Journal: Immunity, Inflammation and Disease

Article Title: Bone marrow stromal cell‐derived exosomes improve oxidative stress and pyroptosis in doxorubicin‐induced myocardial injury in vitro by regulating the transcription of GSDMD through the PI3K‐AKT‐Foxo1 pathway

doi: 10.1002/iid3.810

Figure Lengend Snippet: BMSC‐Exos regulates GSDMD transcription through the PI3K‐AKT‐Foxo1 pathway in DOX‐induced H9C2 cells. (A) Western blot to examine the expression of p‐PI3K, p‐AKT, total‐AKT, and p‐mTOR in cells. * p < .05; ** p < .01; *** p < .001 versus the DOX + BMSC group. (B) Western blot to measure the protein expression levels of total‐Foxo1 in cell nucleus and cytoplasm. (C) Western blot to determine the expression of acetylated Foxo1, phosphorylated Foxo1, and total‐Foxo1,  *** p < .001 versus the DOX + BMSC group. (D) Public database to analyze the binding site sequence of Foxo1 to GSDMD. (E) Dual‐luciferase reporter gene assay to validate binding of Foxo1 to GSDMD promoter,  *** p < .001 versus the OE‐NC group. (F) ChIP assay to test the enrichment level of Foxo1 in the promoter region of GSDMD, *** p < .001 versus the anti‐IgG group. The two‐tailed test was employed to confirm p values. Except for special statements, one‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. AKT, protein kinase B; BMSC, bone marrow stromal cells; ChIP, chromatin immunoprecipitation; DOX, doxorubicin; Exo, exosome; GSDMD, gasdermin D; IgG, immunoglobulin G; mTOR, mechanistic target of rapamycin kinase; OS, oxidative stress; p, phosphorylated; PI3K, phosphatidylinositol 3‐kinase.

Article Snippet: The membranes were supplemented respectively with diluted primary antibodies against Alix (ab275377, 1:1000; Abcam), CD63 (PA5‐92370, 1:1000; Thermo Fisher Scientific), GM130 (PA5‐95727, 1:2000; Thermo Fisher Scientific), cytochrome c (ab133504, 1:1000; Abcam), calnexin (ab133615, 1:1000; Abcam), nucleotide‐binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3, ab263899, 1:1200; Abcam), apoptosis‐associated speck‐like protein containing A CARD (ASC, 307560, 1:1000; Abcam), cleaved caspase‐1 (89332S, 1:1000; Cell Signaling Technologies [CST]), interleukin (IL)‐18 (67775S, 1:1000; CST), cleaved IL‐1β (63124S, 1:1000; CST), active N‐terminal fragment of GSDMS (NT‐GSDMD, 10137S, 1:1000; CST), phosphorylated‐phosphatidylinositol 3‐kinase (p‐PI3K, ab278545, 1:1000; Abcam), p‐protein kinase B (p‐AKT, 4060T, 1:1000; CST), total‐AKT (4685S, 1:1000; CST), p‐mechanistic target of rapamycin kinase (p‐mTOR, ab109268, 1:1000; Abcam), p‐Foxo1 (ab259337, 1:1000; Abcam), acetyl‐Foxo1 (PA5‐104560, 1:1000; Thermo Fisher Scientific), and β‐actin (ab8226, 1:2500; Abcam) for overnight probing at 4°C.

Techniques: Western Blot, Expressing, Binding Assay, Sequencing, Luciferase, Reporter Gene Assay, Two Tailed Test, Chromatin Immunoprecipitation

Journal: Immunity, Inflammation and Disease

Article Title: Bone marrow stromal cell‐derived exosomes improve oxidative stress and pyroptosis in doxorubicin‐induced myocardial injury in vitro by regulating the transcription of GSDMD through the PI3K‐AKT‐Foxo1 pathway

doi: 10.1002/iid3.810

Figure Lengend Snippet: Inactivation of the PI3K‐AKT pathway abrogates the relieving impacts of BMSC‐Exos on DOX‐induced OS and pyroptosis in H9C2 cells. (A, B) DCFH‐DA staining to measure ROS levels in cells. (C, D) JC‐1 immunofluorescence to monitor the changes of potential difference of mitochondrial membrane in cells. (E, F) Western blot to test the expression of NLRP3, ASC, cleaved caspase‐1, NT‐GSDMD, cleaved IL‐1β, and cleaved IL‐18 in cells. (G, H) Western blot to measure the phosphorylation levels and total protein levels of Foxo1 in cell nucleus and cytoplasm; *** p < .001 versus the control group; ## p < .01; ### p < .001 versus the DOX group; & p < .05; && p < .01; &&& p < .001 versus the DOX + BMSC‐Exo group. One‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. AKT, protein kinase B; ASC, apoptosis‐associated speck‐like protein containing A CARD; BMSC, bone marrow stromal cells; DCFH‐DA, 20,70‐dichlorofluorescein diacetate; DOX, doxorubicin; Exo, exosome; Foxo1, forkhead box O1; GSDMD, gasdermin D; IL, interleukin; NLRP3, nucleotide‐binding oligomerization domain, leucine rich repeat and pyrin domain containing 3; OS, oxidative stress; PI3K, phosphatidylinositol 3‐kinase; ROS, reactive oxygen species.

Article Snippet: The membranes were supplemented respectively with diluted primary antibodies against Alix (ab275377, 1:1000; Abcam), CD63 (PA5‐92370, 1:1000; Thermo Fisher Scientific), GM130 (PA5‐95727, 1:2000; Thermo Fisher Scientific), cytochrome c (ab133504, 1:1000; Abcam), calnexin (ab133615, 1:1000; Abcam), nucleotide‐binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3, ab263899, 1:1200; Abcam), apoptosis‐associated speck‐like protein containing A CARD (ASC, 307560, 1:1000; Abcam), cleaved caspase‐1 (89332S, 1:1000; Cell Signaling Technologies [CST]), interleukin (IL)‐18 (67775S, 1:1000; CST), cleaved IL‐1β (63124S, 1:1000; CST), active N‐terminal fragment of GSDMS (NT‐GSDMD, 10137S, 1:1000; CST), phosphorylated‐phosphatidylinositol 3‐kinase (p‐PI3K, ab278545, 1:1000; Abcam), p‐protein kinase B (p‐AKT, 4060T, 1:1000; CST), total‐AKT (4685S, 1:1000; CST), p‐mechanistic target of rapamycin kinase (p‐mTOR, ab109268, 1:1000; Abcam), p‐Foxo1 (ab259337, 1:1000; Abcam), acetyl‐Foxo1 (PA5‐104560, 1:1000; Thermo Fisher Scientific), and β‐actin (ab8226, 1:2500; Abcam) for overnight probing at 4°C.

Techniques: Staining, Immunofluorescence, Western Blot, Expressing, Binding Assay

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antitumor Effects of Delta (9)-Tetrahydrocannabinol and Cannabinol on Cholangiocarcinoma Cells and Xenograft Mouse Models

doi: 10.1155/2022/6477132

Figure Lengend Snippet: Effects of THC and CBN on CCA cell signaling. (a) The human phosphokinase array detected phosphorylated proteins in the control group, in HuCCT1 cells treated with 20 μ M of THC and in cells treated with 20 μ M of CBN after an 18 h incubation. Corresponding signals: (i) GSK-3 α / β (phosphorylated at Ser21/9), (ii) ERK1/2 (phosphorylated at Thr202/Tyr204), and (iii) AKT (phosphorylated at Ser473). (b) Relative changes in phosphorylated kinase proteins between THC- or CBN-treated HuCCT1 cells and untreated HuCCT1 cells were demonstrated. (c) Selected phosphorylated kinase proteins in HuCCT1 cells after 10 μ M to 20 μ M THC or CBN treatment for 18 h were determined by western blotting. At a concentration of 20 μ M, THC and CBN inhibited the phosphorylation of AKT, GSK-3 α / β , and ERK1/2. β -Actin was used as a loading control. (d) The relative levels of phosphorylation of AKT, GSK-3 α / β , and ERK1/2 from western blot analyses were normalized to β -actin level ( ∗ P < 0.01 versus control).

Article Snippet: Polyclonal antibodies against protein kinase B (AKT) (phosphorylated at Ser473), glycogen synthase kinase 3 alpha/beta (GSK-3 α / β ) (phosphorylated at Ser21/9), p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) (phosphorylated at Thr202/Tyr204), caspase 3, cleaved caspase 3, poly (ADP-ribose) polymerase (PARP [46D11]), cleaved PARP (Asp214), caspase 3, cleaved caspase 3 (Asp175), and β -actin were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Western Blot, Concentration Assay