Additional file 1 of Functional crosstalk between mTORC1/p70S6K pathway and heterochromatin organization in stress-induced senescence of MSCs
Creators: name: Liu, Hailong
name: Huang, Biao
name: Xue, Shaolong
name: U, Kin Pong
name: Tsang, Lai Ling
name: Zhang, Xiaohu
name: Li, Gang
name: Jiang, Xiaohua
Publisher: name: figshare
Year: 2020
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Evolutionary Biology
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplementary Fig. S1. Knockdown of p70S6K ameliorates H2O2-induced senescence in hUC-MSCs. β-gal staining of control siRNA- or p70S6K-siRNA- treated hUC-MSCs in the presence or absence of H2O2. 1x105 hUC-MSCs (p6-p7) were seeded in 6-well plates and transfected with siRNAs. 24 hours later, the cells were treated with H2O2 and collected 2 days afterward. Quantification data is shown below, Data are presented as the mean ± SEM. **p&lt;0.01;***p&lt;0.001 by One way Anova with Tukey’s post hoc test. Supplementary Figure S2. Heterochromatin organization is involved in Dox or H2O2-induced senescence. (A) Representative Western blot shows that the expression levels of heterochromatin marks were dramatically reduced at the late stage of senescence. 2x105 hUC-MSCs (p5-p7) were seeded in 6-well plates and treated with Dox (10-8M) for 24h or H2O2(300nM) for 3h, then washed with PBS and grown for another 2-4 days. After that, the cell lysates were collected for Western blot; (B) Representative photos and quantification of immunofluorescence staining of H3K9me3 and HP1γ in H2O2-induced hUC-MSCs at different time points (scale bar=10μm). 1x104 hUC-MSCs (p5-p7) were seeded on coverslip and treated with H2O2. The cells were washed with PBS and incubated in the fresh media for 24 hours. Quantification is shown at the right panel, mean±SEM of values from three independent experiments with triplicate wells analyzed on 6-8 cells/field from five different fields. *p&lt;0.05; **p&lt;0.01;***p&lt;0.001 by One way Anova with Tukey’s post hoc test; (C) 2x105 hUC-MSCs (p5-p7) were seeded in 6-well plates and treated with Dox with or without Chaetocin. The cells were collected 2 days afterward. β-Gal staining shows that Chaetocin aggravates Dox-induced hUC-MSC senescence (scale bar=100μm). Supplementary Figure S3. Rapamycin promotes heterochromatin organization in H2O2-induced senescence. 1x104 hUC-MSCs (p8-p9) were seeded in coverslips and treated with H2O2 with or without Rapamycin. Cells were collected after 24 hours. Representative images and quantification of immunofluorescence staining of HP1γ and H3K9me3 in control or Rapamycin-treated hUC-MSCs in response to Dox (scale bar=10μm). Quantification is shown on the right panel, mean±SEM of values from three independent experiments with triplicate wells analyzed on 6-8 cells/field from five different fields.*p&lt;0.05; **p&lt;0.01 by One way Anova with Tukey’s post hoc test. Supplementary Figure S4. Illustration of the crosstalk between mTORC1/p70S6K pathway and heterochromatin organization in stress-induced senescence. Inhibition of the mTORC1/p70S6K pathway promotes heterochromatin reorganization and maintains the steady state of heterochromatin, which may prevent DNA from further damage during stress-induced cellular senescence. We propose this could be another underlying mechanism behind the anti-senescence effects of rapamycin or other methods to suppress mTORC1/p70S6K pathway. On the other hand, heterochromatin reorganization functions as an inherent and protective mechanism to restrain DNA damage and delay the senescence process. Impairment of this protective mechanism might further activates mTORC1 pathway to deepen the senescence process.