Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

Cell culture and transfections

Human embryonic kidney 293T (HEK293T) and human glioblastoma cell lines LN229 were purchased from the American Type Culture Collection (ATCC) and reauthenticated by Short Tandem Repeat (General Biosystems (Anhui) Corporation Limited). Cells were grown in DMEM (Gibco, C11995500BT) media supplemented with 10% fetal bovine serum (FBS; Hyclone), and penicillin-streptomycin (100 U/mL penicillin, 100 µg/mL streptomycin, Thermo Fisher Scientific, 15140163), a humidified atmosphere of 5% CO2 at 37 °C.

Cells were transfected with plasmids or siRNA using Lipofectamine 2000 reagent (Invitrogen) or Lipofectamine® RNAiMax Reagent (Invitrogen) following the supplier’s instructions. The cells were collected and analyzed by western blot.

Cells were treated with dimethyl sulfoxide (DMSO, Solarbio, D8371), Z-Leu-Leu-Leu-al (MG132, MCE, HY-13259), EBSS (Gibco,14155-063), Bafilomycin (MCE, HY-100558), Rapamycin (Selleckchem, AY-22989) or H2O2 (Beijing Tong Guang Fine Chemicals Company, 7722-84-1).

The following human siRNAs were used: si-Control was purchased from JTS scientific; si-Smurf1: 5ʹ-GCGUUUGGAUCUAUGCAAATT-3ʹ; si-p62: 5ʹ-GGCUGAAGGAAGCUGCCUU-3ʹ; si-Nrf2: 5ʹ-GAAUGGUCCUAAAACACCATT-3ʹ; si-NBR1: 5ʹ-AAACCUGACUUUGGCUUCCACAGAA-3ʹ.

Quantitative RT-PCR

Total RNA was extracted from each sample using TRNzol Universal reagent (Tiangen, DP424) according to the manufacturer’s instructions. 500 ng of RNA was used as a template to generate cDNA using the ABScript II RT Mix for qRT-PCR with gDNA Remover (ABclonal, RK20403) according to the manufacturer’s instructions. For PCR amplification of cDNA, 5 μl of the reverse transcription reaction solution was used with sense and anti-sense primers of the gene of interest using 2 × Universal SYBR Green Fast qRT-PCR Mix (ABclonal, RK21203), and primers corresponding to β-actin were included as an internal control. Primer sequences were NBR1, 5ʹ-AGGAGCAAAACGACTAGCTGC-3ʹ(forward) and 5ʹ-TCTGGGGTCTTCATGTCTGAT-3ʹ(reverse); p62, 5ʹ-GACTACGACTTGTGTAGCGTC-3ʹ(forward) and 5ʹ-AGTGTCCGTGTTTCACCTTCC-3’(reverse); β-actin, 5ʹ-CATGTACGTTGCTATCCAGGC-3ʹ(forward) and 5ʹ-CTCCTTAATGTCACGCACGAT-3ʹ(reverse); Smurf1, 5ʹ-AGATCCGTCTGACAGTGTTATGT-3’(forward) and 5ʹ-CCCATCCACGACAATCTTTGC-3ʹ(reverse); NQO1, 5ʹ-GAAGAGCACTGATCGTACTGGC-3ʹ(forward) and 5ʹ-GGATACTGAAAGTTCGCAGGG-3’(reverse); HO1, 5ʹ-AAGACTGCGTTCCTGCTCAAC-3ʹ(forward) and 5ʹ-AAAGCCCTACAGCAACTGTCG-3ʹ(reverse).

Western blot

Western blot was used to analyze protein expression as described. In brief, cells were collected and lysed with RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER). Protein lysates with 5 × loading buffer (60 mM Tris-HCl, pH 6.8; 2% SDS; 0.1% Bromophenol blue; 25% Glycerol; 14.4 mM β-Mercaptoethanol) were sonicated and boiled for 10 min. Protein samples were first resolved by SDS-PAGE, then transferred to a nitrocellulose membrane, and subsequently incubated with the primary antibody. After incubation with peroxidase-conjugated secondary antibodies, the signals were visualized by enhanced chemiluminescence according to the manufacturer’s instructions. The following primary antibodies were used: p62 (ENZO, BML-PW9860; MBL, PM045; MBL, M162-3), p-p62 Ser403 (Cell Signaling Technology, #39786), p-p62 Ser349 (Cell Signaling Technology, #16177), β-actin (Sigma, A1978), Nrf2 (Proteintech, 16396-1-AP), NDP52 (Proteintech, 12229-1-AP), NBR1 (Proteintech, 160004-1-AP), LC3B (Sigma, L7543), OPTN (Santa, sc-271549), Smurf1 (Santa, sc100616; Abcam, ab57573), GFP (Proteintech, 66002-1-lg), HA (MBL, M180-3), Flag (Sigma, F1804), c-Myc (Proteintech, 16286-1-AP), Keap1 (Proteintech, 10503-2-AP), BAG3 (Santa, sc-136467), p-mTOR (Ser2448) (Cell Signaling Technology, #2971), mTOR (Cell Signaling Technology, #2983), p-p70S6K (Thr389) (Cell Signaling Technology, #9205), p70S6K (Santa, sc-8418). The following secondary antibodies were used: Horseradish Peroxidase-conjugated goat anti-rabbit IgG (BOSTER, BA1054), Horseradish Peroxidase-conjugated goat anti-mouse IgG (BOSTER, BA1050).

Cytoplasmic and nuclear protein extraction

Cells were collected and lysed with sucrose buffer (320 mM Sucrose; 3 mM CaCl2; 2 mM MgAc; 0.1 mM EDTA; 1 mM DTT; 0.5 mM PMSF) with 0.5% NP40 for 40 min on ice. The sample was fractionated after centrifugation at 600 × g for 15 min at 4℃. The supernatant was collected as cytoplasmic fraction. The precipitate was washed using sucrose buffer and centrifugated at 600 × g for 10 min at 4℃ three times. The precipitate was resuspended using RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER), and used as nuclear fraction after sonication.

Immunoprecipitation

Cells were lysed with lysis buffer (50 mM Tris, pH8.0; 150 mM NaCl; 1% NP40; 0.5% sodium deoxycholate) supplemented with 1 mM PMSF and 1% phosphatase inhibitor (BOSTER), and then sonicated with bath sonicator, centrifuged at 12,000 rpm for 10 min at 4 ℃. The supernatant of lysates was incubated with primary antibody and protein G agarose beads (Solarbio, R8300) at 4 ℃. The immunocomplexes were washed with ice-cold 1 × PBS four times and then boiled in the SDS sample buffer for 10 min. Both lysates and immunoprecipitates were identified by western blot.

Fluorescence recovery after photobleaching (FRAP)

Cells were grown in glass base dishes. FRAP assay was conducted using the FRAP module of the NIKON N-SIM E confocal microscopy system. Proteins with fluorescent label GFP and RFP were bleached using a 488 nm and a 555 nm laser beam separately. Bleaching was focused on a circular region of interest (ROI) using 70% laser power (3 s) and time-lapse images were collected. Fluorescence intensity was measured using Image J software.

Immunofluorescence microscopy

Cells were cultured on coverslips, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 (PBS) for 5 min, blocked with 1% (w/v) BSA (PBS) for 1 h, and then incubated with primary antibodies overnight. After washing, cells were incubated with fluorescence-labeled secondary antibody for 1 h at room temperature. The coverslips with cells were sealed with Fluor shield mounting medium with DAPI and imaged using a Super-Resolution Microscope (NIKON, N-SIM E). The number and size of p62 bodies were analyzed with the Image J software.

The primary antibodies were used: Smurf1 (Santa, sc100616; Abcam, ab57573), Keap1 (Proteintech, 10503-2-AP), Keap1 (Abcam, ab118285), p62 (Enzo, BML-PW9860), p62 (MBL, PM045), p62 (MBL, M162-3), LC3B (Sigma, L7543), LC3B (Novus, NB100-2220), NBR1 (Proteintech, 160004-1-AP), HA (MBL, M180-3), Nrf2 (Proteintech, 16396-1-AP), Flag (Sigma, F1804).

The secondary antibodies were used: Alexa Fluor® 555 goat anti-mouse IgG (Life Technologies, A21425). Alexa Fluor® 488 goat anti-rabbit IgG (Life technologies, A11008), Alexa Fluor® 555 goat anti-rabbit IgG (Life technologies, A21428), Alexa Fluor® 488 goat anti-mouse IgG (Life technologies, A11001), Alexa Fluor® 647 goat anti-mouse IgG (Abcam, ab150115), Alexa Fluor® 647 donkey anti-goat IgG (Abcam, ab150131), Alexa Fluor® 405 goat anti-rabbit IgG (Abcam, ab175652).

Preparing mouse embryonic fibroblasts (MEFs) from mouse embryos

Primary MEFs were obtained from embryos at 14-16 days post-coitum. The uterine horns of anesthetized pregnant mice were rinsed. The embryos were separated from placenta and placed in a 5 cm plate containing ice-cold sterile PBS. Remove the head, liver, and gut from the embryo and place the remaining portion in a 5 cm plate containing trypsin EDTA and cut into small pieces (~ 1 mm3) using a sterile razor blade and scissors. The chopped materials were incubated with 5 ml of trypsin EDTA for 30 min at 37 ℃ and then added 1 mL DMEM medium with 10% FBS to the chopped embryos, followed by centrifuging at 1000 rpm speed for 5 min. The pellets were resuspended and transferred to a 60 mm dish containing DMEM (with FBS and Penicillin/Streptomycin).

Mice

Smurf1+/+, Smurf1+/−, and Smurf1−/− mice were a kind gift from Dr. Lingqiang Zhang (Beijing Institute of Lifeomics, China). Mice were housed in specific pathogen‐free facilities, and the Ethics Review Committee for Animal Experimentation of Beijing Institute of Technology University approved the experimental protocol.

Plasmids constructs

Full-length Smurf1 cDNA was amplified and then separately inserted into PKH3-3 × HA, 3 × Flag, pEGFP-N3, RFP and PGEX-5X-1 vector. Similarly, full-length p62 cDNA was amplified and then inserted into RFP-GFP, pEGFP-N3 and RFP vector. Full-length LC3 was amplified from RFP-GFP-LC3 (Addgene, #21074) and then inserted into PET-21a-Flag vector. Smurf1-C2, Smurf1-ΔC2, Smurf1-WW, Smurf1-HECT, Smurf1-ΔHECT was amplified from Flag-Smurf1 and then separately inserted into 3 × Flag. Flag-Smurf1-CS, Flag-Smurf1-ΔWW, Flag-Smurf1-CS-ΔWW, Flag-Smurf1C699A, GFP-p62K7A/D69A and GFP-p62S349A site mutant were generated by the site-directed mutagenesis. p62K7A/D69A was amplified from GFP-p62K7A/D69A and then inserted into RFP vector. Full-length Nrf2 was amplified from pCDNA3-Myc3-Nrf2 (Addgene, #21555) and then inserted into RFP vector. Full-length NBR1 was amplified from pMXs-IP GFP-NBR1 (Addgene, #38283) and then inserted into PKH3-3 × HA and pEGFP-N3 vector.

Pull-down analysis

His-Flag, His-Flag-LC3, or GST-Smurf1 protein is extracted from E. coli strain BL21 using ProBond™ Purification System (Invitrogen, K85001) according to the manufacturer’s instructions. 20 μl of Flag beads (Sigma, A2220) were incubated with His-Flag or His-Flag-LC3 overnight at 4 °C and then washed with ice-cold sterile PBS three times. The beads were incubated with GST-Smurf1 for 6 h at 4 °C followed by washing with ice-cold PBS four times, then boiled in 2 × loading buffer (60 mM Tris-HCl, pH 6.8; 2% SDS; 0.1% Bromophenol blue; 25% Glycerol; 14.4 mM β-Mercaptoethanol) for 10 min and identified by western blot.

留言 (0)

沒有登入
gif