Reduced NRF2/Mfn2 activity promotes endoplasmic reticulum stress and senescence in adipose-derived mesenchymal stem cells in hypertrophic obese mice

Figure 2 Nuclear factor erythroid-derived 2 knockdown promotes senescence phenotype and endoplasmic reticulum stress in adipose-derived mesenchymal stem cells. A: Immunofluorescence staining showed nuclear factor erythroid-derived 2 (NRF2) nuclear localization in control adipose-derived mesenchymal stem cells (ASCs), bar = 50 μm; B: Long-axis NRF2 fluorescence intensity of ASCs in control and hypertrophic obesity groups analyzed by ImageJ software; C: Dual-luciferase assay for NRF2 activity in ASCs (n = 3); D: Western blot analysis of NRF2 in nuclear, cytoplasmic, and total cell fractions of ASCs; E: Relative quantitative analysis of nuclear NRF2 relative to H3 and cytoplasmic NRF2 relative to GAPDH for Western blot by ImageJ software (n = 3); F: Relative expression of Nrf2 and its downstream genes relative to β-actin was detected by quantitative polymerase chain reaction in the Nrf2 RNA interference assay of ASCs (n = 3); G: SA-B-Gal staining and Oil Red O staining of control and Nrf2 knockdown ASCs, bar = 100 μm; H: Percentage of SA-B-Gal staining positive cells was analyzed by ImageJ software (n = 3); I: Percentage of positive Oil Red O stained area to cell number (n = 3); J: Western blot of NRF2 and its target glutathione peroxidase 4 (GPX4), senescent proteins p16, p21, and interleukin-1β (IL-1b) and endoplasmic reticulum stress-related proteins inositol-requiring enzyme-1 alpha (IRE1a), binding immunoglobulin protein (BIP) in control and Nrf2 knockdown ASCs; K: Relative quantitative analysis of NRF2, senescent proteins and endoplasmic reticulum stress-related proteins relative to β-actin (ACTB) for Western blotting by ImageJ software (n = 3). ^aP < 0.05; ^bP < 0.01. Gclc: Glutamate cysteine ligase catalytic; Nqo1: NAD(P)H:quinone oxidoreductase 1.