Fig 1.
IDN5706 reduces the levels of EDEM1 in a time-dependent manner.
(A) H4 cells were left untreated or treated for different periods of time with 250 μM IDN5706, and subsequently analyzed by Western blotting with an EDEM1 antibody. The antibody specificity was validated on protein extracts from cells stably expressing either luciferase shRNA, used as positive control (shLuc), or EDEM1 shRNA (shEDEM1). Specific antibodies against ER proteins including Calnexin, Gp78 and Bip were tested. (B) Densitometric quantification of the levels of EDEM1 in cells left untreated or treated with IDN5706 for 16 h, as shown in A. Bars represent the mean ± SD of three independent experiments (***, P < 0.001). (C) H4 cells were either left untreated (lane 1) or transfected with a plasmid encoding HA-epitope-tagged EDEM1 (lanes 2 and 3). After 16 h cells were left untreated or treated with 250 μM IDN5706 for 8 h, and cellular extracts were subjected to SDS-PAGE followed by immunoblot with mouse antibody anti-HA-epitope. In A and C, Western blotting with antibody to β-actin was used as loading control. The position of molecular mass markers is indicated on the left.
Fig 2.
IDN5706 increases LC3-II levels in different cell types.
Normal rat kidney (NRK) cells stably expressing GFP-LC3 were left untreated (A) or treated with 250 μM IDN5706 for 16 h (B) and analyzed by fluorescence microscopy. Bar, 10 μm. (C) H4 cells were treated with the indicated concentration of IDN5706 for 16 h. (D) H4 cells were treated with 250 μM IDN5706 for the indicated periods of time. (E) The indicated cell lines were left untreated or treated with 250 μM IDN5706 for 16 h. (C-E) Cell extracts were subjected to Western blot analysis with an antibody to LC3. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3. The asterisk indicates a band detected only in H4 cells (C and D). The position of a molecular mass marker is indicated on the left.
Fig 3.
H4 cells stably expressing mRFP-EGFP-LC3 were left untreated (A) or treated with either EBSS (B), 0.1 mM Chloroquine (C) or 250 μM IDN5706 (D) for the indicated time, and analyzed by fluorescence microscopy. Merging of the images in the green and red channels generated the third image in each row; in merged images red indicates presence of LC3 in acidic compartments (mRFP+EGFP-), and yellow indicates presence of LC3 in non-acidic compartments (mRFP+EGFP+). Bar, 10 μm.
Fig 4.
IDN5706 reduces phosphorylation in S2448 of mTOR.
(A) N2a cells were left untreated (lane 1) or treated 4 h with 250 μM IDN5706 (lane 2), and cellular extracts were subjected to SDS-PAGE followed by immunoblot with rabbit antibody to phosphorylated mTOR at serine 2448 (p-mTOR (S2448)), or rabbit antibody to total mTOR. Western blotting with antibody to β-actin was used as loading control. The position of a molecular mass marker is indicated on the left. (B) Densitometric analysis of the levels of p-mTOR (S2448) shown in (A). Values were normalized to the levels of total mTOR, and presented as the mean ± SD of three independent experiments. ***, P < 0.001.
Fig 5.
Depletion of Atg5 inhibits IDN5706-induced autophagy, but not EDEM1 degradation.
H4 cells stably expressing either luciferase shRNA (control; shLuc) or Atg5 shRNA (shAtg5) were treated with 250 μM IDN5706 for different periods of time. Cellular extracts were subjected to SDS-PAGE followed by Western blot with antibody to LC3, EDEM1 or Atg5-Atg12 complex. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3; mEDEM1, mature EDEM1. The asterisk indicates a band detected only in H4 cells. Western blotting with antibody to β-actin was used as loading control. The position of molecular mass markers is indicated on the left.
Fig 6.
IDN5706 inhibits proteolytic processing of APP to CTFs, and production of Aβ species.
(A) Schematic representation of APP and carboxy-terminal fragments (CTFs) indicating their topological domains and the position of the proteolytic cleavage sites by α, β and γ secretases. The cytosolic region, recognized by the anti-tail antibody, and the γ-secretase inhibitor, DAPT, are indicated. (B) H4 cells were left untreated (lane 1) or treated for 16 h either with 250 μM IDN5706 (lane 2), 1 μM DAPT (lane 3), or with a combination of 250 μM IDN5706 and 1 μM DAPT (lane 4). Cell extracts were subjected to Western blot analysis using the anti-tail antibody to the cytosolic C-terminal region of APP. Western blotting with antibody to β-actin was used as loading control. mAPP, mature APP; iAPP, immature APP. The position of molecular mass markers is indicated on the left. (C-D) CHO 7AP2 cells were cultured in DMEM containing low glucose and without fetal bovine serum, in the absence or presence of 250 μM IDN5706 for 16 h. The amount of Aβ40 and Aβ42 peptides in the culture medium was analyzed by ELISA. (E) Ratio of the amount of Aβ42 and Aβ40 peptides as an indicator of toxicity. (C-E) Values are presented as the mean ± SD of three independent experiments. *, P < 0,05 and **, P < 0,005.
Fig 7.
IDN5706 disrupts glycosylation of APP.
(A) H4 cells stably expressing an amyloidogenic version of APP tagged to GFP (APP-GFP) were treated with 250 μM IDN5706 for the indicated periods of time. Cell extracts were subjected to Western blot analysis with an antibody to GFP. Western blotting with antibody to β-actin was used as loading control. (B) H4 cells stably expressing APP-GFP, were left untreated (lanes 1–3) or treated for 16 h with 250 μM IDN5706 (lanes 4–8). Cell extracts were subjected to immunoprecipitation with a an antibody to GFP, followed by denaturation and digestion with the indicated glycosidases for 1 h at 37°C. Immunoprecipitated proteins were subjected to Western blot analysis with anti-GFP-HRP. (A-B) The position of molecular mass markers is indicated on the left. mAPP, mature APP; iAPP, immature APP.
Fig 8.
Depletion of Atg5 increases the accumulation of APP in response to IDN5706.
H4 cells stably expressing an amyloidogenic version of APP tagged to GFP, and stably expressing either luciferase shRNA (control; shLuc) (A and B) or Atg5 shRNA (shAtg5) (C and D), were left untreated (Control; A and C) or treated with 250 μM IDN5706 for 8 h (IDN5706; B and D). Cells were fixed, and labeled with a mouse monoclonal antibody to Calnexin, followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel; A-D). Stained cells were analyzed by fluorescence microscopy. Merging of the images in the green and red channels generated the third image in each row; yellow indicates overlapping localization of the green and red channels. Arrows indicate puncta of colocalization. Bar, 10 μm.
Fig 9.
Accumulated immature APP in response to IDN5706 is degraded by Atg5-dependent autophagy.
(A) H4 cells stably expressing an amyloidogenic version of APP tagged to GFP, and stably expressing either luciferase shRNA (control; shLuc) or Atg5 shRNA (shAtg5), were treated with 250 μM IDN5706 for the indicated periods of time, or (C) were left untreated (lanes 1 and 6) or treated with 250 μM IDN5706 (lanes 2–5 and lanes 7–10) for 12 h, followed by cycloheximide-chase with 150 μg/ml cycloheximide and 40 μg/ml chloramphenicol for 1–3 h in the presence of 250 μM IDN5706 (lanes 2–5 and lanes 7–10). Equivalent amounts of cell extracts were subjected to SDS-PAGE in 7.5% acrylamide gels, followed by Western blotting with an antibody to GFP. Western blotting with antibody to β-actin was used as loading control. mAPP, mature APP; iAPP, immature APP. The position of molecular mass markers is indicated on the left. (B and D) Densitometric quantification of the levels of iAPP shown either in A or C. The values depicted in the graphs represent the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01.
Fig 10.
Effects of IDN5706 on APP turnover at the ER.
(A) Similar to unfolded luminal glycoproteins that are recruited by EDEM1 from the Calnexin/Calreticulum (CNX/CLR) folding cycle (1), a fraction of newly-synthesized, immature APP (iAPP) is substrate of the endoplasmic reticulum-associated protein degradation (ERAD) (2). (B) The levels of EDEM1 are reduced upon IDN5706 treatment by a process independent on the levels of Atg5. This is a mechanism that needs further elucidation (?). Accumulation of iAPP in the ER elicited by IDN5706 activates an alternative early degradation of iAPP by Atg5-dependent autophagy (3).