Fig 1: Chemotherapy induces MCF-7 and LS174T cell senescence and AGR2 is overexpressed during CIS escape. (A) Senescence was induced in MCF-7 breast cancer cell line following doxorubicin treatment (25 ng/ml) for 96 h and in the colorectal cell line LS174T following sn38 treatment (5 ng/ml) for 96 h. The senescent state was evaluated by p21 expression using western blotting and β-galactosidase staining (MCF-7 n=3 ± standard deviation, *P-value=0.0152); LS174T n=2 ± standard deviation; magnification, ×400) and DNA damage using FACS quantification of γ-H2AX staining (n=3 ± standard deviation, *P-value<0.0001). AGR2 expression was evaluated using (B) western blotting (MCF-7 n=3; LS174T n=3) and (C) quantitative PCR (n=3 ± standard deviation, *P-value=0.385, **P-value=0.0085) in non-treated, senescent and emergent cells. (D) The kinetic of AGR2 expression during cell emergence was assessed by western blotting (n=3). AGR2, anterior gradient 2; CIS, chemotherapy-induced senescence.
Fig 2: AGR2 acts through the AKT signaling pathway during CIS escape. (A) Cell emergence was measured by colony counting after iAKT treatment (100 µM; n=3 ± standard deviation; **P-value=0.0011) and Torin treatment (10 nM; n=3 ± standard deviation; *P-value=0.0231) (B) The phosphorylation of AKT protein on emergent cells transfected with siAGR2 or treated with soluble AGR2 (CM and rAGR2) was assessed after two days of emergence using western blotting (n=3). (C) The phosphorylation of S6 and RICTOR after AGR2 suppression or eAGR2 and rAGR2 treatment in emergent cells was evaluated using western blotting following two days of emergence (n=3). AGR2, anterior gradient 2; CIS, chemotherapy-induced senescence; si, small interfering; iAKT, inhibitor of AKT; eAGR2, extracellular AGR2; rAGR2, recombinant AGR2; NT, non-treated; p-, phosphorylated.
Fig 3: Soluble AGR2 favors CIS escape. (A) Conditioned media were generated from MCF-7 and 293 transfected with either pcDNA3.0 or pcDNA3.0 AGR2 wt. eAGR2 expression in the media was assessed through western blotting. (B) Emerging MCF-7 were treated at day two of emergence with the conditioned media. The number of emerging clones was evaluated with crystal violet staining after eleven days of emergence (CM from MCF-7: n=6 ± standard deviation, **P-value=0.0022; CM from 293: n=4 ± standard deviation). (C) Emerging MCF-7 were treated at day two of emergence with recombinant human AGR2 at 200 ng/ml. The emerging clones were revealed by crystal violet staining after 11 days of emergence (n=4 ± standard deviation, *P-value=0.0286). AGR2, anterior gradient 2; iAGR2, intracellular AGR2; CIS, chemotherapy-induced senescence; wt, wild type; eAGR2, extracellular AGR2; rAGR2, recombinant AGR2; NT, non-treated.
Fig 4: AKT and mTOR pathways are deregulated following AGR2 suppression during CIS escape. GSEA was performed on the proteomes obtained from emergent cells transfected with control siRNA (right part of the plots) or siAGR2 (left part of the plots). mTOR, mammalian target of rapamycin; AGR2, anterior gradient 2; CIS, chemotherapy-induced senescence; GSEA, Gene Set Enrichment Analysis; si, small interfering; Ctrl, control.
Fig 5: AGR2 does not regulate the p53/p21 pathway during cell emergence. (A) The expression of senescence markers phosphoSer15-p53 and p21 was evaluated using western blotting on emergent cells transfected with siRNA directed against AGR2 (n=3). (B) The expression of senescence markers phosphoSer15-p53 and p21 was evaluated using western blot on emergent cells following treatment with conditioned media expressing AGR2 (n=3) or with recombinant AGR2 (200 ng/ml; n=3). AGR2, anterior gradient 2; si, small interfering; rAGR2, recombinant AGR2; wt, wild type; si, small interfering; Ctrl, control; p-, phosphorylated.
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