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    Addgene inc caspase 3
    (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. (d) Top: schematic of the behavioral apparatus (head-fixed mice licking for grape or apple juice). Bottom: timeline of the experiment. (e) Quantification of lick preference index for each day of the conditioned taste preference experiment (n = 6 mice). Bars show mean ± SEM across mice. As expected from the immediate efect of stimulation to reduce licking, there was a substantial reduction in preference for the juice paired with non-invasive NG-OxtR + optogenetic activation during conditioning from 0.057 to 0.55 (adj.P = 0.03, Wilcoxon with FDR correction). When comparing day 1 and day 5 (pre stimulation vs. post stimulation), there was a very small yet significant decrease from 0.06 to −0.06, likely reflecting our initial biased choice of pairing stimulation with the mildly preferred juice (adj. P = 0.03, Wilcoxon with FDR correction). (f) Left: learning of the vagal opto NoGo task in ChRmine + mice (n=9), but not in control mice (n=5). Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using <t>Caspase</t> <t>3</t> hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . In contrast, previous work using the OxtR-t2a-Cre knock-in mouse line we use here, observed no change in body temperature and blood pressure . We therefore also verified this in our preparation. We measured body temperature during performance of the vagal opto NoGo task and did not observe a reduction in body temperature upon NG-OxtR + stimulation, nor other evidence of torpor (j). We also conducted heart rate measurements in anesthetized mice, previously trained on both the NoGo and Go vagal opto tasks (k). We observed heart rate reductions in response to optogenetic activation in some mice but not in others. Most importantly, mice with or without a change in heart rate showed similar high performance in the NoGo and Go tasks. (j) Performance in the vagal opto NoGo task does not induce changes in body temperature. Temperature was measured during the vagal opto NoGo task every 5 min (n = 3 mice). Each mouse’s measurements are indicated in gray, the black line is the mean across mice, and the red rectangle indicates the time of the vagal opto NoGo session. (k) Performance in the vagal opto NoGo and Go tasks is independent of potential changes in heart rate. Heart rate measurements in anesthetized mice during NG-OxtR + optogenetic stimulation (same stimulation parameters as in the behavioral task). Top: example of two mice without a change in heart rate in response to the optogenetic stimulation. Bottom: example of two mice with a reduction in heart rate in response to the optogenetic stimulation. In the electrocardiogram traces, each peak indicates one heartbeat, and each red horizontal line indicates 4 sec of vagal optogenetic stimulation. All mice had similarly high performance in the vagal opto NoGo and vagal opto Go tasks, regardless of whether there was a change in heart rate. Red rectangles indicate optogenetic stimulation time, and gray rectangles indicate odor cue presentation. Gray ticks indicate licks.
    Caspase 3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A cortical basis for perception of internal gut sensations"

    Article Title: A cortical basis for perception of internal gut sensations

    Journal: bioRxiv

    doi: 10.64898/2026.02.11.705298

    (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. (d) Top: schematic of the behavioral apparatus (head-fixed mice licking for grape or apple juice). Bottom: timeline of the experiment. (e) Quantification of lick preference index for each day of the conditioned taste preference experiment (n = 6 mice). Bars show mean ± SEM across mice. As expected from the immediate efect of stimulation to reduce licking, there was a substantial reduction in preference for the juice paired with non-invasive NG-OxtR + optogenetic activation during conditioning from 0.057 to 0.55 (adj.P = 0.03, Wilcoxon with FDR correction). When comparing day 1 and day 5 (pre stimulation vs. post stimulation), there was a very small yet significant decrease from 0.06 to −0.06, likely reflecting our initial biased choice of pairing stimulation with the mildly preferred juice (adj. P = 0.03, Wilcoxon with FDR correction). (f) Left: learning of the vagal opto NoGo task in ChRmine + mice (n=9), but not in control mice (n=5). Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using Caspase 3 hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . In contrast, previous work using the OxtR-t2a-Cre knock-in mouse line we use here, observed no change in body temperature and blood pressure . We therefore also verified this in our preparation. We measured body temperature during performance of the vagal opto NoGo task and did not observe a reduction in body temperature upon NG-OxtR + stimulation, nor other evidence of torpor (j). We also conducted heart rate measurements in anesthetized mice, previously trained on both the NoGo and Go vagal opto tasks (k). We observed heart rate reductions in response to optogenetic activation in some mice but not in others. Most importantly, mice with or without a change in heart rate showed similar high performance in the NoGo and Go tasks. (j) Performance in the vagal opto NoGo task does not induce changes in body temperature. Temperature was measured during the vagal opto NoGo task every 5 min (n = 3 mice). Each mouse’s measurements are indicated in gray, the black line is the mean across mice, and the red rectangle indicates the time of the vagal opto NoGo session. (k) Performance in the vagal opto NoGo and Go tasks is independent of potential changes in heart rate. Heart rate measurements in anesthetized mice during NG-OxtR + optogenetic stimulation (same stimulation parameters as in the behavioral task). Top: example of two mice without a change in heart rate in response to the optogenetic stimulation. Bottom: example of two mice with a reduction in heart rate in response to the optogenetic stimulation. In the electrocardiogram traces, each peak indicates one heartbeat, and each red horizontal line indicates 4 sec of vagal optogenetic stimulation. All mice had similarly high performance in the vagal opto NoGo and vagal opto Go tasks, regardless of whether there was a change in heart rate. Red rectangles indicate optogenetic stimulation time, and gray rectangles indicate odor cue presentation. Gray ticks indicate licks.
    Figure Legend Snippet: (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. (d) Top: schematic of the behavioral apparatus (head-fixed mice licking for grape or apple juice). Bottom: timeline of the experiment. (e) Quantification of lick preference index for each day of the conditioned taste preference experiment (n = 6 mice). Bars show mean ± SEM across mice. As expected from the immediate efect of stimulation to reduce licking, there was a substantial reduction in preference for the juice paired with non-invasive NG-OxtR + optogenetic activation during conditioning from 0.057 to 0.55 (adj.P = 0.03, Wilcoxon with FDR correction). When comparing day 1 and day 5 (pre stimulation vs. post stimulation), there was a very small yet significant decrease from 0.06 to −0.06, likely reflecting our initial biased choice of pairing stimulation with the mildly preferred juice (adj. P = 0.03, Wilcoxon with FDR correction). (f) Left: learning of the vagal opto NoGo task in ChRmine + mice (n=9), but not in control mice (n=5). Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using Caspase 3 hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . In contrast, previous work using the OxtR-t2a-Cre knock-in mouse line we use here, observed no change in body temperature and blood pressure . We therefore also verified this in our preparation. We measured body temperature during performance of the vagal opto NoGo task and did not observe a reduction in body temperature upon NG-OxtR + stimulation, nor other evidence of torpor (j). We also conducted heart rate measurements in anesthetized mice, previously trained on both the NoGo and Go vagal opto tasks (k). We observed heart rate reductions in response to optogenetic activation in some mice but not in others. Most importantly, mice with or without a change in heart rate showed similar high performance in the NoGo and Go tasks. (j) Performance in the vagal opto NoGo task does not induce changes in body temperature. Temperature was measured during the vagal opto NoGo task every 5 min (n = 3 mice). Each mouse’s measurements are indicated in gray, the black line is the mean across mice, and the red rectangle indicates the time of the vagal opto NoGo session. (k) Performance in the vagal opto NoGo and Go tasks is independent of potential changes in heart rate. Heart rate measurements in anesthetized mice during NG-OxtR + optogenetic stimulation (same stimulation parameters as in the behavioral task). Top: example of two mice without a change in heart rate in response to the optogenetic stimulation. Bottom: example of two mice with a reduction in heart rate in response to the optogenetic stimulation. In the electrocardiogram traces, each peak indicates one heartbeat, and each red horizontal line indicates 4 sec of vagal optogenetic stimulation. All mice had similarly high performance in the vagal opto NoGo and vagal opto Go tasks, regardless of whether there was a change in heart rate. Red rectangles indicate optogenetic stimulation time, and gray rectangles indicate odor cue presentation. Gray ticks indicate licks.

    Techniques Used: Expressing, Labeling, Control, Activation Assay, Injection, Comparison, Transgenic Assay, Knock-In



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    (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. 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Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using <t>Caspase</t> <t>3</t> hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . 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    Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) <t>GSK3β</t> for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.
    Stat3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/stat3/product/Addgene inc
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    h ras g12v  (Addgene inc)
    93
    Addgene inc h ras g12v
    Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) <t>GSK3β</t> for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.
    H Ras G12v, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    β catenin insert  (Addgene inc)
    93
    Addgene inc β catenin insert
    Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) <t>GSK3β</t> for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.
    β Catenin Insert, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. (d) Top: schematic of the behavioral apparatus (head-fixed mice licking for grape or apple juice). Bottom: timeline of the experiment. (e) Quantification of lick preference index for each day of the conditioned taste preference experiment (n = 6 mice). Bars show mean ± SEM across mice. As expected from the immediate efect of stimulation to reduce licking, there was a substantial reduction in preference for the juice paired with non-invasive NG-OxtR + optogenetic activation during conditioning from 0.057 to 0.55 (adj.P = 0.03, Wilcoxon with FDR correction). When comparing day 1 and day 5 (pre stimulation vs. post stimulation), there was a very small yet significant decrease from 0.06 to −0.06, likely reflecting our initial biased choice of pairing stimulation with the mildly preferred juice (adj. P = 0.03, Wilcoxon with FDR correction). (f) Left: learning of the vagal opto NoGo task in ChRmine + mice (n=9), but not in control mice (n=5). Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using Caspase 3 hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . In contrast, previous work using the OxtR-t2a-Cre knock-in mouse line we use here, observed no change in body temperature and blood pressure . We therefore also verified this in our preparation. We measured body temperature during performance of the vagal opto NoGo task and did not observe a reduction in body temperature upon NG-OxtR + stimulation, nor other evidence of torpor (j). We also conducted heart rate measurements in anesthetized mice, previously trained on both the NoGo and Go vagal opto tasks (k). We observed heart rate reductions in response to optogenetic activation in some mice but not in others. Most importantly, mice with or without a change in heart rate showed similar high performance in the NoGo and Go tasks. (j) Performance in the vagal opto NoGo task does not induce changes in body temperature. Temperature was measured during the vagal opto NoGo task every 5 min (n = 3 mice). Each mouse’s measurements are indicated in gray, the black line is the mean across mice, and the red rectangle indicates the time of the vagal opto NoGo session. (k) Performance in the vagal opto NoGo and Go tasks is independent of potential changes in heart rate. Heart rate measurements in anesthetized mice during NG-OxtR + optogenetic stimulation (same stimulation parameters as in the behavioral task). Top: example of two mice without a change in heart rate in response to the optogenetic stimulation. Bottom: example of two mice with a reduction in heart rate in response to the optogenetic stimulation. In the electrocardiogram traces, each peak indicates one heartbeat, and each red horizontal line indicates 4 sec of vagal optogenetic stimulation. All mice had similarly high performance in the vagal opto NoGo and vagal opto Go tasks, regardless of whether there was a change in heart rate. Red rectangles indicate optogenetic stimulation time, and gray rectangles indicate odor cue presentation. Gray ticks indicate licks.

    Journal: bioRxiv

    Article Title: A cortical basis for perception of internal gut sensations

    doi: 10.64898/2026.02.11.705298

    Figure Lengend Snippet: (a) NG-OxtR + terminals expressing ChRmine-mScarlet innervating the intestine and forming intra-ganglionic-laminar-endings (IGLEs, labeled with white arrows) in both male and female mice. Scale bar = 100 µm. (b) Quantification of lick reduction index during the sucrose consumption task, showing similar responses between male (n = 12 ChRmine+, 5 controls) and female (n = 5 ChRmine+, 2 controls) mice. Bars show mean ± SEM across mice. Males vs. females within the ChRmine+ group: p = 0.8 (Whitney U), males vs. females within the control group: p = 0.38 (Whitney U), and ChRmine+ vs. control overall: p = 1.05×10 −7 (Welch t). (c) Quantification of lick reduction index during the sucrose consumption task in water restricted ChRmine+ (n = 3) and control (n = 5) mice, p = 0.00013 (independent t-test). Bars show mean ± SEM across mice. (d-e) Activation of NG-OxtR + does not cause substantial conditioned taste preference/aversion. (d) Top: schematic of the behavioral apparatus (head-fixed mice licking for grape or apple juice). Bottom: timeline of the experiment. (e) Quantification of lick preference index for each day of the conditioned taste preference experiment (n = 6 mice). Bars show mean ± SEM across mice. As expected from the immediate efect of stimulation to reduce licking, there was a substantial reduction in preference for the juice paired with non-invasive NG-OxtR + optogenetic activation during conditioning from 0.057 to 0.55 (adj.P = 0.03, Wilcoxon with FDR correction). When comparing day 1 and day 5 (pre stimulation vs. post stimulation), there was a very small yet significant decrease from 0.06 to −0.06, likely reflecting our initial biased choice of pairing stimulation with the mildly preferred juice (adj. P = 0.03, Wilcoxon with FDR correction). (f) Left: learning of the vagal opto NoGo task in ChRmine + mice (n=9), but not in control mice (n=5). Values are d’ (discriminability index) over days showing similar performance between vagal opto NoGo (dark red) and vagal opto Go (light red) tasks in ChRmine mice. In contrast, control mice failed to learn the NoGo task over multiple training days (gray). Thicker lines indicate mean ± SEM across mice. Right: d’ calculated for each mouse at the first half of the first session, compared to the last session, shows improved discriminability in the NoGo task with training P = 0.012 (Wilcoxon test). (g-i) NG-OxtR + specific ablation using Caspase 3 hinders behavioral performance in the vagal opto NoGo task. (g) Schematic of the surgical procedure. Retro-AAV-Ef1a-DIO-ChRmine-mScarlet was injected bi-laterally into the NTS of OxtR-Cre mice. Additionally, AAV9-flex-taCasp3-TEVp was injected bi-laterally into the NG to specifically ablate NG-OxtR + neurons. (h) Example histology from two diferent mice demonstrating partial ablation of NG-OxtR + neurons (right), and successful, near-complete, ablation (left). Scale bar = 200 µm. (i) Summary of Hits and FAs in the vagal opto NoGo task of mice without ablation (same plot as , shown for comparison), partial ablation, and near-complete ablation of NG-OxtR + neurons. p = 3.6×10 − (paired t-test, n=19). Middle: mice that underwent bi-lateral NG injections of Caspase 3 but showed partial ablation, p = 8.1×10 −4 (paired t-test, n=3). Right: mice with successful, near-complete ablation of NG-OxtR + neurons, p = 0.129 (paired t-test, n=3). Bars show mean ± SEM across mice (j-k) Previous work using a diferent OxtR-Cre mouse line (BAC transgenic mice) reported that chemogenetic activation of NG-OxtR + neurons induced significant reductions in body temperature, resulting in torpor (also accompanied by changes in blood pressure and heart rate) . In contrast, previous work using the OxtR-t2a-Cre knock-in mouse line we use here, observed no change in body temperature and blood pressure . We therefore also verified this in our preparation. We measured body temperature during performance of the vagal opto NoGo task and did not observe a reduction in body temperature upon NG-OxtR + stimulation, nor other evidence of torpor (j). We also conducted heart rate measurements in anesthetized mice, previously trained on both the NoGo and Go vagal opto tasks (k). We observed heart rate reductions in response to optogenetic activation in some mice but not in others. Most importantly, mice with or without a change in heart rate showed similar high performance in the NoGo and Go tasks. (j) Performance in the vagal opto NoGo task does not induce changes in body temperature. Temperature was measured during the vagal opto NoGo task every 5 min (n = 3 mice). Each mouse’s measurements are indicated in gray, the black line is the mean across mice, and the red rectangle indicates the time of the vagal opto NoGo session. (k) Performance in the vagal opto NoGo and Go tasks is independent of potential changes in heart rate. Heart rate measurements in anesthetized mice during NG-OxtR + optogenetic stimulation (same stimulation parameters as in the behavioral task). Top: example of two mice without a change in heart rate in response to the optogenetic stimulation. Bottom: example of two mice with a reduction in heart rate in response to the optogenetic stimulation. In the electrocardiogram traces, each peak indicates one heartbeat, and each red horizontal line indicates 4 sec of vagal optogenetic stimulation. All mice had similarly high performance in the vagal opto NoGo and vagal opto Go tasks, regardless of whether there was a change in heart rate. Red rectangles indicate optogenetic stimulation time, and gray rectangles indicate odor cue presentation. Gray ticks indicate licks.

    Article Snippet: For specific ablation of NG-OxtR + neurons using Caspase 3, we directly injected AAV9-flex-taCasp3-TEVp (plasmid #45580 from Addgene, produced by the Hebrew University ELSC Virus Core Facility) bilaterally into the NG.

    Techniques: Expressing, Labeling, Control, Activation Assay, Injection, Comparison, Transgenic Assay, Knock-In

    SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

    Journal: Cell Communication and Signaling : CCS

    Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

    doi: 10.1186/s12964-025-02640-y

    Figure Lengend Snippet: SIN1 controls AKT activity and TYMS levels following genotoxic stress. A Lysates from MDA-MB-231 cells stably expressing Sh control or small hairpin against SIN1, treated or not with 50 µM 5-FuDR or 100 µM 5-FU for 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B-D Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (5, 12) = 16.02, P < 0.001), p-P70SK (F(5,12) = 2.86, p = 0.06), P53 (F (5, 12) = 3.498, P < 0.05, P21 (F (5, 12) = 26.09, P < 0.0001), TYMS (F (5, 12) = 19.39, P < 0.001). E Bar graphs showing the percentage of Sh Ctrl and Sh SIN1 MDA-MB-231 cells at SubG1 after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 8) = 45.1, P < 0.0001). F Lysates of MCF7 cells stably expressing Sh control or small hairpin against SIN1 treated or not with 50 µM 5-FuDR for 24 h, then stained with the indicated antibodies. G-I Bar graphs showing the results of densitometric analysis of the protein levels under different conditions obtained in F. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 483.1, P < 0.0001), p-P70SK (F (3, 8) = 11.58, P < 0.01), P53 (F (3, 8) = 616.3, P < 0.0001), P21 (F (3, 8) = 251.3, P < 0.0001), TYMS (F (3, 8) = 97.82, P < 0.0001). J Bar graphs showing the percentage of MCF7 Sh Ctrl and Sh SIN1 cells at different phase of the cell cycle after 72 h of treatment with FuDR. Data are presented as the means ± SEMs. ANOVA (F (3, 18) = 11.89, ** P < 0.001, * P < 0.05)

    Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

    Techniques: Activity Assay, Stable Transfection, Expressing, Control, Staining

    P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

    Journal: Cell Communication and Signaling : CCS

    Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

    doi: 10.1186/s12964-025-02640-y

    Figure Lengend Snippet: P53 controls AKT phosphorylation and TYMS levels following genotoxic stress. A , C Lysates from MDA-MB-231 and MCF7 cells stably expressing Sh control or small hairpin against p53, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. B-D Bar graphs showing the results of densitometric analysis of AKT phosphorylation and TYMS protein levels under different conditions obtained in A and C. The data are presented as the means ± SEMs. B ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 5.51, P < 0.05), TYMS (F (3, 8) = 240.97, P < 0.0001). D ANOVA, multiple comparisons: Dunnett test. p-AKT1 (F (3, 8) = 291.1, P < 0.001), TYMS (F (3, 8) = 25.51, P < 0.0001)

    Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

    Techniques: Phospho-proteomics, Stable Transfection, Expressing, Control, Staining

    SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

    Journal: Cell Communication and Signaling : CCS

    Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

    doi: 10.1186/s12964-025-02640-y

    Figure Lengend Snippet: SIN1 controls P53 transcriptional activity following genotoxic stress. A-D Bar graphs showing mRNA levels of TYMS and P53 target genes under the indicated conditions in MCF7 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. TYMS F (5, 12) = 17.18, P < 0.0001. CDKN1A F (5, 12) = 274.7, P < 0.0001. GAD45A F (5, 12) = 205.0, P < 0.0001. SESN2 F (5, 11) = 12.43, P = 0.0003. E–G Bar graphs showing mRNA levels of P53 target genes under the indicated conditions in MDA-MB-231 cells. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. CDKN1A F(3,8) = 6.94, P = 0.012. GAD45A F (3, 8) = 22.4, P < 0.001. SESN2 F (3, 8) = 2.22, P = 0.16

    Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

    Techniques: Activity Assay

    Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

    Journal: Cell Communication and Signaling : CCS

    Article Title: The mTORC2 component SIN1 post-transcriptionally regulates TYMS levels and modulates P53 activity in response to 5-FU chemotherapy

    doi: 10.1186/s12964-025-02640-y

    Figure Lengend Snippet: Increase in TYMS levels alters P53 activity upon genotoxic stress. A Lysates from MCF7 cells stably expressing Sh control or small hairpin against SIN1 transiently transfected with TYMS expressing plasmid, treated or not with 50 µM during 24 h, were stained with the indicated antibodies. n = 3 separate biological replicates. B Bar graphs showing the results of densitometric analysis of P53 protein levels under different conditions obtained in A. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. P53 F(7,17) = 44.62, P < 0.0001. C Bar graph displaying the proportion of cells at different stages in the cell cycle analyzed by flow cytometry. MCF7 sh control and sh SIN1 transfected as in A, then treated with or without 50 µM during 72 h. ANOVA, multiple comparisons: Dunnett test. G1 phase F (F (7, 24) = 25.65, P < 0.0001. See supplementary Table 2 for detailed statistics. D Lysates from MCF7 cells treated or not with FuDR for the indicated durations (hours). N = 3 biological replicates per condition. E Bar graph displaying the amount of SIN1 isoforms under the condition obtained in D. The data are presented as the means ± SEMs. ANOVA, multiple comparisons: Dunnett test. Long F(3,8) = 39.67, P < 0.001. Short Long F (3,8) = 26.06, P < 0.001

    Article Snippet: Empty vector c-Flag pcDNA3 was a gift from Stephen Smale (Addgene plasmid # 20011; http://n2t.net/addgene:20011 ; RRID: Addgene_20011). pcDNA3 flag p53 was a gift from Thomas Roberts (Addgene plasmid # 10838; http://n2t.net/addgene:10838 ; RRID:Addgene 10838). p53 (dominant negative R175H mutant)-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64638; http://n2t.net/addgene:64638 ; RRID: Addgene_64638).

    Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Plasmid Preparation, Staining, Flow Cytometry

    Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) GSK3β for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

    Journal: bioRxiv

    Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

    doi: 10.64898/2026.01.15.699680

    Figure Lengend Snippet: Wild-type MEFs were transfected with adenovirus encoding βGal (control), wild-type (WT) or kinase-active (K85R, DN) GSK3β for 48hrs. A ) Western blotting for GSK3β demonstrating equivalent expression of the WT-GSK3β and DN-GSK3β. GAPDH was used as a loading control. B ) βGal, WT-GSK3β and DN-GSK3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, WT-GSK3β and DN-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

    Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

    Techniques: Transfection, Control, Western Blot, Expressing, Infection, Staining, Fluorescence

    Wild-type MEFs were transfected with non-targeting (CONsi) or GSK3β-specific (GSK3βsi) siRNAs (100nM) for 48hrs. A ) Western blotting for GSK3β confirmed depletion of the protein in GSK3βsi-transfected MEFs. GAPDH was used as a loading control. B ) CONsi and GSK3βsi-transfected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of CRC measurements using Calcium Green-5N in digitonin-permeabilized CONsi and GSK3βsi-transfected MEFs. D ) Quantification of CRC data in the CONsi and GSK3βsi-transfected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. CONsi.

    Journal: bioRxiv

    Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

    doi: 10.64898/2026.01.15.699680

    Figure Lengend Snippet: Wild-type MEFs were transfected with non-targeting (CONsi) or GSK3β-specific (GSK3βsi) siRNAs (100nM) for 48hrs. A ) Western blotting for GSK3β confirmed depletion of the protein in GSK3βsi-transfected MEFs. GAPDH was used as a loading control. B ) CONsi and GSK3βsi-transfected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. C ) Representative traces of CRC measurements using Calcium Green-5N in digitonin-permeabilized CONsi and GSK3βsi-transfected MEFs. D ) Quantification of CRC data in the CONsi and GSK3βsi-transfected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. CONsi.

    Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

    Techniques: Transfection, Western Blot, Control, Staining

    A ) Upper panels, Western blotting for GSK3β, lactate dehydrogenase (LDH, cytosolic), and succinate dehydrogenase (SDHB, mitochondrial) in cardiac mitochondrial and cytosolic subfractions. Lower panels, Western blotting for GSK3β, VDAC (membrane), and CypD (soluble) in alkali extracted membrane and soluble fractions of cardiac mitochondria. B ) Upper panels, recombinant GST-rat GSK3β was incubated with His-tagged human CypD, purified on Co 2+ -agarose column, and the complexes Western blotted for GST and CypD. Lower panels, recombinant GST-GSK3β was incubated with His-tagged human CypD in kinase buffer supplemented with ATP, purified on Co 2+ -agarose column, and the complexes Western blotted for phosphoserine/threonine, GST and CypD. C ) Mass analysis of phosphorylated recombinant CypD demonstrating the presence of two phosphorylation sites. D ) Example of MS-MS spectra indicating S101 as a putative GSK3β phosphorylation site on CypD. E ) Amino acid sequence of the recombinant His-tagged human CypD depicting canonical GSK3β consensus sites (red), the putative phosphorylation sites identified in our analyses (blue), and a site previously hypothesized to be a key GSK3β phosphorylation residue (purple). F ) Surface representations of human CypD illustrating the positions of the various putative phosphorylation sites and their proximity to the catalytic CsA-binding domain (CsA-BD).

    Journal: bioRxiv

    Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

    doi: 10.64898/2026.01.15.699680

    Figure Lengend Snippet: A ) Upper panels, Western blotting for GSK3β, lactate dehydrogenase (LDH, cytosolic), and succinate dehydrogenase (SDHB, mitochondrial) in cardiac mitochondrial and cytosolic subfractions. Lower panels, Western blotting for GSK3β, VDAC (membrane), and CypD (soluble) in alkali extracted membrane and soluble fractions of cardiac mitochondria. B ) Upper panels, recombinant GST-rat GSK3β was incubated with His-tagged human CypD, purified on Co 2+ -agarose column, and the complexes Western blotted for GST and CypD. Lower panels, recombinant GST-GSK3β was incubated with His-tagged human CypD in kinase buffer supplemented with ATP, purified on Co 2+ -agarose column, and the complexes Western blotted for phosphoserine/threonine, GST and CypD. C ) Mass analysis of phosphorylated recombinant CypD demonstrating the presence of two phosphorylation sites. D ) Example of MS-MS spectra indicating S101 as a putative GSK3β phosphorylation site on CypD. E ) Amino acid sequence of the recombinant His-tagged human CypD depicting canonical GSK3β consensus sites (red), the putative phosphorylation sites identified in our analyses (blue), and a site previously hypothesized to be a key GSK3β phosphorylation residue (purple). F ) Surface representations of human CypD illustrating the positions of the various putative phosphorylation sites and their proximity to the catalytic CsA-binding domain (CsA-BD).

    Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

    Techniques: Western Blot, Membrane, Recombinant, Incubation, Purification, Phospho-proteomics, Tandem Mass Spectroscopy, Sequencing, Residue, Binding Assay

    Wild-type MEFs were infected with adenoviruses encoding βGal (control) or mitochondrially-targeted HA-tagged WT-GSK3β (miWT-3β) or DN-GSK3β (miDN-3β) for 48hrs. A ) Western blotting for HA and GSK3β confirmed equivalent expression of the two GSK3β proteins. GAPDH was used as a loading control. B ) Representative images of immunocytochemistry performed on MEFs infected with miWT-3β stained for the HA tag confirmed mitochondrial localization of the protein. Mitochondria were visualized by staining for CypD. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, miWT-3β and miDN-3β-infected MEFs. Cells were treated with 2.5μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, miWT-3β and miDN-3β-infected MEFs. E ) βGal, miWT-3β and miDN-3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. Each individual point represents one independent cell isolate. Bar represents the mean.

    Journal: bioRxiv

    Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

    doi: 10.64898/2026.01.15.699680

    Figure Lengend Snippet: Wild-type MEFs were infected with adenoviruses encoding βGal (control) or mitochondrially-targeted HA-tagged WT-GSK3β (miWT-3β) or DN-GSK3β (miDN-3β) for 48hrs. A ) Western blotting for HA and GSK3β confirmed equivalent expression of the two GSK3β proteins. GAPDH was used as a loading control. B ) Representative images of immunocytochemistry performed on MEFs infected with miWT-3β stained for the HA tag confirmed mitochondrial localization of the protein. Mitochondria were visualized by staining for CypD. C ) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, miWT-3β and miDN-3β-infected MEFs. Cells were treated with 2.5μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. D ) Quantification of CRC data in the βGal, miWT-3β and miDN-3β-infected MEFs. E ) βGal, miWT-3β and miDN-3β-infected MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. Each individual point represents one independent cell isolate. Bar represents the mean.

    Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

    Techniques: Infection, Control, Western Blot, Expressing, Immunocytochemistry, Staining, Fluorescence

    A) Cardiac mitochondria were exposed to increasing concentrations of proteinase K and then Western blotted for hexokinase-2 (HK2, mitochondrial surface), apoptosis-inducing factor (intermembrane space), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2, inner membrane), and CypD (matrix). B) MEFs were infected with adenoviruses encoding βGal, WT-GSK3β and/or FLAG-tagged CypD for 48hrs. CypD was immunoprecipitated using an anti-FLAG antibody and the complexes Western blotted for GSK3β and FLAG. C) CypD KO MEFs were infected with adenoviruses encoding βGal, WT-GSK3β for 48hrs. Western blotting for GSK3β confirmed overexpression of GSK3β. GAPDH was used as a loading control. D) βGal and WT-GSK3β-infected CypD KO MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. E) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, and WT-GSK3β -infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. F) Quantification of CRC data in the βGal and WT-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

    Journal: bioRxiv

    Article Title: Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

    doi: 10.64898/2026.01.15.699680

    Figure Lengend Snippet: A) Cardiac mitochondria were exposed to increasing concentrations of proteinase K and then Western blotted for hexokinase-2 (HK2, mitochondrial surface), apoptosis-inducing factor (intermembrane space), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2, inner membrane), and CypD (matrix). B) MEFs were infected with adenoviruses encoding βGal, WT-GSK3β and/or FLAG-tagged CypD for 48hrs. CypD was immunoprecipitated using an anti-FLAG antibody and the complexes Western blotted for GSK3β and FLAG. C) CypD KO MEFs were infected with adenoviruses encoding βGal, WT-GSK3β for 48hrs. Western blotting for GSK3β confirmed overexpression of GSK3β. GAPDH was used as a loading control. D) βGal and WT-GSK3β-infected CypD KO MEFs were treated with H 2 O 2 (100, 250 or 500μM) for 4hrs and cell death was measured by Sytox green staining. E) Representative traces of Ca 2+ retention capacity (CRC) measured using Calcium Green-5N in digitonin-permeabilized βGal, and WT-GSK3β -infected MEFs. Cells were treated with 2.5 μM Ca 2+ boluses every minute until the peak of fluorescence indicative of MPT was observed. F) Quantification of CRC data in the βGal and WT-GSK3β-infected MEFs. Each individual point represents one independent cell isolate. Bar represents the mean. * P <0.05 vs. βGal.

    Article Snippet: To generate the mitochondrial-targeted viruses, we obtained clones for HA-tagged human wild-type and K85A GSK3β with HA-tags as a gift from Dr. James Woodgett at (Addgene plasmid #14753 http://n2t.net/addgene:147553 ; RRID:Addgene_14753 and #14755 http://n2t.net/addgene:14755 ; RRID:Addgene_14755, respectively).

    Techniques: Western Blot, Membrane, Infection, Immunoprecipitation, Over Expression, Control, Staining, Fluorescence