Fig. 1Increase in glycogen synthase kinase 3 (GSK3) phosphorylation and mature sterol regulatory element binding protein 1 (SREBP1) in stress-induced senescence. Chang cells were treated with 200 µM deferoxamine (DFO) or H2O2 for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Flow cytometric analysis of the cell distribution after treatment of the stressors (200 µM DFO or H2O2) for 3 days. Cell size and cell granularity were analyzed by comparing forward scatter (FSC; R2+R4) and side scatter (SSC; R1+R2). Representative profiles (left) and their analyses for cell granularity (SCC analysis, right upper) and cell size (FSC analysis, right lower) are shown. (B) Representative images of senescence-associated β-galactosidase-positive cell populations are shown. (C) Western blot analysis. Quantitative analyses of the expression levels of SREBP1 mature form are shown in the lower panels. mSREBP1, mature-form SREBP1; pGSK, phosphor-GSK. aP<0.01 vs. V (DMSO control).
Fig. 2Glycogen synthase kinase 3 (GSK3) inhibition using SB415286, a specific GSK3 inhibitor, enhances cellular lipogenesis. Chang cells were treated with 7.5 µg/mL SB415286 for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Western blot analysis of protein expressions of mature sterol regulatory element binding protein 1 (SREBP1). Quantitative analyses of the expression levels of SREBP1 mature form are shown in the lower panels. (B) Western blot analyses for protein expressions of lipogenic enzymes, fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACL), and cardiolipin synthase (CRLS). (C) Cellular lipid profile of SB415286-treated cells was obtained by thin layer chromatography (TLC) as described in Methods. Representative TLC image (Ca) and quantitative estimation (Cb) of cellular lipids extracted from different numbers of cells are shown. Standard lipid mixture (Std) containing 10 µg each was used. Cholesteryl palmitate (CP, 1) belongs to nonpolar storage lipid, and cholesterol (CL, 2), cardiolipin (CA, 3), phosphatidyl choline (PC, 4) and phosphatidyl serine (PS, 5) belong to membrane lipids. 'd' in the x-axis stands for day. mSREBP1, mature-form SREBP1; PA, palmitate; PE, phosphatidyl ethanolamine. aP<0.01 vs. V (DMSO control).
Fig. 3Glycogen synthase kinase 3 (GSK3) inhibition induces senescence of Chang cell, accompanying an increase in cellular membranous organellar mass. Chang cells were treated with 7.5 µg/mL SB415286 (SB) for the indicated periods. Dimethyl sulfoxide (DMSO) was used as vehicle control (V). (A) Subcellular organellar masses were estimated by comparing the fluorescence intensities with flow cytometry after staining cells with organelle-specific dyes as described in Methods. Representative images of the fluorescence stained cells are shown in the lower panels. (B) Cell size and cell granularity were analyzed by comparing forward scatter (FSC; R2+R4) and side scatter (SSC; R1+R2). (Ba) Quantitative analyses for cell size (FSC analysis, left) and cell granularity (SSC analysis, right). (Bb) Representative cell distribution profiles are shown. (C) Quantitative analysis for senescence-associated β-galactosidase-positive cell populations (left) and representative images (right) are shown. ER, endoplasmic reticulum; BODIPY, boron-dipyrromethene. aP<0.01 vs. V (DMSO control).
Fig. 4Glycogen synthase kinase 3 (GSK3) knockdown induces senescence, accompanying an increase of mature-form sterol regulatory element binding protein 1 (mSREBP1) expression. Chang cells were transfected with si-GSK3α, si-GSK3β, and both si-GSK3α/β for 3 days. (A) Western blot analysis (Aa) and quantitative analysis (Ab) of mSREBP1 (mature form) levels. (B) (Ba) Cell growth rate. (Bb) Representative images (upper) and quantitative analysis of senescence-associated β-galactosidase-positive cell populations (lower) are shown. (C) Quantitative analyses for cell size (Ca, forward scatter; FSC analysis, left) and cell granularity (Cb, side scatter; SSC analysis, right). aP<0.05; bP<0.01 vs. negative control (NC).
Fig. 5Sterol regulatory element binding protein 1 (SREBP1)-mediated lipogenesis is the key event of glycogen synthase kinase 3 (GSK3) inhibition-induced senescence. Quantitative analysis of senescence-associated β-galactosidase-positive cell populations was used to evaluate the extent of senescence. (A) Chang cells (1×104) seeded on 6-well plates were transfected with siRNAs for SREBP1 and then treated with 7.5 µg/mL SB415286 for 4 days. (B) Chang cells were transfected with siRNAs for ATP citrate lyase (ACL) and then treated with 7.5 µg/mL SB415286 for 4 days. (C) Chang cells were pretreated with the indicated concentrations of cerulenin (Cer; Sigma C2389) and C75 (Sigma 5490) for 24 hours and then further treated with 7.5 µg/mL SB415286 for 4 days. siRNA with a random sequence was used as the negative control (NC), and dimethyl sulfoxide was used as the vehicle control (V). aP<0.05; bP<0.01 vs. NC or V.