Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 1 Synthesis and purification of ANG2002. (A) A sulfo-N- [ε-maleimidocaproyloxy]succin- imide ester (sulfo-EMCS) linker was attached to the lysine in position 6 of NT and to the C-ter- minally modified cysteine of An2 to maintain separation of both moieties for functionality pur- poses. In step 1, 1.3 equivalents of sulfo-EMCS were added to NT. Within 30 minutes, the reac- tion was acidified and the [Lys6- MHA]-NT intermediate purified by HPLC. In step 2, 1.3 equiva- lents of An2-cysteine were added to the reaction at RT. (B) Differ- ent retention times were found by analytical RP-UPLC for (i) NT, (ii) [Lys6-MHA] NT, (iii) An2-Cys, and (iv) ANG2002. (C) Analytical RP-UPLC profile of the purified ANG2002 after the 2-step conju- gation reaction. (D) Multicharged ESI-TOF MS spectrum of puri- fied ANG2002.

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Purification, Modification

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 2 Molecular interaction of An2 and ANG2002 with LRP1. (A) An2 inhi- bition of specific α2M-MA binding to LRP1 on MEF cells. Cells were incu- bated with 0.1 nM α2M-MA in the presence or absence of increasing concentrations of An2. (B) Uptake of Alexa Fluor 488–labeled An2 in LRP1-positive MEF-1 fibroblasts and LRP1-deficient PEA-13 fibroblasts. (C) Binding of 125I-An2 to Fc, CCR-2 (Cluster II), and CCR-4 (Cluster IV) with and without preincubation with 0.5 μM RAP. (D) Binding of 125I-An2 to CCR-4 performed in the absence and presence of excess unlabeled An2 or ANG2002 (500 μM). Data represent mean ± SD. **P < 0.01, ***P < 0.001 versus respective con- trol; #P < 0.05 versus An2; Student’s t test (B), 2-way ANOVA followed by Bonferroni post-test (C), or 1-way ANOVA followed by Bonferroni cor- rection (D).

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Binding Assay, Labeling

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 3 Brain uptake of ANG2002 and NT measured by in situ mouse brain perfusion. (A) Time course of brain uptake of [125I]-ANG2002 and [125I]-NT. Results represent apparent Vd in total brain homogenate. Lines represent best fits to the data by least-squares regression. (B) After a 2-minute perfusion of [125I]-ANG2002 (black bars) and [125I]-NT (white bars), brain capillary depletion was performed, and radioactivity was quantified in total brain homogenate, brain capillary fractions, and brain parenchymal fractions. Results represent apparent Vd for the radiolabeled drugs in the indicated compartments. Data represent mean ± SD (n = 4–6 mice per time point). *P < 0.05, **P < 0.01 vs. NT, Student’s t test.

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: In Situ, Radioactivity

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 4 Antinociceptive responses to ANG2002 in acute pain models. (A) Hot- plate test performed on CD-1 mice after administration of ANG2002 (10 and 20 mg/kg i.v.), NT (8 mg/kg i.v.), or buprenorphine (Bupe; 1 mg/kg s.c.). (B) Analgesic effects of ANG2002 and morphine sulfate (MS; 5 mg/kg i.p.), assessed by mouse radiant heat tail-flick assay. MPE was calculated at the time of peak antinociceptive response. *P < 0.05, **P < 0.01, ***P < 0.001 versus saline control, 1-way ANOVA followed by Dunnett multiple comparison test.

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Hot Plate Test, Tail Flick Test, Saline, Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 5 Effect of NT receptor inactivation on ANG2002-induced analgesia. (A) Influence of the NT receptor antagonist SR142948A on NT- and ANG2002-induced antinociceptive responses. MPE was calculated 60 minutes after i.t. injection. In the presence of 10 μg/kg SR142948A, antinociceptive responses to NT (20 μg/kg) and ANG2002 (50 μg/kg) were significantly reduced. No change in tail-flick latencies was seen after administration of SR142948A alone. ***P < 0.001 versus saline vehicle; ###P < 0.01 versus no SR142948A; 1-way ANOVA followed by Bonferroni post-test. (B and C) WT, NTS1-deficient (B), and NTS2- deficient (C) C57BL/6 mice submitted to the tail-immersion test after i.v injection of ANG2002 (5 mg/kg). In both NTS1-deficient and NTS2- deficient mice, ANG2002 conserved its analgesic properties. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle-treated WT, 2-way ANOVA fol- lowed by Bonferroni post-test.

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Injection, Tail Flick Test, Saline

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 6 Analgesic efficacy of ANG2002 in the formalin model of tonic nociceptive pain. (A) ANG2002 dose-response analgesic effect on reducing forma- lin-induced nociceptive pain behaviors after i.v. administration. (B) Analgesic effect of i.v. morphine sulfate (0.05 mg/kg) compared with ANG2002 (0.05 mg/kg) and An2 (5 mg/kg). (C and D) ANG2002 ED50 in acute (0–9 minutes; C) and inflammatory (21–60 minutes; D) phases. (E) Effect of ANG2002 on time spent flinching, licking, and biting in the acute phase of the formalin test. (F and G) Time spent flinching, licking, and biting in the inflammatory phase of the formalin test. n = 6–10 rats per group. ***P < 0.001 versus control, 1-way ANOVA followed by Dunnett multiple-comparison test (E and F) or Kruskal-Wallis test followed by Dunn correction (G).

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 7 Antiallodynic effects of ANG2002 in 2 chronic pain models. (A) Effect of ANG2002 on tactile allodynia induced by CCI of the sciatic nerve. PWT after automated von Frey hair stimulation was measured at various time points after induction of neuropathic pain. On day 21, rats were given i.v. administration of ANG2002 (0.05 mg/kg) or vehicle. The antiallodynic effect was evaluated 45 minutes after administration. BL, baseline. (B) The antiallodynic effect was monitored at additional time points (75 and 120 minutes) after ANG2002 injection and calculated as AUC over the 2-hour period. (C) Effect of ANG2002 on mechanical allodynia induced by the inoculation of the syngeneic mammary tumor cell line MRMT-1 into the femoral bone. The time course of tactile allodynia was examined in cancer-bearing and sham-operated rats during the 3-week period after the surgery. PWT was determined at day 18, 45 minutes after acute i.v. injection of either ANG2002 (0.05 mg/kg) or vehicle. (D) The effect of ANG2002 was monitored at additional time points (75 and 120 minutes) after ANG2002 injection and calculated as AUC over the 2-hour period. n = 8–10 rats per group. (A and C) ***P < 0.01, ###P < 0.001 versus vehicle, 1-way ANOVA followed by Dunnett multiple comparison test. (B and D) *P < 0.05, **P < 0.01, Student’s unpaired t test.

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Injection, Comparison

Journal: Journal of Clinical Investigation

Article Title: Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

doi: 10.1172/jci70647

Figure Lengend Snippet: Figure 8 Effects of ANG2002 on physiological parameters. (A) MAP was determined for 60 minutes after i.v. adminis- tration of ANG2002 (0.05, 0.5, or 5 mg/kg) or NT (2 mg/kg) in Wistar male rats. ***P < 0.001, NT vs. vehicle; ###P < 0.001, 5 mg/kg ANG2002 vs. vehicle; †††P < 0.001, 0.5 mg/kg ANG2002 vs. vehicle; 2-way ANOVA followed by Bonferroni post-test. (B) Change in body temperature (ΔTb) over a 6-hour time span after i.v. ANG2002 injec- tion in Sprague-Dawley rats. *P < 0.05, **P < 0.01, ***P < 0.001 versus 5 mg/kg ANG2002; #P < 0.05, ###P < 0.001 versus 0.05 mg/kg ANG2002; 2-way ANOVA followed by Bonferroni multiple comparison test. (C) Spon- taneous locomotor activity assessed using the open field. Rats treated with ANG2002 (0.05 mg/kg) traveled total distances similar to those of saline-treated animals (unpaired t test). (D) Motor balance and coordination were evaluated using the Rotarod test after ANG2002 (0.05 mg/kg) treatment. The time the animal spent on the Rotarod (e.g., latency to fall) was measured (1-way ANOVA followed by Bonferroni post-test).

Article Snippet: NT or ANG2002 (10–11 to 10–5 M) were added for 20 minutes, then completed with coelenterazine-400A to a final concentration of 5 μM (Goldbio).

Techniques: Comparison, Activity Assay, Saline

Journal: BMC Cancer

Article Title: MTA2 enhances colony formation and tumor growth of gastric cancer cells through IL-11

doi: 10.1186/s12885-015-1366-y

Figure Lengend Snippet: Differential gene expression in MTA2 overexpression and knockdown cells

Article Snippet: Recombinant human interleukin-11 (rhIL-11, R&D systems) was reconstituted following user manual at a concentration of 50 μg/ml.

Techniques: Expressing, Over Expression, Knockdown

Journal: BMC Cancer

Article Title: MTA2 enhances colony formation and tumor growth of gastric cancer cells through IL-11

doi: 10.1186/s12885-015-1366-y

Figure Lengend Snippet: IL-11 expression related with MTA2 in SGC-7901 and BGC-823 cells. A : IL-11 expression was detected by real-time PCR. B : IL-11 protein was detected by western blot and ELISA. C : IL-11 expression in SGC-7901 and BGC-823 xenografts was detected by IHC. D : IL-11 mRNA expression was reduced by SAHA in both SGC-7901/NC cell and BGC-823/MTA2 cell.

Article Snippet: Recombinant human interleukin-11 (rhIL-11, R&D systems) was reconstituted following user manual at a concentration of 50 μg/ml.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: BMC Cancer

Article Title: MTA2 enhances colony formation and tumor growth of gastric cancer cells through IL-11

doi: 10.1186/s12885-015-1366-y

Figure Lengend Snippet: IL-11 recovered colony formation capacity of SGC-7901/shMTA2 cell. A : Growth curves of SGC-7901/NC and SGC-7901/shMTA2 cells were not affected by rhIL-11 treatment. B : Colony formation in soft agar was assessed after rhIL-11 treatment. C : Number of colonies in SGC-7901/shMTA2/IL-11 group was more than it in SGC-7901/shMTA2/PBS group, and similar with SGC-7901/NC/PBS group. D : Size of colonies in SGC-7901/shMTA2/IL-11 group was larger than it in SGC-7901/shMTA2/PBS group.

Article Snippet: Recombinant human interleukin-11 (rhIL-11, R&D systems) was reconstituted following user manual at a concentration of 50 μg/ml.

Techniques:

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Schematic showing signalling pathways by which IL11 induces LKB/AMPK inactivation and mTOR activation. b Western blots (WB) of IL11, GAPDH, p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP in livers from 12, 25, 50, 75, and 110-week-old (w/o) male mice (n=5/group). c Heat map showing densitometry of IL11 protein expression normalised to GAPDH in gastrocnemius and visceral white adipose tissues (vWAT) from 12 to 110 w/o male mice (n=5/group). d Representative immunofluorescence images (scale bars, 100 µm) of EGFP and SLC10A1 expression in the livers of 10 and 110 w/o Il11 - EGFP mice (representative dataset from n=3/group). e WB of p-ERK1/2, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, p16, p21, and GAPDH in livers from 10 and 110 w/o male wild-type (WT) and Il11ra1 -/- mice (n=3/group). f Body weights (BW), g percentages of fat and lean mass (normalised to BW), h body temperatures, and the levels of i serum cholesterol, and j serum triglycerides (TG) of 110 w/o male and female WT and Il11ra1 -/- mice (male WT for ( f-h ), n=12; male WT for ( i-j ), n=11; male Il11ra1 -/- , n=16; female WT, n=15; female Il11ra1 -/- , n=13). Liver k telomere length and l mitochondria DNA (mtDNA) copy number from young (10 w/o) and old (110 w/o) male and female WT and Il11ra1 -/- mice (young male WT, n=8; young male Il11ra1 -/- , n=7; old male WT, n=11; old male Il11ra1 -/- , n=17; young female WT, n=7; young female Il11ra1 -/- , n=8; old female WT, n=15; old female Il11ra1 -/- , n=13). m Estimated liver DNA methylation age from male 110 w/o WT and Il11ra1 -/- mice (n=8/group). n Effects of U0126 (10 µM) and rapamycin (10 nM) on p16, p21, Cyclin D1, and PCNA protein expression in IL11 (5 ng/ml)-stimulated HCFs by WB (n=6/group). o-u Data for HCF passage 4 (P4), 7, 10, and 14 that had been passaged in the presence of either IgG or anti-IL11RA (X209; 2µg/ml) from P2. o WB of p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, p-NFκB, p-STAT3, p16, p21, PCNA, Cyclin D, and GAPDH, p IL6 and IL8 levels in the supernatant based on ELISA, q telomere length, and r mtDNA copy number (n=6/group). Seahorse assay showing s mitochondrial oxygen consumption rate (OCR), t changes in OCR during basal respiration and ATP production states, and u oxidative and glycolytic energy phenotypes at baseline (n=8/group). f-m, p-u Data are shown as meanLJ±LJSD. f-l Two-way ANOVA with Sidak’s correction; m two-tailed Student’s t-test; p two-way ANOVA, q, r, t one-way ANOVA with Tukey’s correction. FC: fold change.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Activation Assay, Western Blot, Expressing, Immunofluorescence, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Western blots (WB) of IL11 and GAPDH in visceral gonadal white adipose tissue (vWAT) and gastrocnemius from 12, 25, 50, 75, and 110-week-old (w/o) male mice (n=5/group). b WB of IL11 and GAPDH in the liver, vWAT and gastrocnemius from 12, and 110-week-old (w/o) male and female mice (n=3/group). c WB of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, and S6RP in livers from 12, 25, 50, 75, and 110-week-old (w/o) male mice (n=5/group) for the respective phopsho proteins shown in . d WB of p-ERK1/2, p-p90RSK, p-LKB1, p-AMPK, p-mTOR, p-p70S6K, p-S6RP, and their respective total protein in gastrocnemius from 12, 25, 50, 75, and 110 w/o male mice (n=5/group). e-g Representative immunofluorescence images (scale bars, 100µm) of EGFP expression in the livers, vWAT, and gastrocnemius, colocalized with parenchymal cell markers Adiponectin (AdipoQ) in vWAT and Four and a half LIM domains (FHL1) in gastrocnemius, endothelial cells (CD31), smooth muscle transgelin (SM22lJ), and pan-fibroblast marker (PDGFRA) of 10 and 110-week old Il11 - EGFP mice (representative dataset from n=3/group).

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Western Blot, Immunofluorescence, Expressing, Marker

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: Effects of U0126 and rapamycin on a the activation status of ERK1/2 and mTOR by WB, and on the levels of secreted b IL6 and c IL8 by ELISA (n=6/group) from IL11 (5 ng/ml)-stimulated primary human cardiac fibroblasts. d Relative levels of IL6, IL8, LIF, VEGFA, HGF, CCL2, CXCL1, CXCL5, CXCL6, and CCL20 in the supernatant of IL11-stimulated primary human hepatocytes (6 and 24 hours) as measured by Olink proximity extension assay (n=4/group). e-g Data for IL11 (10 ng/ml)-stimulated primary human hepatocytes in the presence of either DMSO, U0126, or rapamycin (n=6/group). e WB showing the activation status of ERK1/2, mTOR, p70S6K, S6RP, and the protein expression levels p16, p21, PCNA, Cyclin D, and GAPDH. Concentrations of f IL6 and g IL8 in the supernatant (as measured by ELISA). a-c, e-g U0126 (10 µM), rapamycin (10 nM). h Immunofluorescence images (scale bars, 100 μm; representative datasets from n=7/group) and quantification of intensity/area (n=14/group) for p16 and p21 staining, and i concentrations of IL11 in the supernatant (by ELISA) of HCF passage 4 (P4), 7, 10, and 14 that had been passaged in the presence of either IgG or anti-IL11RA (X209; 2µg/ml) from P2. j WB showing the expression levels of p16, p21, and GAPDH from HCFs P4 that were stimulated for 8, 24, 48, and 72 hours with media collected from HCFs P14 that had been grown and passaged in the presence of either IgG or anti-IL11RA (X209; 2µg/ml) from P2 (representative datasets from n=4/group). b-d, f-i Data are shown as meanL±LSD. b, c, f, g one-way ANOVA with Tukey’s correction; d one-way ANOVA with Dunnett’s correction, h two-way ANOVA with Sidak’s correction, i two-way ANOVA.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a WB of IL11 and GAPDH expression from the indicated organs of young (12 w/o) and old (105 w/o) male WT and Il11 -/- mice (n=2/group). b Representative picture of 108 w/o female WT and Il11 -/- mice. c Body weights (BW), d percentages of fat and lean mass (normalised to BW), e frailty scores, f body temperatures, g full body grip strength measurements, h serum cholesterol and TG levels, i glucose and insulin tolerance tests (GTT and ITT) from young (12 w/o) and old (105 w/o) female WT and Il11 -/- mice. Weights of j skeletal muscle (gastrocnemius and soleus), and k liver (indexed to BW), l liver TG levels, m indexed weights of vWAT and inguinal subcutaneous WAT (scWAT), n relative vWAT mRNA expression levels of Acc1 , Fasn , and Srebp1c , o WB showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP and protein expression levels of p16, p21, and GAPDH (representative datasets from n=6/group) in vWAT, p telomere length and q mtDNA copy number, r serum IL6 levels from young and old female WT and Il11 -/- mice. s Respiratory exchange ratio (RER) measurements in 68-70 w/o male WT and Il11 -/- mice (n=10/group). c-n, p-r Data are shown as meanLJ±LJSD (young WT, n=8; young Il11 -/- , n=9; old WT, n=16; old Il11 -/- , n=18). c-h, j-n, p-r Two-way ANOVA with Sidak’s correction; i two-way ANOVA. FC: fold change.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Expressing, Activation Assay

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Front paw grip strength, serum levels of b ALT, AST, c BHB, d area under the curves (AUC) of glucose tolerance tests (GTT) and insulin tolerance tests (ITT), e body lengths, f indexed brown adipose tissues (BAT) weight, g WB of total proteins for the respective phospho proteins shown in (representative datasets from n=6/group), h WB showing ERK1/2, mTOR, p70S6K, and S6RP activation and p16, p21, and GAPDH protein expression levels (representative datasets from n=6/group), i relative pro-inflammatory gene expression ( Ccl2, Ccl5, Tnf lZ , Il1 □ and Il6 ) levels in vWAT from young and old female WT and Il11 -/- mice. a-f, i Data are shown as meanL±LSD, two-way ANOVA with Sidak’s correction. a-e, i young WT, n=8; young Il11 -/- , n=9; old WT, n=16; old Il11 -/- , n=18; f young WT, n=5; young Il11 -/- , n=7; old WT and Il11 -/- , n=16/group. FC: fold change; AU: arbitrary units.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Activation Assay, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Representative image of 108 w/o mice, b body weights (BW), c percentages of fat and lean mass (normalised to BW), d frailty scores, e body temperatures, f full body and forepaw grip strength measurements, g glucose and insulin tolerance tests (GTT and ITT) from young (12 w/o) and old (105 w/o) male WT and Il11 -/- mice. h RER measurements, cumulative food intake, and locomotive activities as measured by phenomaster for 5 days (n=10/group) in 68-70 w/o male WT and Il11 -/- mice (n=10/group). i Body length, j indexed weight of skeletal muscle (gastrocnemius and soleus), k liver, l vWAT, subcutaneous WAT (scWAT), and BAT. b-g, i-l Data are shown as mean☐±☐SD (young WT and Il11 -/- , n=6-9/group; old WT, n=15; old Il11 -/- , n=12-14), two-way ANOVA with Sidak’s correction. FC: fold change; AU: arbitrary units.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques:

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Schematic of anti-IL11 (X203) therapeutic dosing experiment in old male mice for experiments shown in ( b-p ). Mice were either aged naturally (untreated) or given either X203 or an IgG control antibody (40 mg/kg, every 3 weeks) starting from 75 weeks of age for a duration of 25 weeks. b Body weights across time. c-f Changes (Δ; values at end-point (100 w/o) - values at starting point (75 w/o)) in c fat and lean mass percentage, and d area under the curve (AUC) of GTT and ITT. e Frailty scores at starting and end-point. f Full body grip strength. g RER measurements in young (14 w/o) and IgG/X203-treated old (81 w/o) mice - 6 weeks after IgG/X203 administration was started (n=10/group). h Body temperatures, i serum ALT level, j liver TG levels. k indexed weights of and l total collagen content (by hydroxyproline assay) in liver, gastrocnemius, and vWAT. m WB showing activation status of ERK1/2, p90RSK, LKB1, AMPK, mTOR, p70S6K, S6RP and protein expression levels of IL11, p16, p21, and GAPDH in vWAT (representative datasets from n=6/group). n Estimated liver and gastrocnemius DNA methylation age, o telomere length and p mtDNA copy number. b-d, f, h-l, n-p Data are shown as meanLJ±LJSD, e data are shown as values recorded at starting and end-point (75 w/o control, n=14; untreated 100 w/o, n=6; IgG-treated 100 w/o n=13; X203-treated 100 w/o, n=12). b Two-way ANOVA, c-f, h-l, o-p one-way ANOVA with Tukey’s correction, n two-tailed Student’s t-test. FC: fold change; AU: arbitrary units.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Control, Hydroxyproline Assay, Activation Assay, Expressing, DNA Methylation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a Front paw grip strength, b RER measurements, cumulative food intake, and locomotive activities as measured by phenomaster for 5 days on IgG/X203-treated old (81 w/o) mice - 6 weeks after IgG/X203 administration was started (n=10/group), serum levels of c cholesterol, TG, and IL6, d BHB, and e AST, indexed weight of f soleus, g subcutaneous white adipose tissues (scWAT) and BAT, and h WB of total proteins for the respective phospho proteins shown in (representative datasets for n=6/group) for anti-IL11 therapeutic dosing experiment in old mice as shown in Schematic . a, c-g (75 w/o control, n=6-14; untreated 100 w/o, n=6; IgG-treated 100 w/o n=13; X203-treated 100 w/o, n=12). a, c-g Data are shown as meanL±LSD; one-way ANOVA with Tukey’s correction.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: Control

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a-e, g-j Data for X203 therapeutic experiments in old male mice as shown in . a Bubblemap showing results of hallmark gene set enrichment analysis for differentially expressed genes in the vWAT, liver, gastrocnemius of mice receiving anti-IL11 therapy as compared to IgG. Normalized enrichment scores (NES) are represented by colors, (black: negative NES, suggesting down-regulation of the gene set; yellow: positive NES, suggesting up-regulation). Dot size indicates significance (the larger the dot, the smaller the adjusted p-value). b Heatmap of row-wise scaled Transcripts per million (TPM) values of senescence genes in vWAT, liver, gastrocnemius, c abundance of Ucp1 reads in vWAT (TPM), and d log2 FC heatmap of beijing genes in vWAT from IgG or anti-IL11 treated 100 w/o mice based on RNA-seq. e WB of Ucp1, Pgc1⍺ and GAPDH expression in vWAT (representative datasets from n=6/group). f Relative expression levels of Ucp1 mRNA (young WT, n=8; young Il11 -/- , n=9; old WT, n=16; old Il11 -/- , n=18) as well as Ucp1 and Pgc⍺ protein rsin (representative datasets from n=6/group) in vWAT isolated from young and old female WT and Il11 -/- mice. g Abundance of Clstn3b and S100b reads and h log2 FC heatmap of pro-inflammatory markers (from RNA-seq) in vWAT. i H&E-stained vWAT (scale bars, 100 µm) and quantification of lipid droplet size (mean of lipid droplet area), and j immunohistochemistry staining of CD68 in vWAT (scale bars, 50 µm). a-d, f-h Liver and gastrocnemius (n=8/group), vWAT IgG, n=7; vWAT anti-IL11, n=6. c, f-g, i Data are shown as meanLJ±LJSD. c, g, i Two-tailed Student’s t-test; f two-way ANOVA with Sidak’s correction.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: RNA Sequencing, Expressing, Isolation, Staining, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a. Violin plot of Transcripts per million (TPM) values of senescence genes (based on Tabula Muris Senis consortium) in vWAT, liver, gastrocnemius samples from mice receiving either IgG or anti-IL11 as shown in schematic . b. Relative Ucp1 mRNA from young (10 w/o) and old (110 w/o) male and female WT and Il11ra1 -/- mice. c. Heatmap showing row-wise scaled TPM values for the gene-list in Mitocarta 3.0. (no. of genes = 1,019 with TPM>=5 in at least one condition). d. A lollipop plot for top 50 Mitocarta 3.0 pathways found significant (p-adj < 0.05) in enrichment analysis using fgsea R package. No negative NES was found to be significant. e. Distribution of RNA-seq reads at the Clstn3 locus from IgG or anti-IL11-treated vWAT. Relative Ucp1 mRNA expression levels in BAT from f therapeutic anti-IL11 dosing group (left; 75 w/o control, n=6; untreated 100 w/o, n=6; IgG-treated 100 w/o n=13; X203-treated 100 w/o, n=12) and from g WT and Il11 -/- mice (right; young WT, n=5; young Il11 -/- , n=7; old WT and Il11 -/- , n=16/group). H Relative vWAT mRNA expression of pro-inflammatory markers ( Ccl2 , Ccl5 , Tnf⍺, Il1flJ, Il6 ) in young (10 w/o) and old (110 w/o) male and female WT and Il11ra1 -/- mice. a, c, d, e Liver and gastrocnemius (n=8/group), vWAT IgG, n=7; vWAT anti-IL11, n=6; b, h young male WT, n=8; young male Il11ra1 -/- , n=7; old male WT, n=11; old male Il11ra1 -/- , n=13-14; young female WT, n=7; young female Il11ra1 -/- , n=8; old female WT, n=15; old female Il11ra1 -/- , n=12. a Data are shown as violin plots with medianL±Lmin-max; b, f-h data are shown as meanL±LSD. b, g, h Two-way ANOVA with Sidak’s correction; e one-way ANOVA with Tukey’s correction.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: RNA Sequencing, Expressing, Control

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a VVB011 interaction with human IL11 (top) or mouse IL11 (bottom) as determined by surface plasmon resonance (SPR). b Dose-response curve and half maximal inhibitory concentration value of VVB011 (61 pg/mL to 4 μg/mL) in inhibiting matrix metalloproteinase 2 (MMP2) secretion by hIL11-stimulated human cardiac fibroblasts, mIL11-stimulated mouse atrial fibroblasts, and cIL11-stimulated monkey dermal fibroblasts. c Blood pharmacokinetics of VVB011 in rodents (n=3). d Binding affinity and kinetics of humanised VVB011 to hIL11 by SPR. e STAT3-luciferase reporter activity of IL11-stimulated HEK293 in the presence of varying concentration of IgG control or humanised VVB011 clone (n=2). c,e Data are shown as meanLJ±LJSD.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: SPR Assay, Concentration Assay, Drug discovery, Binding Assay, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Inhibition of an immunometabolic axis of mTORC1 activation extends mammalian healthspan

doi: 10.1101/2023.07.09.548250

Figure Lengend Snippet: a VVB011 interaction with cynomolgus IL11 as determined by surface plasmon resonance (SPR). b Dose-response curve and half maximal inhibitory concentration value of VVB011 (61 pg/mL to 4 μg/mL; 4-fold dilution) in inhibiting STAT3 activation (by WB) by hIL11-stimulated A549.

Article Snippet: Recombinant cynomolgus IL 11 (cIL11, 90925-CNCE, Sino Biological), human IL11 (hIL11, Z03108, Genscript), mouse IL11 (mIL11, Z03052, Genscript).

Techniques: SPR Assay, Concentration Assay, Activation Assay