Figure 3.
Induction of oxidative stress–induced cellular senescence by ARL6IP1 deficiency. (A) Representative dendritic arborization of primary rat hippocampal neurons after transfection with ARL6IP1-shRNA (shARL6IP1) and scramble shRNA (shControl) plasmids (600×; scale bar: 20 μm). (B) Sholl profiles show that dendritic arborization was attenuated by transfection of shARL6IP1. Means ± SEM of data from shControl and shARL6IP1 are shown (n = 10/group). (C and D) Numbers of primary and secondary dendrites were analyzed. Means ± SEM of data from shControl and shARL6IP1 neurons are shown (n = 10/group) and are analyzed using Student’s t test. (E) ReNcell CX was transduced with 100 MOI of adenovirus expressing ARL6IP1 shRNA (Ad-shARL6IP1) or scramble shRNA (Ad-shControl) 2 d after incubation in differentiation media (differentiated), and apoptotic cell death was assessed by Annexin V staining using MUSE cell analyzer. (F) Iodixanol density gradient analysis of ReNcell CX transduced with 100 MOI of adenovirus expressing ARL6IP1 (Ad-ARL6IP1) and Ad-shARL6IP1 (left). Quantitative data of intensity values of each protein using ImageJ v1.57 (right). (G) MMP was determined fluorometrically with JC-1 probe in ARL6IP1 WT or KO MEFs at three passages (left, 600×; scale bar: 20 μm). The ratio of red to green JC-1 fluorescence intensity was measured using a fluorescence microplate reader (right, green fluorescent JC-1 monomers, Ex/Em = 485/529 nm; red fluorescent JC-1 aggregates, Ex/Em = 485/590 nm). Data represent mean ± SD of triplicates and are analyzed using Student’s t test. (H) mRNA level of ROS-related genes quantified using RT-qPCR in ARL6IP1 WT and KO MEFs at three passages. Data represent mean ± SEM of triplicates. (I) Protein levels of apoptosis-related genes from ARL6IP1 WT and KO MEFs at three passages. (J) Representative bright field images of passage 3 and 5 cells are shown in ARL6IP1 WT and KO MEFs (200×; scale bar: 100 μm). (K) SA-β-Gal activity was measured in ARL6IP1 WT and KO MEFs at passages 3 and 5 using a fluorescence microplate reader (Ex/Em = 360/465 nm). Data represent mean values of triplicates ± SD. *P < 0.05; **P < 0.01; ns, not significant. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F3.

Induction of oxidative stress – induced cellular senescence by ARL6IP1 deficiency. (A) Representative dendritic arborization of primary rat hippocampal neurons after transfection with ARL6IP1-shRNA (shARL6IP1) and scramble shRNA (shControl) plasmids (600×; scale bar: 20 μm). (B) Sholl profiles show that dendritic arborization was attenuated by transfection of shARL6IP1. Means ± SEM of data from shControl and shARL6IP1 are shown (n = 10/group). (C and D) Numbers of primary and secondary dendrites were analyzed. Means ± SEM of data from shControl and shARL6IP1 neurons are shown (n = 10/group) and are analyzed using Student’s t test. (E) ReNcell CX was transduced with 100 MOI of adenovirus expressing ARL6IP1 shRNA (Ad-shARL6IP1) or scramble shRNA (Ad-shControl) 2 d after incubation in differentiation media (differentiated), and apoptotic cell death was assessed by Annexin V staining using MUSE cell analyzer. (F) Iodixanol density gradient analysis of ReNcell CX transduced with 100 MOI of adenovirus expressing ARL6IP1 (Ad-ARL6IP1) and Ad-shARL6IP1 (left). Quantitative data of intensity values of each protein using ImageJ v1.57 (right). (G) MMP was determined fluorometrically with JC-1 probe in ARL6IP1 WT or KO MEFs at three passages (left, 600×; scale bar: 20 μm). The ratio of red to green JC-1 fluorescence intensity was measured using a fluorescence microplate reader (right, green fluorescent JC-1 monomers, Ex/Em = 485/529 nm; red fluorescent JC-1 aggregates, Ex/Em = 485/590 nm). Data represent mean ± SD of triplicates and are analyzed using Student’s t test. (H) mRNA level of ROS-related genes quantified using RT-qPCR in ARL6IP1 WT and KO MEFs at three passages. Data represent mean ± SEM of triplicates. (I) Protein levels of apoptosis-related genes from ARL6IP1 WT and KO MEFs at three passages. (J) Representative bright field images of passage 3 and 5 cells are shown in ARL6IP1 WT and KO MEFs (200×; scale bar: 100 μm). (K) SA-β-Gal activity was measured in ARL6IP1 WT and KO MEFs at passages 3 and 5 using a fluorescence microplate reader (Ex/Em = 360/465 nm). Data represent mean values of triplicates ± SD. *P < 0.05; **P < 0.01; ns, not significant. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal