Journal: Cancer Biology & Medicine

Article Title: Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy

doi: 10.20892/j.issn.2095-3941.2020.0262

Figure Lengend Snippet: HSP90 inhibits the autophagic degradation of DDX5 protein. (A) The mRNA level of DDX5 after HSP90 knockdown with shRNA (Hsp90-K.D.), tested by qRT-PCR, using GAPDH as a reference gene. (B) The protein level of DDX5 after HSP90 knockdown (Hsp90-K.D.) (mean ± SD, * P = 0.0238). (C) The mRNA level of DDX5 after STA9090 treatment, tested by qRT-PCR, using GAPDH as a reference gene. (D) The protein level of DDX5 after STA9090 treatment (mean ± SD, * P = 0.0294). (E) The inhibitory effect of DDX5 expression in the presence of STA9090, an inhibitor of HSP90, was ameliorated in HepG2 cells treated with an inhibitor for autophagy, MRT68921, but not by MG132, an inhibitor of proteasomes. (F) Confocal microscopy of intracellular localization analysis of DDX5 and autophagosomes after treatment with STA9090, chloroquine, and AICAR (Scale bar: 10 μm). Chloroquine: inhibitor of lysosome; AICAR: agonist of autophagy. Arrow: DDX5 combined with autophagosomes.

Article Snippet: MG132 (ubiquitin-proteasome inhibitor, working concentration: 10 μM), MRT68921 (unc-51-like kinase 1 inhibitor to block autophagy, working concentration: 1 μM), 5-amino-4-imidazolecarboxamide ribofuranoside (AICAR, autophagy inducer, working concentration: 100 μM), and STA9090 (heat shock protein 90, HSP90, inhibitor, working concentration: 10 μM) were purchased from Shanghai Selleck Chemicals (Shanghai, China).

Techniques: Knockdown, shRNA, Quantitative RT-PCR, Expressing, Confocal Microscopy

Journal: Bone Research

Article Title: Simultaneous augmentation of muscle and bone by locomomimetism through calcium-PGC-1α signaling

doi: 10.1038/s41413-022-00225-w

Figure Lengend Snippet: Sequential screening identified 17b (locamidazole: LAMZ) as a candidate therapeutic drug for impaired locomotion. a Primary screening for extracting compounds that enhance the proliferation and/or differentiation of myocytes. The relative proliferation and myotube formation are plotted in the scattergram. Each dot represents the corresponding compound. Compounds A–H are as follows: A, AICAR; B, monastrol; C, PARP inhibitor XII; D, AMI-5; E, 17b; F, IWR-1-endo; G, apocynin; and H, L-165,041. The dots depicted in red underwent further screening. b Representative immunocytofluorescence images of C2C12 cells. Myosin heavy chain (red); nuclei (blue). c mRNA expression of myogenic genes. d Secondary screening for extracted compounds enhancing osteoblastogenesis based on alkaline phosphatase (ALP) activity. e Representative ALP staining images of osteoblasts. f Representative images of the mineralization of osteoblasts. g mRNA expression of osteoblastic genes. h Tertiary screening for extracting compounds that suppress osteoclastogenesis based on Ctsk expression indicated by LacZ activity. i Representative images of tartrate–resistant acid phosphatase (TRAP) staining. j Number of TRAP + multinucleated cells. N.D. not detected. k Apoptosis ratio of osteoclasts detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay. The data of the effects of 17b were obtained from 3 independent experiments with replicates of three wells. Scale bar, 100 μm. Statistical analyses were carried out using one-way ANOVA followed by Dunnett’s multiple-comparison test or Brown–Forsythe ANOVA test and Dunnett’s T3 test. The error bars show the mean ± s.e.m. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.000 1

Article Snippet: The compounds used in the experiments other than primary screening were purchased individually: AICAR (Calbiochem), monastrol (Santa Cruz Biotechnology), PARP inhibitor XII (Calbiochem), AMI-5 (Calbiochem), 17b (Calbiochem), IWR-1-endo (Calbiochem), apocynin (Santa Cruz Biotechnology), L-165, 041 (Abcam Limited), linifanib (Cayman Chemical Company), SR18292 (Cayman Chemical Company), FK506 (Cayman Chemical Company) and KN93 (Cayman Chemical Company).

Techniques: Expressing, Activity Assay, Staining, End Labeling, TUNEL Assay

Journal: Frontiers in Physiology

Article Title: Empagliflozin Inhibits Hepatic Gluconeogenesis and Increases Glycogen Synthesis by AMPK/CREB/GSK3β Signalling Pathway

doi: 10.3389/fphys.2022.817542

Figure Lengend Snippet: 5-Aminoimidazole-4-carboxamide1-b-D-ribofuranoside (AIACR) and compound C could regulate the AMPK signalling pathway under empagliflozin treatment in vitro . HL7702 cells were treated with the AMPK agonist AICAR and empagliflozin and PA. (A) Glucose production levels ( n = 3 samples/group). (B) PEPCK and G6Pase activities ( n = 3 samples/group). (C) Relative protein expression levels of p-AMPK, AMPK, p-GSK3β, GSK3β, p-CREB, CREB, PEPCK, and G6PASE were determined by western blotting. (D) Protein quantitative statistics ( n = 3 samples/group). * P < 0.05, PA vs. PA+Empa; # P < 0.05, PA vs. PA+AIACR. HL7702 cells were treated with the AMPK inhibitor compound C and empagliflozin and PA. (E) Glucose production levels ( n = 3 samples/group). (F) PEPCK and G6Pase activities ( n = 3 samples/group). (G) Relative protein expression levels of p-AMPK, AMPK, p-GSK3β, GSK3β, p-CREB, CREB, PEPCK, and G6PASE were determined by western blotting. (H) Protein quantitative statistics ( n = 3 samples/group). * P < 0.05, PA vs. PA+Empa; # P < 0.05, PA+Empa vs. PA+Empa+Compound C.

Article Snippet: In the 5-Aminoimidazole-4-carboxamide1-b-D-ribofuranoside (AIACR) treatment experiment, HL7702 cells were divided into four groups: normal control group, PA intervention group, PA combination with empagliflozin intervention group, and PA combination with 10 μmol/L AIACR (MCE, HY-13417, Trenton, NJ, United States) intervention group.

Techniques: In Vitro, Expressing, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: Increased Afterload Augments Sunitinib-Induced Cardiotoxicity in an Engineered Cardiac Microtissue Model

doi: 10.1016/j.jacbts.2017.12.007

Figure Lengend Snippet: Characterizing Changes in Mitochondrial Function and Cell Energetics With Sunitinib Treatment (A) Flow cytometry histogram showing levels of tetramethylrhodamine (TMRM). Neonatal rat ventricular myocytes grown in flat culture were treated with 0.1% dimethyl sulfoxide (vehicle), 1 μmol/l sunitinib, or 50 μmol/l carbonyl cyanide m -chlorophenyl hydrazine (CCCP) for 30 min (CCCP) or 4 h (dimethyl sulfoxide and sunitinib) and then were labeled with 10 nmol/l TMRM. (B) Time-dependent decreases in mitochondria membrane potential following treatment with 1 μmol/l sunitinib. Neonatal rat ventricular myocytes grown in flat culture were treated with 0.1% dimethyl sulfoxide or 1 μmol/l sunitinib for 30 min to 24 h and labeled with TMRM to assess mitochondria membrane potential. *p < 0.05 (n = 3 experiments). (C) Decreases in adenosine triphosphate (ATP) levels in neonatal rat ventricular myocytes following 24 h treatment with 1 μmol/l sunitinib. *p < 0.05 (n = 3 experiments). (D) Upstream activation of adenosine monophosphate-activated protein kinase with molecule 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) did not reverse sunitinib-induced caspase activation in rat cardiac microtissues (n = 2 experiments). NS = not significant.

Article Snippet: In a subset of experiments, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (Sigma Aldrich) was administered concurrently with sunitinib at a concentration of 1 mmol/l.

Techniques: Flow Cytometry, Labeling, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Metformin Protects against NMDA-Induced Retinal Injury through the MEK/ERK Signaling Pathway in Rats

doi: 10.3390/ijms22094439

Figure Lengend Snippet: The AMPK activator, AICAR, attenuates NMDA-induced cell loss in the ganglion cell layer (GCL). The number of cells in the GCL and thickness of the inner plexiform layer (IPL) were measured seven days after intravitreal injection. ( A ): Saline ( a ); NMDA (200 nmol) + vehicle ( b ); NMDA (200 nmol) + AICAR (20 nmol) ( c ); NMDA (200 nmol) + AICAR (50 nmol) ( d ). Scale bar, 30 µm. ( B ): Bar graph showing the number of cells in the GCL and thickness of the IPL. n = 5–6. ** p < 0.01; Kruskal–Wallis test.

Article Snippet: In the second series of experiments, we examined the effect of AICAR (Tocris Bioscience), an activator of AMPK, on NMDA (200 nmol)-induced retinal neuronal cell loss in the GCL.

Techniques: Injection, Saline