Figure 1.
Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in Hek293 cells.
A) Western-blot analysis of homogenates from Hek293 cells treated with different siRNA against laforin (#2198 and #2108) or control siRNA (C-). Thirty hours after silencing, cells were cultured either in presence or absence of 1 µM thapsigargin for 18 h. A representative blot of three independent experiments is shown. Right panel shows quantification (mean±SEM) of different blots expressed in percentage respect to cells treated with control siRNA (100%). Tubulin was used as a loading control. B) Similar analysis as in A) but in cells treated with tunicamycin (2 µg/ml, 18 h). C) Proteasome activity was assayed in Hek293 cells under different treatments as above by using a luminogenic proteasome substrate (succinyl-leucine-leucine-valine-tyrosine-aminoluciferine) and referred to the values obtained in control cells treated with thapsigargin. The effect of laforin was assayed by either depleting endogenous laforin (#2108) or by overexpressing laforin and malin (Laf/Mal). Cell extracts (50 µg) were also analyzed by western blotting using antibodies against two different subunits of the 26S proteasome (anti-S1 and anti-β2) (lower panel).
Figure 2.
Lack of laforin enhances cell sensitivity to endoplasmic reticulum stress in human neuroblastoma SH-SY5Y cells.
SH-SY5Y cells were stably transfected with empty pSuper.neoGFP vector or pSUPER-laforin expressing a shRNA against laforin, as described in Materials and Methods. A) Stable transfectants were treated with 1 µM thapsigargin for 18 h and analyzed by western-blot using anti-BIP/Grp78, anti-laforin and anti-tubulin antibodies. B) The expression of additional ER-stress markers such as CHOP and pEIF2α (phospho-EIF2alpha) was analyzed in the stable transfectants at different times after the treatment with 1 µM thapsigargin (see Supplementary Fig. S1 for time course appearance of different ER-stress markers); right panel shows quantification (mean±SEM) of different blots expressed as percentage respect to tubulin lebels. C) Quantitative real time PCR analysis of the expression of BIP/Grp78 in stable transfectants treated with 1 µM thapsigargin for 18 h; expression of target gene was normalized using GAPDH as an internal control; data are expressed as fold induction over untreated control (mean±SEM) of four independent measurements. D) Proteasome activity was measured in extracts from transfectants used in A) treated with 1 µM thapsigargin. E) Growth of control and laforin depleted transfectants was assessed by flow cytometry as described in Materials and Methods. The percentage of apoptotic cells in the sub-G1 population was measured in cells treated or not with 1 µM thapsigargin for 18 h. The left panel shows a representative analysis of three independent experiments of cells treated with thapsigargin; the right panel shows the corresponding mean±SEM. F) Extracts from laforin-depleted and control cells treated or not with 1 µM thapsigargin for 18 h were analyzed by western-blotting using anti-caspase 3 and anti-activated caspase 3 antibodies; tubulin was used as a loading control. G) Cell sensitivity of laforin depleted and non-depleted cells to thapsigargin treatment. Stable laforin depleted SH-SY5Y and control cells were cultured in 96-well plates and treated with different amounts of thapsigargin for 24 hr. Then, cell viability was assessed using the AlamarBlue assay as described in Materials and Methods. Each point represents mean±SEM of three independent measurements and expresses the percentage of viability respect to the corresponding untreated cells.
Figure 3.
Laforin deletion results in increased ER stress in the liver of Epm2a-/- mice.
A and B) Western blot analyses of laforin and ER-stress markers (BIP/Grp78, CHOP, SOD2) in liver (A) and whole brain (B) biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2). A representative blot of four different animals of each type is shown. Right panels show normalized intensities (mean±SEM; n: 4) of different markers expressed as a percentage with respect to control mice. Tubulin was used as a loading control. * In brain samples, a band of 94 kDa is recognized by the anti-BIP/Grp78 antibody instead of the corresponding 78 kDa band.
Figure 4.
Proteasome activity in biopsies of Epm2a+/+ and Epm2a-/- mice.
A) Proteasome activity was assessed by using the luminogenic proteasome substrate (succinyl-leucine-leucine-valine-tyrosine-aminoluciferine) in extracts from whole brain and liver of Epm2a+/+ and Epm2a-/- mice as described in Materials and Methods. Data are presented as luciferase activity (mean±SEM; n: 4), and represent the average of two luciferase activity determinations for each sample. B) Fifty micrograms of total lysates from liver and whole brain biopsies of two Epm2a+/+ (A and B) and two Epm2a-/- mice (1 and 2) were analyzed by SDS–PAGE and western blotting using anti-S1 (19S regulatory particle), anti-β2 (20S proteasome) and anti-tubulin antibodies.
Figure 5.
Expression of ER-stress markers is increased in brain necropsies of a LD patient.
A) Quantitative-real time PCR analysis of ER-stress markers in brain necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements. B) Extracts from brain necropsies from both control and LD patient were analyzed by western blot using the indicated antibodies. Membranes were probed with anti-tubulin antibodies to ensure equal protein loading. * non-specific band. C) Quantitative-real time PCR analysis of ER-stress markers in skeletal muscle necropsies from both control and LD patient. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of four independent measurements.
Figure 6.
Malin expression is induced under conditions of ER-stress.
A) SH-SY5Y cells were treated or not with 1 µM thapsigargin for 18 h and levels of mRNAs were measured by quantitative-real time PCR. Expression of target genes was normalized using GAPDH as an internal control. Data are expressed as fold induction over untreated cells (mean±SEM) of three independent measurements. B) Quantitative-real time PCR analysis of EPM2B gene expression in brain necropsies from both control and LD patient normalized using GAPDH as an internal control. Data are expressed as fold induction over control (mean±SEM) of two independent measurements.
Table 1.
Sequences of the primers and probes used in this work.