Journal: Aging Cell

Article Title: Ribosylation triggering Alzheimer’s disease-like Tau hyperphosphorylation via activation of CaMKII

doi: 10.1111/acel.12355

Figure Lengend Snippet: AGE formation, viability of cells, and Tau phosphorylation following the treatment of D-ribosylation in N2a cells. Different concentrations of D-ribose (final concentration 0, 5, 10, 20, 50, and 100 m m ) were added to N2a cells before incubation for 24 h. AGEs were detected by Western blotting (panel a,a′, 20 μg cell lysate loaded). Cell viability was measured using CCK-8 assay (panel c). D-ribose (final concentration 10 m m ) was added to N2a cells and incubated for different time lengths (0, 4, 8, 24, and 48 h), and AGE production (panel b,b′) and cell viability (panel d) were measured. n = 5. N2a cell lysate extracts were separated by 12% SDS-PAGE, and levels of phosphorylated Tau and AGEs were determined by Western blotting using antibodies directed at pThr181, pSer214, pSer396, or total Tau at different concentrations of D-ribose (panel e) and for different cell culture times (panel f). Panels e′ and f′ show quantitative analyses for data from panel e and f, respectively. n = 6. For each phospho-epitope, relative immunoreactive band intensities are expressed as ratios in comparison with total Tau. For total Tau, the relative values are expressed as a ratio to β-actin. Data are expressed as mean ± SD; * and ** denote P < 0.05 and P < 0.01 vs. control, respectively.

Article Snippet: Mouse neuroblastoma cell line Neuro 2A (N2a) cells (obtained from Cell Resource Center, Beijing, Amresco, Solon, OH, China) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 IU mL −1 penicillin and 100 μg mL −1 streptomycin and 10% fetal bovine serum at 37 °C in a humidified incubator maintained at 5% CO 2 .

Techniques: Phospho-proteomics, Concentration Assay, Incubation, Western Blot, CCK-8 Assay, SDS Page, Cell Culture, Comparison, Control

Journal: Aging Cell

Article Title: Ribosylation triggering Alzheimer’s disease-like Tau hyperphosphorylation via activation of CaMKII

doi: 10.1111/acel.12355

Figure Lengend Snippet: Changes in the levels of Tau kinases in N2a cells following the treatment of D-ribose. N2a cells were first treated with different concentrations of D-ribose for 24 h. Then, cell lysate extracts were separated by 12% SDS-PAGE and levels of Tau kinases were determined using antibodies directed at activated or total kinases as follows: phospho-ERK/ total ERK (panel a), phospho-p38/ total p38 (panel b), phospho-JNK/ total JNK (panel c), phospho-GSK-3β/ total GSK-3β (panel d), phospho-CaMKII/ CaMKII (panel e), and p35/25 (panel f). N2a cells were incubated with 10 m m D-ribose for different lengths of time (0, 4, 8, 24, and 48 h). N2a cell lysate extracts were separated by SDS-PAGE, and levels of Tau kinases were determined using antibodies directed at activated or total kinases as follows: phospho-ERK/ total ERK (panel g), phospho-p38/ total p38 (panel h), phospho-JNK/ total JNK (panel i), phospho-GSK-3β/ total GSK-3β (panel j), phospho-CaMKII/ CaMKII (panel k), and p35/25 (panel l). Relative immunoreactive band intensities are expressed as a ratio to control. n = 3. Data are expressed as mean ± SD; ** denotes P < 0.01 vs. control.

Article Snippet: Mouse neuroblastoma cell line Neuro 2A (N2a) cells (obtained from Cell Resource Center, Beijing, Amresco, Solon, OH, China) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 IU mL −1 penicillin and 100 μg mL −1 streptomycin and 10% fetal bovine serum at 37 °C in a humidified incubator maintained at 5% CO 2 .

Techniques: SDS Page, Incubation, Control

Journal: Aging Cell

Article Title: Ribosylation triggering Alzheimer’s disease-like Tau hyperphosphorylation via activation of CaMKII

doi: 10.1111/acel.12355

Figure Lengend Snippet: Effect of CK59 or aminoguanidine (AG) on D-ribose-induced Tau hyperphosphorylation. CK59 (10, 100 μ m ) was added to N2a cells together with D-ribose (10 m m ), and cells were cultured for 24 h. Extracts of N2a cell lysate were separated by SDS-PAGE, and levels of Tau phosphorylation were determined using antibodies directed at Tau pThr181, pSer214, pSer396, and total Tau (panel a). Data from panel (a) were quantitatively analyzed (panel b). AG (1, 5 m m ) was added with D-ribose (10 m m ) to N2a cells and cultured for 24 h. N2a cell lysate extracts were separated by SDS-PAGE, and levels of Tau phosphorylation and AGEs were determined using antibodies directed at Tau pThr181, pSer214, pSer396, total Tau (Tau-5), or AGEs (panel c). The same data from panel (c) were quantitatively analyzed (panels d, e). For each phospho-epitope, relative immunoreactive band intensities are expressed as a ratio to total Tau. For total Tau and AGEs, relative values are expressed as the ratio to β-actin. n = 3. Data are expressed as mean ± SD; *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001 vs. control, respectively. # denotes P < 0.05 vs. D-ribose treatment.

Article Snippet: Mouse neuroblastoma cell line Neuro 2A (N2a) cells (obtained from Cell Resource Center, Beijing, Amresco, Solon, OH, China) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 IU mL −1 penicillin and 100 μg mL −1 streptomycin and 10% fetal bovine serum at 37 °C in a humidified incubator maintained at 5% CO 2 .

Techniques: Cell Culture, SDS Page, Phospho-proteomics, Control