Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Oncostatin M Induces IFITM1 Expression to Inhibit Hepatitis B Virus Replication Via JAK-STAT Signaling.

doi: 10.1016/j.jcmgh.2023.10.003

Figure Lengend Snippet: Figure 1. OSM is the most effective among the examined IL6 family cytokines for suppression of HBV replication. (A) HepG2.2.15, (B) Huh7-1.3, and (C) HepAD38 cells were treated with IL6, LIF, OSM, CNTF, cardiotrophin-1 (CT-1), IL11, or IL31 at a concentration of 20 ng/mL for 72 hours. The levels of extracellular HBsAg, HBeAg, and HBV DNA were quantitated. One- way analysis of variance. (D) Quantitation of OSM concentration by enzyme-linked immunosorbent assay in serum from health control subjects and patients with chronic HBV infection in different phases. Kruskal-Wallis test. (E) Serum OSM, HBsAg, and HBeAg levels in HBeAg-positive CHB patients. Mann-Whitney. HC, health control; I, HBeAg-positive chronic HBV infection; II, HBeAg-positive CHB; III, HBeAg-negative chronic HBV infection; IV, HBeAg-negative CHB. *P < .05, **P < .01, and ***P < .001. PBS, phosphate-buffered saline.

Article Snippet: The recombinant human cytokines IL6, IL11, IL31, IL35, LIF, OSM, CNTF, and cardiotrophin-1 were purchased from R&D Systems and Novoprotein (Shanghai, China).

Techniques: Concentration Assay, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Control, Infection, MANN-WHITNEY, Saline

Journal: Cell Death & Disease

Article Title: CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

doi: 10.1038/s41419-018-1258-6

Figure Lengend Snippet: a Microarray analysis showed the fold changes of CXCL8 and IL11 in the CUL1 knockdown group compared with control groups. b Real-time PCR was used to validate the mRNA expressions of CXCL8 and IL11 in CUL1 knockdown and vector control MDA-MB-231 cells. c MDA-MB-231 cells were transiently transfected with CUL1 siRNA (si-CUL1) and vector siRNA control (si-Ctrl) for 48 h, and the serum-free medium was added to the cells for 24 h, then the conditioned medium was collected. ELISA analysis was used to test the secreted CXCL8 and IL11 expression levels in the 24 h conditioned medium. d The correlation of CUL1 mRNA expressions with CXCL8 and IL11 mRNA expressions in TCGA breast cancer dataset. CUL1 expression was divided into high and low groups based on the average value. e MDA-MB-231 cells were seeded in serum-free medium in the upper chamber and the 24 h conditioned medium from CUL1 knockdown and vector control MDA-MB-231 cells containing either supplementary recombinant CXCL8 and IL11 proteins or nothing was placed in the lower chamber, then the representative migration images of MDA-MB-231 cells were showed. f The number of cell migration per field was counted in five random fields ( n = 3/group). g The tube formation by HUVECs in the conditioned medium from MDA-MB-231 cells with CUL1 knockdown and vector control which was complemented recombinant CXCL8 and IL11 proteins. f The number of tubes formed per field was counted in five random fields ( n = 3/group) * P < 0.05, ** P < 0.001 (Student’s t -test)

Article Snippet: Secreted CXCL8 and IL11 protein in the 24 h conditioned medium were measured by the human CXCL8 and IL11 ELISA kits from Elabscience Biotechnology according to the manufacturer’s instructions.

Techniques: Microarray, Knockdown, Control, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Expressing, Recombinant, Migration

Journal: Cell Death & Disease

Article Title: CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

doi: 10.1038/s41419-018-1258-6

Figure Lengend Snippet: a The activation Z -score arithmetic was used to predict the upstream transcriptional regulators of CXCL8 and IL11. b Western blotting was used to test the protein expression levels of predicted common upstream regulators ZEH2 and NFKBIA. c The correlation of CUL1 mRNA expressions with EZH2 mRNA expressions in 1104 TCGA breast invasive carcinomas from starBase v3.0 project. d Western blotting was used to test the expression of CUL1 and EZH2 in the MDA-MB-231 cells transfected with pCMV-EZH2 or vector plasmid, together with either si-CUL1 or si-Ctrl. e Real-time PCR was used to explore the CXCL8 and IL11 expressions in the MDA-MB-231 cells transfected with pCMV-EZH2 or vector plasmid, together with either si-CUL1 or si-Ctrl. f MDA-MB-231 cells were transiently transfected with pCMV-EZH2 or PCMV vector plasmid, together with either si-CUL1 or si-Ctrl for 48 h, and the serum-free medium was added to the cells for 24 h, then the conditioned medium was collected. ELISA analysis was used to test the protein expression levels of CXCL8 and IL11 in the 24 h conditioned medium. * P < 0.05, ** P < 0.001 (Student’s t -test)

Article Snippet: Secreted CXCL8 and IL11 protein in the 24 h conditioned medium were measured by the human CXCL8 and IL11 ELISA kits from Elabscience Biotechnology according to the manufacturer’s instructions.

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

Journal: Cell Death & Disease

Article Title: CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

doi: 10.1038/s41419-018-1258-6

Figure Lengend Snippet: a The expressions of PI3K–AKT–mTOR signaling pathway-related proteins, such as PI3K, p-AKT, AKT, p-mTOR, mTOR, p-P 70S6K , and P 70S6K in MDA-MB-231 cells with CUL1 knockdown or vector control. b MDA-MB-231 cells were transiently transfected with si-CUL1 and si-Ctrl for 48 h and these cells were continued culturing in serum-free medium with or without supplementary recombinant CXCL8 and IL11 proteins for 24 h, then western blotting was used to test the expressions of p-AKT, AKT, p-mTOR, mTOR, p-P 70S6K , and P 70S6K

Article Snippet: Secreted CXCL8 and IL11 protein in the 24 h conditioned medium were measured by the human CXCL8 and IL11 ELISA kits from Elabscience Biotechnology according to the manufacturer’s instructions.

Techniques: Knockdown, Plasmid Preparation, Control, Transfection, Recombinant, Western Blot

Journal: Cell Death & Disease

Article Title: CUL1 promotes breast cancer metastasis through regulating EZH2-induced the autocrine expression of the cytokines CXCL8 and IL11

doi: 10.1038/s41419-018-1258-6

Figure Lengend Snippet:

Article Snippet: Secreted CXCL8 and IL11 protein in the 24 h conditioned medium were measured by the human CXCL8 and IL11 ELISA kits from Elabscience Biotechnology according to the manufacturer’s instructions.

Techniques:

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