human aortic smooth muscle cells haosmc  (Innoprot Inc)

Journal: Cells

Article Title: MitoQ Has Diverse Effects on H 2 O 2 -Induced Oxidative Stress and the NRF2 Signalling Pathway in Aortic Smooth Muscle Cells of Different Origins

doi: 10.3390/cells15161499

Figure Lengend Snippet: Comparison of baseline NFR2 protein levels ( A ) NFR2 activity ( B ) and NRF2 transcript expression without additional oxidative stress induction. ( A ) NRF2 protein levels were determined by ELISA and normalized to the total protein level of each sample. Data are shown as pg NRF2 per µg protein. Each color circle represents a normalized value derived from n = 4 AAA-SMC, n = 3 healthy VSMC samples and one immortalized cell line (IM), analysed in duplicate. ( B ) NRF2 binding activity to DNA was assayed from nuclear extracts by using a colorimetric NRF2 activity assay. Data were normalized to the protein content of the corresponding nuclear extracts. Each color circle represents a normalized value derived from n = 2 AAA-SMC, n = 3 healthy VSMC and one immortalized cell line (IM), analysed in duplicate. ( C ) NFE2L2 transcript expression was analysed by qPCR using specific primers. Data show the 2 −(ΔCt) values. Each colour circle represents a normalized value of n = 2 AAA-SMC (AAA), n = 3 healthy VSMC (healthy), and one immortalized cell line (IM), analysed in triplicate. Bars represent the mean ± SEM. Data were statistically analysed by ordinary one-way ANOVA and Tukey’s multiple comparison test; ns: not significant. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The immortalized VSMC line (iHAoSMC) was obtained from InnoProt ( P10456 -IM, Derio, Spain).

Techniques: Comparison, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Binding Assay

Journal: Cells

Article Title: MitoQ Has Diverse Effects on H 2 O 2 -Induced Oxidative Stress and the NRF2 Signalling Pathway in Aortic Smooth Muscle Cells of Different Origins

doi: 10.3390/cells15161499

Figure Lengend Snippet: Dose response of AAA-SMC ( A ), healthy VSMC ( B ), and iHAoSMC ( C ) to acute oxidative stress induced by H 2 O 2 . Cells were exposed to different concentrations of H 2 O 2 for 30 min and analysed for viability using a WST-1 viability assay after recovery times of 0 h, 2 h, 4 h, and 24 h in smooth muscle cell growth medium without H 2 O 2 . Data were statistically analysed by two-way ANOVA and Tukey’s multiple comparison test; ns: not significant, *: p < 0.05, ** p < 0.01, ****: p < 0.0001.

Article Snippet: The immortalized VSMC line (iHAoSMC) was obtained from InnoProt ( P10456 -IM, Derio, Spain).

Techniques: Viability Assay, Comparison

Journal: Cells

Article Title: MitoQ Has Diverse Effects on H 2 O 2 -Induced Oxidative Stress and the NRF2 Signalling Pathway in Aortic Smooth Muscle Cells of Different Origins

doi: 10.3390/cells15161499

Figure Lengend Snippet: Response of AAA-SMC, immortalized iHAoSMC, and healthy VSMC against acute oxidative stress induced by exposure to H 2 O 2 . ( A ) Treatment scheme for induction of acute oxidative stress. Cell lines were treated with 10 nM of MitoQ or vehicle for 7 days before they were challenged with 100 µM H 2 O 2 for 30 min. ( B ) NRF2 binding activity to DNA (antioxidative response element, ARE). Nuclear extracts were harvested immediately after H 2 O 2 treatment and analysed by using an NRF2 activity assay. ( C ) Transcriptional expression of the NRF2 target gene HMOX1 (HO1), 6 h after H 2 O 2 treatment. Data show the 2 −ΔΔCt values derived from qPCR. ( D ) HO1 protein expression was analysed by ELISA. Protein extracts were harvested after 24 h of regeneration. ( B – D ) Data are derived from n = 2 individual AAA-SMC cultures, n = 3 individual healthy VSMC cell cultures, and one iHAoSMC line. Bars represent the mean ± SEM. Data were statistically analysed by two-way ANOVA and Tukey’s multiple comparison test; ns: not significant, *: p < 0.05, ** p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: The immortalized VSMC line (iHAoSMC) was obtained from InnoProt ( P10456 -IM, Derio, Spain).

Techniques: Binding Assay, Activity Assay, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cells

Article Title: MitoQ Has Diverse Effects on H 2 O 2 -Induced Oxidative Stress and the NRF2 Signalling Pathway in Aortic Smooth Muscle Cells of Different Origins

doi: 10.3390/cells15161499

Figure Lengend Snippet: ROS production in AAA-SMC, immortalized iHAoSMC, and in healthy VSMC. Cells were exposed to acute oxidative stress by H 2 O 2 for 30 min, and images were taken after 24 h of regeneration. ( A ) Representative images of ROS staining from a AAA-SMC line. ROS production was examined via CellROX fluorescent dye; nuclei were stained with NucBlue. ( B ) Quantification of ROS fluorescence intensity, normalized to NucBlue fluorescence. Data are derived from n = 2 individual AAA-SMC cultures analysed fivefold ( left panel ) from n = 3 individual healthy VSMC cell cultures analysed in triplicate ( middle panel ) and an iHAoSMC line analysed in six technical replicas ( right panel ). Box plots show the median, maximum, and minimum of values derived from the ROS assay. Each colour circle represents an individual value after normalization. Data were statistically analysed by two-way ANOVA and Tukey’s multiple comparison test; ns: not significant, *: p < 0.05, ** p < 0.01, ***: p < 0.001.

Article Snippet: The immortalized VSMC line (iHAoSMC) was obtained from InnoProt ( P10456 -IM, Derio, Spain).

Techniques: Staining, Fluorescence, Derivative Assay, ROS Assay, Comparison