Journal: Pharmaceutical Biology

Article Title: Deoxyshikonin isolated from Arnebia euchroma inhibits colorectal cancer by down-regulating the PI3K/Akt/mTOR pathway

doi: 10.1080/13880209.2019.1626447

Figure Lengend Snippet: Effects of deoxyshikonin on Akt/PI3K/mTOR signalling pathway in HT29 cells. (A) PI3K, p -PI3K, Akt, p -Akt308 and mTOR proteins in HT29 and DLD-1 cell lines were determined with specific antibodies. β-Actin was used as loading control. The relative intensity of each protein was normalized with β-actin. (B) The expression levels of Akt, PI3K, mTOR mRNA in HT29 and DLD-1 cell lines were measured by RT-PCR. Data were results of three independent experiments with mean ± SD. * p < 0.05, ** p < 0.001, *** p < 0.0001 and **** p < 0.00001 versus control (0 μg/mL).

Article Snippet: Briefly, the PVDF membrane containing the target protein was washed with TBST solution, incubated with 5% nonfat milk solution and primary antibodies: PI3K, p-PI3K, Akt, p-Akt308, mTOR and β-actin (Cell Signaling Technology, Boston, MA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Pharmaceutical Biology

Article Title: Deoxyshikonin isolated from Arnebia euchroma inhibits colorectal cancer by down-regulating the PI3K/Akt/mTOR pathway

doi: 10.1080/13880209.2019.1626447

Figure Lengend Snippet: Deoxyshikonin mediated HT29 cells viability, cell apoptosis and cycle arrest depended on the PI3K/Akt/mTOR signalling pathway. HT29 cells were pretreated with LY294002, or NVP-BEZ235, or MK-2206, then co-treated with 50 μg/mL deoxyshikonin for further 48 h. (A) Cell viability was assessed by MTT assay; (B) cell apoptosis and cell cycle arrest were detected by flow cytometry; (C) Akt and p -Akt308 proteins were determined by Western blotting. Data were presented as mean ± SD of three independent tests. * p < 0.05, ** p < 0.001, *** p < 0.0001 and **** p < 0.00001 versus control (0 μg/mL).

Article Snippet: Briefly, the PVDF membrane containing the target protein was washed with TBST solution, incubated with 5% nonfat milk solution and primary antibodies: PI3K, p-PI3K, Akt, p-Akt308, mTOR and β-actin (Cell Signaling Technology, Boston, MA).

Techniques: MTT Assay, Flow Cytometry, Western Blot

Journal: Pharmaceutical Biology

Article Title: Deoxyshikonin isolated from Arnebia euchroma inhibits colorectal cancer by down-regulating the PI3K/Akt/mTOR pathway

doi: 10.1080/13880209.2019.1626447

Figure Lengend Snippet: Anticancer activity of deoxyshikonin in vivo via PI3K/Akt/mTOR pathway. BALB/c nude mice were injected with DLD-1 cells into the subcutaneous tissue of the right auxiliary region. Xenograft model was established when tumours reached an average size of 62.5 mm 3 , and the treatments were given different drugs by intraperitoneal injection for a total of 13 days: control, DMSO (1% DMSO, every two days), deoxyshikonin (20 mg/kg in 1% DMSO, every two days), irinotecan (66.7 mg/kg, every four days). (A) Body weights and tumour volumes were measured once daily; (B) images, tumour weights and H&E staining (200× magnification) were measured after 13 days of treatment; (C) the proteins of tumour tissues were blotted with antibodies against indicated proteins (PI3K, p-PI3K, Akt, p-Akt308 and mTOR), and β-actin was blotted as a loading control. Data are presented as mean ± SD. * p < 0.05, ** p < 0.001, *** p < 0.0001 and **** p < 0.00001 versus control.

Article Snippet: Briefly, the PVDF membrane containing the target protein was washed with TBST solution, incubated with 5% nonfat milk solution and primary antibodies: PI3K, p-PI3K, Akt, p-Akt308, mTOR and β-actin (Cell Signaling Technology, Boston, MA).

Techniques: Activity Assay, In Vivo, Injection, Staining

Journal: eLife

Article Title: ACE2 pathway regulates thermogenesis and energy metabolism

doi: 10.7554/eLife.72266

Figure Lengend Snippet: Primary brown adipocytes were isolated, cultured, and treated with Ang-(1-7) (10 –6 M) for 24 hr, Akt inhibitor MK2206 (20 μM) for 24 hr, PKA inhibitor H89 (30 μM) for 24 hr, or adenylylcyclase inhibitor SQ-22536 (10 μM) for 24 hours. ( A, B ) Representative western blots showing the changes of p-Akt308 and Akt in BAT of Ace2 -/y ( A ) and Lepr db/db + Ace2 mice ( B ) exposure at 4 °C (n = 3/each group). ( C ) Representative western blots showing the Akt, UCP1 and forkhead box protein O 1 (FoxO1) Changes (n = 3/each group). ( D ) Relative mRNA levels of thermogenic and mitochondrial genes (n = 4–6/each group). ( E ) Continuous measurement of oxygen consumption rate (OCR) in Ang-(1-7) and MK2206 treated primary brown adipocytes. Oxygen consumption was performed under basal conditions, following the addition of oligomycin (1 μM), the pharmacological uncoupler FCCP (1 μM) or the Complex III and I inhibitor antimycin A and rotenone (0.5 μM) (n = 3–4/each group). ( F, G ) Representative western blots showing the p-PKA and PKA changes in BAT of Ace2 -/y ( F ) and Lepr db/db + Ace2 mice ( G ) exposure at 4 °C (n = 3/each group). ( H ) Relative mRNA levels of thermogenic and mitochondrial genes (n = 4–6/each group). ( I ) Representative western blots showing the UCP1 and PGC1α changes (n = 3/each group). ( J ) Continuous measurement of OCR in Ang-(1-7) and H89-treated primary brown Adipocytes (n = 3–5/each group). Data represent mean ± SEM. *p< 0.05, **p < 0.01 vs GFP/WT group by Student’s t -test. *p < 0.05, **p < 0.01 vs control group, # p < 0.05, ## p < 0.01 vs Ang-(1-7) group by one-way ANOVA. ( K ) Mechanisms involved in ACE2 pathway activation-induced improvement of BAT function.

Article Snippet: Antibody , Anti-p-Akt308 (rabbit monoclonal) , Cell signaling , #13038 RRID: AB_2629447 , (1:1000).

Techniques: Isolation, Cell Culture, Western Blot, Activation Assay

Journal: eLife

Article Title: ACE2 pathway regulates thermogenesis and energy metabolism

doi: 10.7554/eLife.72266

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-p-Akt308 (rabbit monoclonal) , Cell signaling , #13038 RRID: AB_2629447 , (1:1000).

Techniques: Transfection, Construct, Sequencing, Software