Figure 6: SIRT1 regulates HIS in BRCA1^mut/+ HMECs.

(a) Western blot analysis and quantification of SIRT1 levels in Prolif and Ag/HIS WT (n=3) and BRCA1^mut/+ (n=3) HMECs, Prolif and senescent (M0/HIS) WT (n=3) and BRCA1^mut/+ (n=3) HDEs, as well as Prolif and senescent (M1) WT (n=3) and BRCA1^mut/+(n=3) HMFs and HDFs. (b) ChIP analysis of SIRT1 abundance at telomeres in WT and BRCA1^mut/+ HMECs. Data are presented as the average of three WT and three BRCA1^mut/+ patient samples±s.e.m. (c) SIRT1 levels in shLuciferase (control) and shBRCA1 HMECs determined using western blot analysis. (d) SIRT1 levels (protein and mRNA) in shScr (control), shSIRT1-#1 and shSIRT1-#2 HMECs. Images of SA-β-galactosidase staining in shScr (control), shSIRT1-#1 and shSIRT1-#2 HMECs. (e, upper panel) Acetyl-pRb (Acetyl-Lysine blot) and total pRb levels in shScr (control), shSIRT1 and shBRCA1 HMECs determined with pRb IP and western blot. (lower panel) Western blot analysis of Acetyl-Lysine, total pRb and tubulin (loading control) levels in total cell lysates from shScr (control), shSIRT1 and shBRCA1 HMECs. (f) Western blot analysis of Acetyl-H3K9, total H3, Acetyl-H4K16 and total H4 levels in shScr (control), shSIRT1 and shBRCA1 HMECs from Patient 1 and Patient 2. (g) ChIP analysis of Acetyl-H4K16 and Acetyl-H3K9 abundance at telomeres in shScr (control), shSIRT1 and shBRCA1 HMECs from Patient 1 and Patient 2 (results were averaged). Student’s two-tailed t-test was used to calculate P values. (*) indicates P value within the 0.05 level of significance. Error bar, s.e. Scale bar, 100 μm.