Journal: bioRxiv

Article Title: The role of Cycloastragenol at the intersection of Nrf-2/ARE, telomerase, and proteasome activity

doi: 10.1101/2022.03.11.483898

Figure Lengend Snippet: (a) HEKn cells were transfected with either oligo duplex specific to Nrf-2, hTERT, or Control siRNA. After 48 h later, cells were harvested, and total protein levels of hTERT, Nrf-2, β1, and β5 were determined by IB. GAPDH was used as a loading control. ( b, c, d ) HEKn cells were transfected with either Nrf-2 or Control siRNA (each of them 2 nM). After 48 h later, cells were treated with indicated concentrations of CA for 24 h. ( b ) The total protein levels of Nrf-2, hTERT, β1, and β5 were determined by IB. ( c ) The telomerase enzyme activity was determined by using ELISA (n:3, *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.005). ( d ) The proteasomal β1 and β5 subunit activities were determined by using fluorogenic substrates. The activity data were normalized to cellular total protein level and presented as a fold change compared to control cells treated with DMSO used as a solvent control (n:3, *p ≤ 0.05, **p. ≤ 0.001, ***p ≤ 0.005).

Article Snippet: When HEKn cells reached at confluency of 60-70%, cells were transfected with 2 and 15 nM siRNA against Nrf-2 (Origene, USA, SR321100) and hTERT (Origene, USA, SR322002), respectively.

Techniques: Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The role of Cycloastragenol at the intersection of Nrf-2/ARE, telomerase, and proteasome activity

doi: 10.1101/2022.03.11.483898

Figure Lengend Snippet: HEKn cells were transfected with either oligo duplex specific to hTERT or Control siRNA. After 48 h later, cells were treated with CA for 24 h. ( a ) The total protein levels of Nrf-2, hTERT, β1, and β5 were determined by IB. ( b ) Before performing the nuclear Nrf-2 activity, cellular fractionation was performed. The Nrf-2 activity was determined by ELISA. The Nuclear activity data was normalized to nuclear protein level and presented as a fold change compared to control cells treated with DMSO used as solvent control. Error bars are presented as standard deviations (n = 3; *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.005). ( c ) The proteasomal β1 and β5 subunit activities were evaluated via fluorogenic substrates. The data was normalized to cellular total protein level and presented as a fold change compared to control cells treated with DMSO used as solvent control. Error bars are presented as standard deviations (n = 3; *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.005).

Article Snippet: When HEKn cells reached at confluency of 60-70%, cells were transfected with 2 and 15 nM siRNA against Nrf-2 (Origene, USA, SR321100) and hTERT (Origene, USA, SR322002), respectively.

Techniques: Transfection, Activity Assay, Cell Fractionation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The role of Cycloastragenol at the intersection of Nrf-2/ARE, telomerase, and proteasome activity

doi: 10.1101/2022.03.11.483898

Figure Lengend Snippet: ( a, b, c ) HEKn cells were transfected with Nrf-2, hTERT, and Control siRNA. After 48 h later, cells were pre-treated with the desired concentration of CA for 8 h, then treated with 6-OHDA for 16 h. The cell viability was assessed by MTT reagent. The absorbance value of cells treated with DMSO as a solvent control for each experiment was considered 100%, and the viabilities of others were calculated. This assay was performed by triplicate samples. Student’s t-test was used to determine the significance of the differences Error bars are presented as standard deviations (n = 3; *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.005).

Article Snippet: When HEKn cells reached at confluency of 60-70%, cells were transfected with 2 and 15 nM siRNA against Nrf-2 (Origene, USA, SR321100) and hTERT (Origene, USA, SR322002), respectively.

Techniques: Transfection, Concentration Assay