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. 2017 Mar 17;9(3):860–876. doi: 10.18632/aging.101197

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Copyright: © 2017 Ejaz et al.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.

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(A) Morphology of ASCs infected with shCntrl and shDIRAS3 was documented using light microscope at 40x magnification. (B and C) ASCs infected with either shDIRAS3 or shControl (shCntrl) expressing lentiviruses were fixed and stained for SA-β-GAL. Percentage of SA-β-GAL positive cells was calculated by scanning 5 low-power magnification fields (n=3). (D) ASCs were transduced by indicated lentiviruses with increasing MOI and cell lysates immunoblotted using phospho-Ser-139 Gamma H2A.X antibody. β-Actin served as a loading control. DIRAS3 was KD in ASCs using lentiviruses expressing specific shRNA at increasing MOI. Cell lysates were immunoblotted with the specific antibodies to investigate accumulation of senescent associated proteins p16^INK4A, p21^CIP1, p53 phosphorylated p53 (S15), Rb and pRb (S807/811). β-Actin served as a loading control. Fold changes in densitometric band intensities for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl was taken as 1. Western blot shown is from replicate from one donor with similar protein expression pattern was observed with 2 different donors. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.

(A) Morphology of ASCs infected with shCntrl and shDIRAS3 was documented using light microscope at 40x magnification. (B and C) ASCs infected with either shDIRAS3 or shControl (shCntrl) expressing lentiviruses were fixed and stained for SA-β-GAL. Percentage of SA-β-GAL positive cells was calculated by scanning 5 low-power magnification fields (n=3). (D) ASCs were transduced by indicated lentiviruses with increasing MOI and cell lysates immunoblotted using phospho-Ser-139 Gamma H2A.X antibody. β-Actin served as a loading control. DIRAS3 was KD in ASCs using lentiviruses expressing specific shRNA at increasing MOI. Cell lysates were immunoblotted with the specific antibodies to investigate accumulation of senescent associated proteins p16^INK4A, p21^CIP1, p53 phosphorylated p53 (S15), Rb and pRb (S807/811). β-Actin served as a loading control. Fold changes in densitometric band intensities for phosphorylated proteins normalized to un-phosphorylated total proteins, acquired by image J were compared. Band intensity of shCntrl was taken as 1. Western blot shown is from replicate from one donor with similar protein expression pattern was observed with 2 different donors. All error bars represents the means ± SEM. p values * = p<0.05, **= p<0.001 and *** = p<.0001.