Figure 3

Sulforaphane (SFN) treatment rescues the Hutchinson–Gilford progeria syndrome (HGPS) cellular phenotypes. (A) Population doubling levels were calculated as described in the Materials and Methods for control and HGPS cells that were mock treated (vehicle DMSO) or treated daily with 1 μ m SFN for a period of 3 or 9 days. (B) Proteasome activity was determined by measuring chymotrypsin-like proteasome activity in four control and four HGPS fibroblast lines using Suc-LLVY-AMC as a substrate. The percentage of activity was calculated relative to the activity in mock-treated control cells. Data are expressed as the mean ± SD (*P < 0.05; n = 4). (C) The same cells and culture conditions as in (B) were used to determine autophagy activity by measuring monodansylcadaverine (MDC) levels using fluorescence photometry, as indicated in the Procedures. Data are presented as the mean ± SD (*P < 0.05; n = 4). (D) Representative Western blots for lamin A/C, proteasome subunit 20S C2, Hsp27, and β-actin in total cell extracts isolated from mock-treated control and HGPS cells and cells treated with 1 μ m SFN daily for a period of 4 or 9 days. (E and F) Quantification of Westerns (D) for lamin A, lamin C, progerin, proteasome subunit 20S C2 and Hsp27 levels normalized to β-actin and presented as the fold change relative to the levels in mock-treated control cells (*P < 0.05; n = 5). (G) The proportions of lamin A, progerin, and lamin C were determined within each sample analyzed by Western blotting with anti-lamin A/C antibody in panel (D). (H) Representative Western blot of HGPS cell lysates from cultures that were mock treated or treated with SFN or SFN plus MG132 (MG) or with chloroquine (CQ). Left panel corresponds to Ponceau red staining of the blot probed sequentially with antibodies specific for progerin, ubiquitin, and β-actin (n = 3). (I) Representative Western blot of the same culture conditions as in (H) probed with antibodies specific for lamin A/C, LC3B-I and LC3B-II and β-actin (n = 3). (J) Intracellular reactive oxygen species (ROS) levels were determined by measuring oxidized dichlorofluorescein (DCF) levels using a 2′,7′-dichlorofluorescein diacetate (DCFDA) cellular ROS detection assay, as described in the Procedures. Data represent the mean ± SD (*P < 0.05; n = 3) compared with mock-treated counterparts. (K) Cellular ATP levels were measured using a CellTiter-Glo luminescence ATP assay, as described in the Procedures. Data represent the mean ± SD (*P < 0.05; n = 3) relative to mock-treated counterparts.