Journal: BMC Pulmonary Medicine

Article Title: Simvastatin attenuates silica-induced pulmonary inflammation and fibrosis in rats via the AMPK-NOX pathway

doi: 10.1186/s12890-024-03014-9

Figure Lengend Snippet: Effects of Sim treatment on the AMPK-NOX pathway in silica-induced rats. ( A ) The western blotting bands of p-AMPK, AMPK and GAPDH in lung tissues among different groups ( n = 3). The gels/blots are corpped, and full-length gels/blots are presented in Fig. . ( B ) The relative protein expression of p-AMPK/AMPK. ( C ) The western blotting bands of NOX4, NOX2, p-p47phox, p47phox, p40phox, p22phox and GAPDH in lung tissues among different groups ( n = 3). The gels/blots are corpped, and full-length gels/blots are presented in Fig. . The relative protein expressions of NOX4 ( D ), NOX2 ( E ), p-p47phox/p47phox ( F ), p40phox ( G ), and p22phox ( H ). Data are presented as the means ± SD, n = 3. * p < 0.05, ** p < 0.01 vs. the control group; # p < 0.05, ## p < 0.01 vs. the silica group

Article Snippet: Other reagents included hydroxyproline (HYP), superoxide dismutase (SOD) and ROS kits (Nanjing Jiancheng, Nanjing, China); malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) detection kits (Shanghai Biotechnology Co., LTD.); tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (Beijing Baizhi Biological Technology Co., LTD.); transforming growth factor (TGF)-β1, E-cad, Vimentin, α-SMA and NOX2 antibodies (Abcam, London, England, 1:1 000); NOX4, p47phox and p40phox antibodies (Proteintech, China, 1:1 000); p-p47phox antibodies (Bioworld, USA, 1:1 000); p22phox antibodies (CST, USA, 1:1 000); GAPDH antibodies (Shanghai Beyotime Biotechnology, China, 1:1 000); IRDye680 Sheep Anti-Rabbit secondary antibodies and aRD Sheep Anti-Mouse secondary antibodies (LI-COR, USA, 1:10 000).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing direct interaction between AMPKγ1 and ULK1. B. Evolutionally conserved sequence alignment of AMPKγ1 in mammals. C. Immunoblot of proteins subjected to a kinase assay using purified trimeric AMPK consisting of AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A mutant with and without ULK1 showing direct phosphorylation of AMPKγ1 at S260/T262 by ULK1. D. Immunoblots of proteins subjected to a kinase assay using purified monomeric AMPKγ1 and ULK1. E. Immunoprecipitation of FLAG-tagged AMPKγ1 and/or Halo-tagged ULK1 expression in HEK293T cells showing AMPKγ1 bound to T-ULK1 is likely to be phosphorylated at S260/T262.

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Immunoprecipitation, Expressing, Sequencing, Western Blot, Kinase Assay, Purification, Mutagenesis

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Immunoprecipitation of FLAG-tagged AMPKγ1 with/without Halo-tagged ULK1 expression in HEK293T cells showing AMPK bound to T-ULK1 is likely to be phosphorylated at P-AMPKα Thr172 . Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression when ULK1 is overexpressed (n = 3). A. B. Immunoprecipitation of FLAG-tagged AMPKγ1 WT or AMPKγ1 S260A/T262A with Halo- tagged ULK1 expression in HEK293T cells. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A when ULK1 is overexpressed (n = 4). B. Immunoprecipitation of FLAG-tagged AMPKγ1 with Halo-tagged ULK1 expression in HEK293T cells treated with/without BL918 (selective ULK1 activator) for 15 minutes. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 (lower) and P-AMPKα Thr172 (upper) expression (n = 3). C. Immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 WT or AMPKγ1 S260A/T262A with/without 15 minutes BL918 (selective ULK1 activator, 5 μM) treatment (n = 3). D. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with/without BL918 for 15 minutes (n = 3). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Immunoprecipitation, Expressing, Western Blot, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in kidneys of wild-type (WT) or Ulk1 −/− mice following starvation (n = 8). B. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 and P-AMPKα Thr172 expression in livers of WT or Ulk1 −/− mice following starvation (n = 6). C. Representative immunoblots and densitometric analysis of P-AMPKγ1 Ser260/Thr262 expression in kidneys of mice subjected to sham operation or 5/6 nephrectomy to induce chronic kidney disease (CKD) (n = 8). P values ( * P < 0.05, ** P < 0.01) were calculated using a two-tailed unpaired t -test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A, B. Representative immunoblots (A) and densitometric analysis (B) evaluating the sensitivity of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or S260A/T262A mutant to 12h-AICAR treatment (n = 3). A. C. Crystal structure revealing the role of Ser260 and Thr262 in maintaining the structure of AMPKγ1. B. D. Fluorescence intensity of Mant-AMP (fluorescent dye) bound to purified trimeric AMPK protein consisting of AMPKγ1 WT or AMPKγ1 S260A/T262A with/without ULK1 (n = 5) P values ( * P < 0.05) were calculated using one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Mutagenesis, Fluorescence, Purification

Journal: bioRxiv

Article Title: ULK1-regulated AMP sensing by AMPK and its application for the treatment of chronic kidney disease

doi: 10.1101/2023.08.09.552390

Figure Lengend Snippet: A. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 complexed with AMPKγ1 wild-type (WT) or AMPKγ1 S260A/T262A treated with/without MK8722 (n = 3). B. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in NRK-52E cells transfected with control siRNA (scramble) or ULK1 siRNA (Si-Ulk1) with or without MK8722 treatment (n = 3). C. Representative immunoblots and densitometric analysis of P-AMPKα Thr172 , P- ACC Ser79 , and P-RAPTOR Ser792 expression in kidneys of WT or Ulk1 −/− mice treated intraperitoneally with or without MK8722 at 30 mg/kg BW (n = 5). P values ( * P < 0.05, ** P < 0.01) were calculated using the two-tailed unpaired t -test or a one-way analysis of variance with Tukey’s honestly significant difference test. Data are presented as the mean ± standard error of the mean. For detailed data, statistical analysis, and exact P values, refer to the Source Data file .

Article Snippet: For protein expression and purification experiments, mouse cDNA was isolated from AMPKγ1 and S260A/T262A-mutant AMPKγ1 by PCR using the aforementioned AMPKγ1, pAMPK- alpha1-full-length (#27297, Addgene), and AMPK-β1-FLAG (#40602, Addgene) vectors.

Techniques: Western Blot, Expressing, Transfection, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Vinblastine resets tumor-associated macrophages toward M1 phenotype and promotes antitumor immune response

doi: 10.1136/jitc-2023-007253

Figure Lengend Snippet: VBL targets NF-κB-Cyba to reprogram macrophages. (A) Gene ontology analysis of RNA-sequencing data collected from BMDMs with or without VBL treatment for 24 hours. (B) A heatmap of genes related to the NFkB pathway from RNA-sequencing data collected from macrophages (Mϕ in vivo), sorted and analyzed from Vehicle/VBL treated tumors. The heat map was created based on the normalized FPKM of genes in the NF-κB pathway. (C) Venn diagrams of upregulated genes enriched in ROS production pathway. (D) Gene expression of Mmp8, Cyba, Cd36, Dcxr, and Alox5 in macrophages sorted from MC38 tumors. n=4. (E) Representative western blot against p-NF-κB, NF-κB, p22 phox , and β-actin in macrophages sorted from indicated tumor samples. (F) Fold change of p-NF-κB, NF-κB, and p22 phox protein level in panel (E). n=3. (G) Representative western blot against p22 phox and β-actin in BMDMs. (H) Fold change of p22 phox protein levels in panel (G). n=3. (I) Flow cytometry analysis for percentages of M1 (F4/80 + CD11b + CD86 + ) and M2 (F4/80 + CD11b + CD206 + ) BMDMs after the corresponding treatment for 24 hours. n=6. (J) Representative flow cytometry results of T cell proliferation after co-culture with BMDMs for 72 hours. (K) Statistical results of T cell proliferation in panel (J). n=6. Data are presented as mean±SD. P values were determined by a two-tailed Student’s t-test. *p<0.05, **p<0.01, ***p<0.001. BMDMs, bone marrow-derived macrophages; ROS, reactive oxygen species; VBL, vinblastine.

Article Snippet: The following commercial antibodies were used: anti-phospho-NF-κB (Cell Signaling Technology, 3033S), anti-NF-κB (Cell Signaling Technology, 8242S), anti-p22 phox (Cell Signaling Technology, 27 297S), anti-β-actin (Sigma A1978), anti-mouse (Abcam ab6728), anti-Rabbit (Abcam ab6721).

Techniques: RNA Sequencing Assay, In Vivo, Expressing, Western Blot, Flow Cytometry, Co-Culture Assay, Two Tailed Test, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Vinblastine resets tumor-associated macrophages toward M1 phenotype and promotes antitumor immune response

doi: 10.1136/jitc-2023-007253

Figure Lengend Snippet: Working model for VBL-mediated antitumor immune response. VBL phosphorylates and activates the transcription factor NF-κB in TAMs, upregulating cyba which encodes p22 phox protein expression. Cytosolic ROS and MitoROS levels were increased by p22 phox -catalyzed synthesis. ROS further facilitated the entry of transcription factor TFEB into the nucleus, accelerating lysosome biogenesis and activation, and thus endowing TAMs with a higher phagocytic capacity. ROS also repolarized TAMs into the M1-like phenotype and abolished their suppressive activity on CD8 + T cells. This graph model was created with biorender.com. ROS, reactive oxygen species; TAMs, tumor-associated macrophage; VBL, vinblastine.

Article Snippet: The following commercial antibodies were used: anti-phospho-NF-κB (Cell Signaling Technology, 3033S), anti-NF-κB (Cell Signaling Technology, 8242S), anti-p22 phox (Cell Signaling Technology, 27 297S), anti-β-actin (Sigma A1978), anti-mouse (Abcam ab6728), anti-Rabbit (Abcam ab6721).

Techniques: Expressing, Activation Assay, Activity Assay