Pdcd4

The procedures of IHC studies were performed as previously described [23 (link)]. In brief, The samples were fixed with 10% formalin, embedded in paraffin and sliced into 5-μm sections. The slides were incubated with primary antibodies against SKP2 (Cell Signaling Technology, 1:100), PDCD4 (Cell Signaling Technology, 1:50), Caspase-3 (Cell Signaling Technology, 1:100), γ-H2AX (Abcam, 1:100) and Ki-67 antibody (Epitomics, 1:160). Staining was observed in 5 randomly selected high-power fields. The staining intensity was based on the average percentage of positive cells. The scoring results were analyzed by 2 investigators.

An IHC assay was used to examine PPARγ and PDCD4 expression in the kidneys. The sacrificed kidney tissues were embedded in paraffin and cut into 4 μm thick sections. The samples were deparaffinised with xylene and rehydrated with pure ethanol. BSA (3%) was added to the circle to evenly cover the tissue sections, which were then sealed for 60 min at room temperature. The sealing solution was gently removed, and the PPARγ (Cell Signalling Technology; USA) and PDCD4 (Cell Signalling Technology; USA) primary antibodies were used at a dilution of 1 : 200 for incubation overnight at 4°C. The sections were placed in PBS and washed by shaking on a decolorizing shaker thrice for 5 min each. After the sections were shaken slightly and dried, the tissues were covered with HRP-labeled secondary antibodies. After incubation with DAB and counterstaining with hematoxylin, the images were collected using light microscopy.

Western blotting was performed as described previously [15 (link)]. The antibodies against EZH2, PDCD4 and GAPDH were purchased from Cell Signaling Technology.

Asiatic acid, purity >98%, was provided by the Chengdu Herbpurify Co., Ltd (Chengdu, China). LPS (Escherichia coli 055:B5), GalN, and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inhibitors of AMPK (Compound C) was offered by Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, Dulbecco’s modified Eagle’s medium, penicillin, and streptomycin were acquired from Invitrogen-Gibco (Grand Island, NY, USA). Antibodies against Nrf2, GCLC, GCLM, HO-1, NQO1, P-AMPK, AMPK, P-PI3K, PI3K, P-AKT, AKT, P-ERK, ERK, PDCD4, P-P65, P65, IκBα, P-IκBα, and β-actin were obtained from Cell Signaling (Boston, MA, USA) or Abcam (Cambridge, MA, USA). Additionally, $O_2^{-}$ , H₂O₂, NO, glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and ROS test kits were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals were offered by Sigma-Aldrich (St. Louis, MO, USA), if not otherwise indicated.

Cells were harvested and whole cell, cytoplasmic and nuclear fractions were prepared as described previously [33 (link)]. Equal amounts of protein lysates were resolved on SDS-PAGE, transferred to nitrocellulose membranes and detected via immunoblotting using antibodies specific for the following proteins: eIF3b (Bethyl, #A301–761A), eIF3c (Bethyl, #A300–377A), 4EBP1 (Cell Signaling, #9644), mTOR (Cell Signaling, #2972), S6 (Cell Signaling, #2317), S6 S240/244 (Cell Signaling, #2215), p70 S6K1 (Cell Signaling, #9202), p70 S6K1 T389 (Cell Signaling, #9205), CAD (Cell Signaling, #12662), Akt S473 (Cell Signaling, #4060), PDCD4 (Cell Signaling, #9535), Fibrillarin (Cell Signaling, #2639), HA (Cell Signaling, #3724), Cyclin A (Santa Cruz, #sc-751), Cyclin D1 (Santa Cruz, #sc-718), p27^(Kip1) (BD Biosciences, #K25020), GAPDH (Trevigen, #2275-pc-100) and αTubulin (Calbiochem, #CP06). The following secondary antibodies were used: anti-rabbit IgG, HRP-linked heavy and light chain antibody from goat (Cell Signaling, #7074) and anti-mouse IgG, HRP-linked heavy and light chain antibody from horse (Cell Signaling, #7076). Signals were detected by chemiluminescence (Pierce, #32106).

Western blot was described previously [6 (link)]. Briefly, cells were lysed using Laemmli buffer containing 10% dithiothreitol (DTT, Thermo Fisher Scientific, Carlsbad, USA), and then denatured by heating at 95 °C for 5 min. Proteins were detected with antibodies against eIF4A (Cell Signaling Technology, Danvers, USA), eIF4E (Cell Signaling Technology), eIF4G (Cell Signaling Technology), PDCD4 (Cell Signaling Technology), and SA11 rotavirus VP4 (provided by professor Harry Greenberg, Stanford University School of Medicine, USA). The bound antibodies were visualized with Odyssey (LI-COR Biosciences, USA). The β-actin (Santa Cruz) was used as a loading control.

Anti-mouse S6K, S6, 4EBP, PDCD4, beta-Actin, phospho-S6K, phosphor-S6, phosphor-4EBP, and phospho-4EBP-Alexa647 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-mouse phoshoS6-PE, anti-mouse CD45.1 Pacific Blue, and anti-mouse Ki67 Brilliant Violet 605 antibodies were purchased from Biolegend (San Diego, CA, USA). Anti-GFP antibody purchased from Abcam (Cambridge, UK). Cytarabine arabinoside (Ara-C) and doxorubicin were purchased form Sigma-Aldrich (St. Louis, MO, USA). Rapamycin, Rad001, Torin2, AZD0855, and MLN(0128) were purchased from Selleckchem (Houston, TX, USA).