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: 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

Journal: Cancer Research

Article Title: SAICAR Drives T Regulatory Cell Differentiation and FOXP3 Maintenance to Promote Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-25-4373

Figure Lengend Snippet: SAICAR promotes TGFβ–SMAD3 signaling by binding PPM1A. A, Summary of gene expression related to TGFβ–SMAD3 signaling and Jak–STAT5 signaling from RNA-seq of naïve CD4 + T from WT mice, which were cultured under Treg differentiation with or without SAICAR for 48 hours ( n = 3). B and C, Statistics showing Foxp3 mRNA expression by qPCR of naïve CD4 + T cells sorted from Adsl f/f - Cd4 Cre or Adsl f/f ( B ) and Paics f/f - Cd4 Cre or Paics f/f ( C ) mice under Treg differentiation conditions. Experiments were repeated four times. D and E, ChIP assay to measure the combination of SMAD3 and Foxp3 enhancer in naïve CD4 + T cells from Adsl f/f - Cd4 Cre or Adsl f/f ( D ) and Paics f/f - Cd4 Cre or Paics f/f ( E ) mice under Treg differentiation. Experiments were repeated four times. Homologous IgG and naïve CD4 T cells from Adsl f/f ( D ) or Paics f/f ( E ) mice were used for negative control. F, Statistics showing the percentage of Th17 cells in CD4 + T cells after naïve CD4 + T cells were present in Th17 differentiation conditions with or without SAICAR. Experiments were repeated five times. G, Statistics showing the percentage of Treg cells in CD4 + T cell of naïve CD4 + T cells, which were present in Treg differentiation conditions under SIS (SMAD3 inhibitor) and STAT5-IN-1 (STAT5 inhibitor) with or without SAICAR. Experiments were repeated three times. H, Naïve CD4 + T cells isolated from indicated mice, which were present in Treg differentiation conditions, and the percentage of Treg cells in CD4 + T cells was analyzed. Experiments were repeated three times. I and J, MC38 tumor cells were implanted into indicated mice, and the tumor growth curves ( n = 5; I ) and the percentage of Treg cells in CD4 + T cells are shown ( n = 5; J ). K, Whole-cell extracts (WCE) of naïve CD4 + T cells were collected and subjected to coimmunoprecipitation assays. L, Docking model of SAICAR and PPIMA (left) and interactions of SAICAR with PPM1A (right) in its pocket. Molecular forces implied in the interactions are hydrogen bonds with Arg33, Asp66, and Ala192 and ionic bonds with His62, Lys165, and Arg186. M, The binding of SAICAR to purified PPM1A was analyzed by SPR. The apparent dissociation constant K d is 244 μmol/L. N and O, PPM1A-KD naïve CD4 + T cells were created by lentivirus. Statistics showing the mRNA expression of Foxp3 ( n = 3; N ) and the percentage of Treg cells in CD4 + T cells ( n = 3; O ) by qPCR and flow cytometry after presence in the Treg differentiation condition. Error bars, means ± SD. Statistical significance was assessed using the Student two-tailed t test, one-way ANOVA, and Tukey HSD test. FDR was used in A . *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: Material used for the experiment was obtained from the following manufacturers: SAICAR (MedChemExpress), PPM1A (MedChemExpress), ultrapure water, 10 mmol/L Glycine-HCl (pH 2), IPA (MedChemExpress), NHS (Thermo Fisher Scientific), EDC (Thermo Fisher Scientific), and Sensor Chip COOH (Nicoya).

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Cell Culture, Expressing, Negative Control, Isolation, Purification, Flow Cytometry, Two Tailed Test

Journal: Molecular biology reports

Article Title: L-Arginine alleviates heat stress-induced intestinal epithelial barrier damage by promoting expression of tight junction proteins via the AMPK pathway.

doi: 10.1007/s11033-019-05090-1

Figure Lengend Snippet: Fig. 5 l-Arginine activates the AMPK pathway after heat stress in rat intestines. a Expression of heat shock protein-related genes in intestines measured by RT-qPCR. b Expression levels of AMPKα and p-AMPKα pro- teins assessed by western blot- ting. l-Arginine increased the expression level of p-AMPKα in intestines. c mRNA expres- sion level of LKB1 assessed by RT-qPCR. d mRNA and protein expression levels of ZO-1 and claudin-1 assessed by RT-qPCR and western blotting, respectively. e The expression level of ZO-1 protein were evaluated in jejunal specimens by immunohistochemistry. f The expression of claudin-1 protein in rats jejunum tissues. Data are presented as the mean ± SD, n = 8. †C control, HS heat stress, LA l-arginine + heat stress, AR AICAR + heat stress, DD dorsomorphin dihydrochlo- ride + heat stress. a–eBars with letters indicate significant differ- ences among various treatments (P < 0.05)

Article Snippet: For heat stress, after 48 h of incubation, IEC-6 cells in HS and ARG groups were exposed to 42 °C with 5% CO2 for 1.5 h. For AMPK activation and inhibition experiments, 0.8 mM AICAR (13417, MCE, NJ, USA) or 5 μM dorsomorphin dihydrochloride (13418, MCE) were applied to IEC-6 cells at 70–80% confluence.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Control