Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of Linc00173 increases BCL2 mRNA stability via the miR-1275/PROCA1/ZFP36L2 axis and induces acquired cisplatin resistance of lung adenocarcinoma

doi: 10.1186/s13046-022-02560-6

Figure Lengend Snippet: Low expression of LINC00173 in DDP-resistant LUAD attributes to the negative regulation of c-Myc. a. Predicted c-Myc binding site in the promoter region of LINC00173. b. Transfection of c-Myc plasmids suppressed LINC00173 expression in A549 and PC9 cells. Student’s two-tailed t-test, ** P < 0.01; *** P < 0.001; **** P < 0.0001. c. ChIP assay and agarose gel electrophoresis verified the binding of c-Myc to LINC00173 promoter in A549 and A549-DDP cells. Student’s two-tailed t-test, ** P < 0.01; **** P < 0.0001. d. Luciferase reporter assay further validated c-Myc was directly binding to LINC00173 promoter. Student’s two-tailed t-test, * P < 0.05; ** P < 0.01; ns, no significance. e. Western blot analysis of PI3K, p-PI3K, AKT, p-AKT and c-Myc expression in A549 and A549-DDP cells. GAPDH was used as a loading control. f. Western blot detection of p-PI3K, p-AKT, c-Myc, PROCA1 and ZFP36L2 expression in A549-DDP and PC9-DDP cells after treated with PI3K inhibitor LY294002. GAPDH was used as a loading control. g. The expression level of LINC00173 was obviously increased in A549-DDP and PC9-DDP cell lines after treated with LY294002. h. The IC 50 values to cisplatin were detected in A549 and PC9 cells transfected with a gradient concentration of c-Myc plasmids. Student’s two-tailed t-test, ** P < 0.01; **** P < 0.0001. Error bars, mean ± SD

Article Snippet: The antibodies against the following proteins were used: Caspase-3 (Cell Signaling Technology #14220, 1:1000), PARP (Cell Signaling Technology #9542, 1:1000), BCL2 (Cell Signaling Technology #15071, 1:1000), PROCA1 (Biorbyt #orb1703, 1:1000), ZFP36L2 (Santa Cruz #sc-365908, 1:1000), c-Myc (Cell Signaling Technology #9402, 1:1000), phosphor-Akt (Cell Signaling Technology #4060, 1:1000), Akt (Cell Signaling Technology #4691, 1:1000), phosphor-PI3K (Cell Signaling Technology #17366, 1:1000), and PI3K (Cell Signaling Technology #4292, 1:1000).

Techniques: Expressing, Binding Assay, Transfection, Two Tailed Test, Agarose Gel Electrophoresis, Luciferase, Reporter Assay, Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: Hepatic Glucose Intolerance Precedes Hepatic Steatosis in the Male Aromatase Knockout (ArKO) Mouse

doi: 10.1371/journal.pone.0087230

Figure Lengend Snippet: Protein phosphorylation analyses of Akt and AMPK levels were performed on protein extracted from insulin stimulated ( a ) gonadal adipose tissue, ( b ) gastrocnemius muscle, ( c ) liver in insulin stimulated liver of 6 month-old male wildtype (WT) and aromatase knockout (KO) and 2.5 µg/day estrogen-replaced KO (KOE) mice. Expression data from 6–8 samples per genotype are shown, and presented from replicate analysis as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001 versus expression in age-matched WT samples and # p<0.05, ### p<0.001 versus KO samples.

Article Snippet: Primary antibodies diluted in 1% BSA in Tris Buffered Saline with 1% Tween were incubated overnight at 4°C (mouse anti-Phosphor-Akt (Ser473), rabbit anti-Akt, rabbit anti-phosphor-AMPK (Thr172), rabbit anti-AMP alpha (Cell Signaling Technologies, Beverly MA, USA).

Techniques: Knock-Out, Expressing

Journal: PLoS ONE

Article Title: Mu-Opioid Receptors Transiently Activate the Akt-nNOS Pathway to Produce Sustained Potentiation of PKC-Mediated NMDAR-CaMKII Signaling

doi: 10.1371/journal.pone.0011278

Figure Lengend Snippet: 10 nmol morphine was icv-injected into CD1 mice. At various intervals post-injection, the mice were sacrificed and the PAG structures were collected. The presence of Akt and nNOS in the PAG synaptosomal membrane was determined. Also, the association of these proteins with the MOR was evaluated. At each time point studied, the PAG structures from four to six mice were pooled. MOR proteins were immunoprecipitated and the co-precipitated Akt and nNOS were evaluated. Immunosignals (average optical density of the pixels within the object area/mm 2 ; Quantity One Software, BioRad) were expressed as the change relative to the control animals (attributed an arbitrary value of 1). Each data point represents the mean of three assays performed on PAG samples obtained from independent groups of mice. Data are presented as the area below the curve (Sigmaplot v11), the upper curve indicates the +SEM. For comparison, we have superimposed the control levels of the pair of curves being compared. Upper panel: The effect of morphine on the presence of Akt in the synaptosomal PAG membrane and the MOR environment. The presence of activating Thr308 and Ser473 phosphorylations in the membrane-associated Akt. Insets: Representative western blots of these data. An absence of phosphorylation in Akt recruited to the MOR was shown but not plotted. MOR-rcted stands for MOR-recruited. Lower panel : A similar study of nNOS was carried out. Enzyme levels associated with PAG synaptosomal membranes and MORs were determined. The presence of Akt-activating Ser1417 phosphorylation and CaMKII-inactivating Ser847 phosphorylation were also determined in membrane-associated nNOS. Insets: Representative nNOS western blots are shown. nNOS that was recruited to the MOR environment lacked Ser1417 and Ser847 phosphorylation.

Article Snippet: Akt (1∶1000; Cell Signaling 4691); Akt phospho-Thr308 (1∶1000; Cell Signaling 2965); Akt phosphor-Ser473 (1∶1000; Cell Signaling 4060); nNOS (1∶1000; Santa Cruz SC-1025); nNOS phospho-Ser1417 (2 µg/mL; Abcam ab5583); nNOS phospho-Ser847 (1 µg/mL; Abcam ab16650) and Actin (1∶3000; Stressgen, CSA-400).

Techniques: Injection, Immunoprecipitation, Software, Western Blot