopy at various time points to monitor spheroid growth in suspension culture. Similar cultures incubated for 48 h were used for determination of transduction efficiency using the FACS method.Primary human hepatocytes were cultured on collagen I-coated plates in complete Hapato-STIM hepatocyte medium containing EGF (Corning). For levitated spheroid cell culture, the GFP + or RFP + human primary hepatocytes were isolated by FACS. Primary human hepatocytes  were resuspended in complete Hepato-STIM medium, mixed with lentiviruses, and incubated in a six-well ultra-low attachment plate for 2 h in a COOne thousand Huh7, Huh7-RFP or Huh7-RFP-DCLK1 cells were cultured in a 24-well low attachment plate containing RPMI glutamax medium plus 1% fetal calf serum in matrigel for one week and total number of spheroids per well were counted manually. For live imaging of the spheroids, the cells were cultured in Lab-Teck II 8-well chambered cover glass system (Thermo Fisher) containing 100 µl matrigel per well in Hepato-STIM hepatocyte defined medium supplemented with EGF(10 ng/mL). The developing spheroids were directly imaged (× 4 magnification) at day 2, 4, 8, and 12 of plating the cells. To perform confocal microscopy, these spheroid cultures were fixed with 10% formamide-PBS for 20 min, blocked with 1% BSA in washing buffer . After washing three times, the spheroids were stained with primary and secondary antibodies. Nuclear staining was carried out with DAPI. The spheroids were subjected to confocal microscopy after washing and addition of one drop of ProLong Gold Anti-fade reagent (Invitrogen) directly into the wells.FRG mice with a C57BL/6 background  were randomized into three groups . The mice were kept healthy with 5LJ5 diet and sterile drinking water containing nitisinone  and 3% dextrose. The mice were subjected to a defined NTBC ON/Off cycle according to Yecuris’s manual, followed by infection with adenovirus expressing urokinase-type plasminogen activator  administered by retro-orbital injection (1.25E + 9 pfu in 100 microL of PBS) under anesthesia one day before hepatocyte transplantation. Human hepatocytes  were manually injected into the spleens. Prophylactic treatment with sulfamethoxazole (640 μg/mL) and trimethoprim (128 μg/mL) in drinking water was administered to the FRG mice once every two weeks to reduce the risk of bacterial infection. The mice were imaged for RFP expression 10 days post-transplantation with a IVIS Spectrum Imager. Upon termination (5 weeks post-transplantation), the livers were subjected to immunohistochemical staining.p-values were calculated using Student’s t-test. Results with p ≤ 0.05 were considered statistically significant.All experiments were performed in biological replicates of at least three and repeated to confirm the results. The graphs were presented as mean ± standard deviation. The Supplementary information


Dlx5 homeobox gene was first implicated as an oncogene in a T-ALL mouse model expressing myristoylated (Myr) Akt2. Furthermore, overexpression of Dlx5 was sufficient to drive T-ALL in mice by directly activating Akt and Notch signaling. These findings implied that Akt2 cooperates with Dlx5 in T-cell lymphomagenesis. To test this hypothesis, Lck-Dlx5;Lck-MyrAkt2 transgenic mice were generated. MyrAkt2 synergized with Dlx5 to greatly accelerate and enhance the dissemination of T-lymphomagenesis. RNA-seq analysis performed on lymphomas from Lck-Dlx5;Lck-MyrAkt mice revealed upregulation of genes involved in the Wnt and cholesterol biosynthesis pathways. Combined RNA-seq and ChIP-seq analysis of lymphomas from Lck-Dlx5;Lck-MyrAkt mice demonstrated that β-catenin directly regulates genes involved in sterol regulatory element binding transcription factor 2 (Srebf2)-cholesterol synthesis. These lymphoma cells had high Lef1 levels and were highly sensitive to β-catenin and Srebf2-cholesterol synthesis inhibitors. Similarly, human T-ALL cell lines with activated NOTCH and AKT and elevated LEF1 levels were sensitive to inhibition of β-catenin and cholesterol pathways. Furthermore, LEF1 expression positively correlated with expression of genes involved in the cholesterol synthesis pathway in primary human T-ALL specimens. Together, these data suggest that targeting β-catenin and/or cholesterol biosynthesis, together with AKT, could have therapeutic efficacy in a subset of T-ALL patients.The  Normal thymic T cells undergo development under the orchestrated and tight control of cellular pathways such as Notch and Wnt signaling. Disturbances of Notch and Wnt pathways result in T-ALL in mice and humans2. Human T-ALL samples frequently have activating NOTCH1 mutations3. Mutant NOTCH1 has a 1- to threefold increase in HES1 reporter activity4. Dysregulation of homeobox genes such as HOXA and HOX11 is also a hallmark of human T-ALL6. In mice, up-regulated expression of active Notch3 has been shown to cause T-ALL7. In some T-ALLs, recombination activating gene (Rag)-induced genomic instability that results in recurring T-cell receptor alpha (Tcra)-Myc translocations can underlie this process8. This same rearrangement has also been observed in some of our Lck-MyrAkt2 transgenic mice, in which the Lck promoter was used to direct expression of myristoylated (Myr), constitutively active Akt2 in immature T lymphocytes9. Recently, ~ 85% of cases of childhood T-ALL have been shown to have upregulation of β-catenin and Wnt target genes10. Also, the active form of β-catenin is sufficient to induce T-ALL without the involvement of NOTCH, by stalling T-cell development at the double-positive (DP) stage11. A recent report demonstrated that leukemic stem cells in T-ALL require activated Wnt signaling12. Although β-catenin transactivates its target genes via binding to TCF and LEF, interestingly, the depletion of Tcf7 causes T-ALL in mice through the upregulation of Lef113. Moreover, a recent study demonstrated that the β-catenin pathway is critical to NOTCH1-initiated T-ALL by binding with NOTCH1 to the MYC enhancer14.T-cell acute leukemia/lymphoma (T-ALL) is thought to be derived from the malignant transformation of immature thymic T cellsPten-null mice develop T-ALL which requires Rag-mediated V(D)J recombination15. β-catenin plays an essential role in Pten loss-induced T-ALL, as haploinsufficiency of the β-catenin gene, Ctnnb1, abolishes Pten-null-induced lymphomagenesis16. We found that expression of constitutively activated Akt2 (MyrAkt2) in immature T lymphocytes can induce T-ALL in mice via chromosome rearrangement-mediated upregulation of Dlx5 or Myc17. Human T-ALL cell lines lacking PTEN expression are highly sensitive to the PI3K pathway inhibitor LY29400218. One investigation uncovered alterations of PI3K, AKT and PTEN genes in about 48% of T-ALL patient samples19. Another study revealed that clinical T-ALL samples have constitutive AKT activity via posttranslational inactivation of PTEN, rather than by gene alteration20. The relationship between NOTCH and PTEN are intertwined. Although human T-ALL cell lines harboring NOTCH1 and PTEN mutations failed to respond to NOTCH inhibitors, primary murine T-ALLs were sensitive to such inhibitors21. Moreover, in mice, Notch cooperates with Akt signaling, as Pten loss accelerates Notch mutation-induced T-ALL21. However, the mechanism of such cooperativity remains unknown.Activation of AKT signaling is another major driving force in T-ALL. Dlx5 induces T-ALL in mice by directly activating Notch1, Notch3 and Irs2 transcription, which results in the upregulated Notch and Akt signaling. Moreover, the resulting tumors frequently acquired Notch1 mutations and were sensitive to Notch inhibitors. Additionally, Pten was frequently inactivated in these tumors, which suggests that Notch activation and Pten loss cooperate tumorigenically in these T-ALLs22. To address whether the Akt pathway cooperates with the Dlx5-Notch pathway in murine T-ALL development, we crossed Lck-MyrAkt2 mice to Lck-Dlx5 mice. We herein report that these doubly transgenic Lck-Dlx5;Lck-MyrAkt2 mice rapidly develop disseminated thymic lymphomas with upregulation of Wnt signaling leading to enhanced cholesterol synthesis. To our knowledge, this is the first report linking Notch and Akt crosstalk directly to β-catenin activation and cholesterol synthesis in T-cell lymphomagenesis.We recently reported that thymocyte-specific overexpression of the homeobox gene Lck-Dlx5 transgenic mice were crossed with Lck-MyrAkt2 mice. Tumor onset was greatly accelerated in Lck-Dlx5;Lck-MyrAkt2 transgenic mice, with median survival being only 8 weeks versus 24 weeks in Lck-MyrAkt2 mice and 39 weeks in Lck-Dlx5 mice  and thymic T cells from three wild-type (WT) mice. The comparison revealed 1,294 up-regulated genes and 2,728 down-regulated genes in tumor cells versus normal thymic cells with > threefold expression change. A representative list of these genes is summarized in the heatmap shown in Fig. Lck-Dlx5;Lck-MyrAkt2 mice, Notch, Myc, and Ccnd1 were previously reported to be upregulated in lymphomas from Lck-Dlx5 mice, and Myc was previously shown to be upregulated in Lck-MyrAkt2 mice22. However, upregulation of genes involved in the Wnt pathway and cholesterol synthesis were unique to the lymphomas from the Lck-Dlx5;Lck-MyrAkt2 mice. Moreover, upregulation of Vegf and down-regulation of many apoptosis-related genes and Jak-Stat genes were seen only in tumor cells from Lck-Dlx5;Lck-MyrAkt2 mice. Reactome pathway analysis identified Wnt, SREBF2 (Sterol Regulatory Element Binding Transcription Factor 2) regulation of cholesterol biosynthesis, Notch, mTOR, cell cycle, and p53 as the major altered signaling pathways in Lck-Dlx5;Lck-MyrAkt2 lymphomas , AKT activation in 11 (69%), and β-catenin activation in 6 (38%); additionally, MYC was expressed, typically abundantly, in all 16 cell lines Fig. . MoreoveTo discover genes and their associated pathways under the control of β-catenin in human T-ALL cells, WNT signaling was blocked in TIB153 and CUTLL1 cells with a β-catenin inhibitor. The cells were treated with 1 µM of PKF118-310 for 24 h, and RNA was subjected to whole transcriptome sequencing. A Venn diagram depicting commonly up- or downregulated genes in these cell lines is shown in Supplementary Fig. LEF1 expression was positively correlated with expression of cholesterol synthesis genes SREBF2, FASN, SQLE, DHCR24 and EBP, but negatively correlated with expression of steroid hormone synthesis genes   were shown to develop T-ALL by directly transactivating Notch and upregulating Akt signaling22. We thus hypothesized that constitutively active Akt2 and overexpression of Dlx5 cooperate to promote T-cell lymphomagenesis. Indeed, in the present study, we found that deaths due to T-ALL occurred 3–5 times faster in Lck-Dlx5;Lck-MyrAkt2 mice than in either Lck-MyrAkt2 mice or Lck-Dlx5 mice. This marked acceleration of tumor onset and progression appears to result from the unique activation of β-catenin signaling in lymphoma cells from Lck-Dlx5;Lck-MyrAkt2 mice, as activation of Akt, Notch, and Myc were common to the lymphomas from mice with all three genotypes. Additionally, treatment of lymphoma cells from Lck-Dlx5;Lck-MyrAkt2 mice with β-catenin/Tcf inhibitors demonstrated that β-catenin signaling plays a critical role in promoting cell survival. RNA-seq analysis revealed that Srebf2-mediated cholesterol synthesis is tightly regulated by β-catenin signaling. Furthermore, lymphoma cells from Lck-Dlx5;Lck-MyrAkt2 mice had elevated expression of genes encoding components of the Srebf2 pathway, and their expression was suppressed when β-catenin/Tcf inhibitors were used.Activating mutations in Lck-Dlx5;Lck-MyrAkt2 mice indicate that Wnt signaling mediates oncogenic synergy between Akt and Dlx5 in T-cell lymphomagenesis by enhancing cholesterol synthesis. Oncogenesis is sustained by cancer anabolism, of which lipid biosynthesis plays an important role. Rewired lipid metabolism is now considered a hallmark of cancer, and enhanced lipid uptake and lipogenesis are well documented in many cancers and facilitate tumorigenesis27. Cholesterol-reducing statins have been reported to suppress tumor cell proliferation28. Moreover, clinical studies have demonstrated reduced cancer-specific mortality in patients taking statins29. Sterol regulatory element binding protein/factors (SREBP/SREBFs) are transcription factors that control lipid homeostasis by transactivating genes encoding enzymes needed for the synthesis of cholesterol, triacylglycerol, fatty acids, and phospholipids. The SREBP-2 isoform is responsible for cholesterol synthesis, whereas the SREBP-1a isoform has been implicated in both cholesterol and fatty acid synthesis30. In breast cancer cells, increased levels of cholesterol and lipids, resulting from upregulation of the SREBP pathway, convey resistance to tamoxifen31. SREBP activity has been found to be regulated by various oncogenic or tumor suppressive mechanisms, with tumor suppressor genes usually inhibiting SREBP function, and oncogenes typically promoting and depending on SREBP activity32. For example, Lats2-p53 can inhibit SREBP transcription and activity32. Additionally, a recent study revealed that SREBP target genes can be controlled by pyruvate dehydrogenase A133. However, a potential link between Wnt/β-catenin and cholesterol synthesis is now beginning to emerge. Mutations in DHCR7, the gene encoding delta-7-sterol reductase, impair the reduction of 7-dehydrocholesterol (7-DHC) to cholesterol and thereby directly downregulate Wnt/β-catenin signaling34. The combinatory RNA-seq and ChIP-seq data presented here have revealed that some key players in the Srebf2-cholesterol pathway are direct targets of Wnt signaling. A working mechanism of such action is depicted in Fig. Collectively, our findings in Lck-Dlx5;Lck-MyrAkt2 lymphoma cells as well as a subset of human T-ALL cells. Together, these findings suggest that targeting β-catenin and/or cholesterol biosynthesis, together with AKT, could have therapeutic efficacy in some T-ALL patients.To our knowledge, this investigation is the first to demonstrate that the SREBF2-cholesterol biosynthesis pathway is transcriptionally regulated by WNT/β-catenin, and that the SREBF2-cholesterol pathway is pivotal to the survival of murine Antibodies against Akt (cat. #9272), Caspase 3 (cat. #9661), Cyclin D3 (cat. #2936), HSP90 (cat. #4874), Myc (cat. #5605), Myc-Tag (cat. #2276), Notch1 (cat. #3608), PARP (cat. #9542), phospho (p)-Akt(Ser473) (cat. #4060), p-S6 ribosomal protein (cat. #2211), and Pten (cat. #9559) were from Cell Signaling . Antibodies against β-Actin (cat. #sc-47778), β-Catenin (cat. #sc-7963), Gapdh (cat. #sc-32233), Hes1 (cat. #sc-13844), Lamin B (cat. #sc-6216), LEF-1 (cat. #sc-374412), Notch3 (cat. #sc-5593), TCF-4 (cat. #sc-166699), as well as mouse anti-goat IgG-horseradish peroxidase (HRP) secondary antibody (cat. # sc-2354) were all from Santa Cruz Biotechnology . Anti-rabbit IgG, peroxidase-linked species-specific whole antibody (from donkey) secondary HRP-linked secondary antibody (cat. #NA 9341) and anti-mouse IgG, peroxidase-linked species-specific whole antibody (from sheep) secondary antibody (cat. #NA 3911) were from GE Healthcare Life Sciences . Immobilon ECL Ultra Western HRP substrate (cat. #WBULS0100), β-Catenin/Tcf inhibitor II, PKF118-310 (cat. #219331), OSC inhibitor, RO48-8071 (cat. #499635), and InSolution Simvastatin, Sodium Salt (cat. #567022) were from Millipore Sigma . JQ1 inhibitor (cat. #S7110), BEZ235 (cat. #S1009), RAD001 (cat. #S1120), FH535 (cat. #S7484), and γ-secretase inhibitor XI (cat. #S0058) were from Selleckchem . Lipofectamine 2000 (cat. #11668019) was from Thermo Fisher .Lck-MyrAkt2 and Lck-Dlx5-Myc-tag were previously reported22. Mice were sacrificed upon evidence of labored breathing, hunched posture, distended abdomen, or upon a 10% change in body weight, under Fox Chase Cancer Center Institutional Animal Care and Use Committee protocol #96-36. This protocol was updated, reviewed and approved annually by the Fox Chase Cancer Center Institutional Animal Care and Use Committee, allowing for the generation, maintenance, and ethical care and monitoring of Lck-MyrAkt2, Lck-Dlx5-Myc-tag, and Lck-Dlx5;Lck-MyrAkt2 transgenic mice. All experiments were performed in accordance with these institutional as well as NIH guidelines and regulations. A portion of each tumor was collected for protein or RNA analysis, and the remainder was placed into culture to establish tumor cell lines.The transgenic mouse models 17. Cells were maintained in Iscove’s MEM complete medium, supplemented with 15% FBS, 100 μg/mL penicillin/streptomycin, 2 mM L-glutamine, 1 mM MEM sodium pyruvate, and non-essential amino acids , and β-mercaptoethanol (Thermo Fisher). Human malignant T cell lines were a gift of Owen O’Connor . Cells were maintained in RPMI1640 medium supplemented with 10% FBS containing 100 μg/mL penicillin/streptomycin and 2 mM L-glutamine. HEK293T cells were from ATCC and maintained in DMEM with the same supplements.Mouse T cell lymphoma cells were collected from thymic tumors and filtered through a 100-gauge mesh, as previously describedPrimary T-ALL samples were provided by the Children's Oncology Group under NCTN Operations Center Grant U10CA180886 and NCTN SDC Grant 180899. Total RNAs were extracted using TRIzol and purified via spin column .ueous One Solution Cell Proliferation Assay , as previously described17. In brief, cells were seeded at 2 × 104 cells/well in 96-well plates overnight and treated with various inhibitors for 24 or 48 h. MTS reagent (20 μL) was added to each well, followed by incubation for 1–4 h for color development. OD value at 490 nm was measured using a 96-well microplate reader .Cell viability was measured by MTS using a CellTiter 96 AQFlow cytometry was performed with a FACS . Cells were fixed with 70% ice-cold ethanol for 5 min, washed with cold PBS, and stained with propidium iodide for 15 min at room temperature. Data were analyzed with FlowJo software.6 human T-ALL cells cultured in single wells of a 6-well plate using Lipofectamine LTX . Luciferase activity was measured 24 h after transfection using a Dual-Luciferase reporter assay kit  on an Enspire alpha plate reader .β-catenin luciferase reporter plasmids were obtained from Addgene (cat. #12457 and #12456). 3 µg of TOP-FLASH or FOP-FLASH plasmids were transfected onto 1 × 1017. In brief, cells were lysed with 1 × cell lysis buffer  supplemented with 2 mM PMSF. Bradford reagent was used to measure protein concentrations. Cell lysates were loaded into Bis–Tris gels  and transferred onto PVDF membranes . Membranes were blocked with 5% non-fat milk in TBST for 1 h and then incubated with primary antibodies at 4 °C overnight. After washing 3 times with TBST, membranes were incubated with secondary antibody at room temperature for 1 h and further washed three times before film exposure with ECL (Millipore).Immunoblotting was performed as previously describedPp1a gene was used as the normalizing control.RNA was extracted using an RNeasy column . RNA quality was checked by running on a BioAnalyzer using RNA nano CHIP . Next, 1000 ng of total RNA with RIN > 8 was used to make cDNA via reverse transcription. cDNA was generated using SuperScript II from Thermo Fisher (cat. #18064014) with random primers at 42 °C for 1 h. qPCR was performed on a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher), using PowerUp SYBR Green Master Mix  and the default program. The 35. ChIP was carried out using an Active Motif  kit. For input DNA, genomic DNA was sonicated to 150–800 bp using a Covaris S2 ultrasonicator . ChIP-grade TCF7 antibody was used to pull down bound DNA fragments. The Illumina library was constructed using a NEBNext Ultra II kit , and ~ 50 million reads/sample were obtained. FASTQ files were aligned to mm10, and peaks were called using the MACS2 algorithm in Chipster. FASTQ files were uploaded to GEO.Total RNA was extracted using TRIzol and purified using an RNA miniprep kit . Construction of sequencing libraries was with a TruSeq stranded mRNA library kit . Next generation sequencing (NGS) was performed on an Illumina HiSeq 2500 system. For each sample, approximately 30 million reads were obtained. FASTQ files were aligned to the mouse reference genome using TopHat algorithm. Differentially expressed genes were analyzed using EdgerR algorithm. The related signaling pathways were viewed on the ConsensusPath database, with analyses performed using ChipsterSupplementary information 1.Supplementary information 2.