Agarose

Bankpeptide Co., Ltd. (Hefei, China) custom-make the peptide (DEN-TAT) (sequence, K4K2KGRKKRRQRRRPPQC). 2-(2-Pyridyldithio)ethylamine hydrochloride (HY-101794-50) was purchased from MedChemExpress (Shanghai, China). Perfluorooctanoyl chloride and Cobalt chloride (CoCl₂) were from Sigma-Aldrich (St. Louis, MO, USA). TrypLETM Express, Opti-MEM^®, and HEPES buffer were from Gibco (Waltham, MA, USA). Lipo8000™, DTT, Agarose, TBE buffer, LysoTracker Green, 100 × Hoechst 33342, Calcein AM cell activity assay kit, and Propidium iodide were obtained from Beyotime (Shanghai, China). Triton X-100 was purchased from Solarbio (Beijing, China). Matrigel^® matrix was from Corning (New York, NY, USA). Sorafenib was obtained from CSNpharm (Arlington Heights, IL, USA). Glutathione was purchased from Adamas-beta (Shanghai, China). DMOG was obtained from TCL. β-Actin antibody, HIF-1α antibody, and secondary antibody were purchased from Proteintech (Rosemont, IL, USA). siRNA-targeting VEGF (siVEGF) (anti-sense strand: 5′-GAUCUCAUCAGGGUACUCCdTdT-3′, sense strand: 5′-GGAGUACCCUGAUGAGAUCdTd-3′), siRNA-targeting HIF-1α (siHIF-1α) (sense strand: 5′-CGAUCAUGCAGCUAC UACAdT dT-3′; anti-sense strand: 5′-UGUAGUAGCUGCAUGAUCGdTdT-3′), Cyanine 5 labeled siRNA (Cy5-siRNA), and negative control scrambled siRNA (siNC) were all from Genepharma (Shanghai, China).

HEK293T cells were transfected with HA-E^RNS and Flag-LC3-II encoding plasmids for 48 h and incubated on ice with immunoprecipitation lysis buffer (Beyotime, P0013). For each sample, 500 μL of lysate was incubated with the appropriate antibodies and protein A/G plus agarose (Santa Cruz Biotechnology, sc-2003) overnight. The agarose beads were washed four times with 1 mL of lysis buffer containing 1% NP-40 (Beyotime, ST366). The precipitates were detected by Western blot.

Cells were harvested at the indicated times, lysed in lysis buffer supplemented with a protease inhibitor cocktail, incubated on ice for 20 min, and cleared by centrifugation at 14,000 rpm at 4 °C for 20 min. Total protein lysate (500 μg) was incubated with agarose-conjugated protein A/G beads for 2 h at 4 °C; the lysate was washed 5X with PBS at 4 °C and centrifuged at 14,000 rpm at 4 °C for 20 min. Then, the lysate was subjected to immunoprecipitation with agarose (Beyotime)-immobilized antibodies or isotype control antibodies overnight at 4 °C. The mix was washed 3X with lysis buffer at 4 °C. The precipitated proteins were subjected to SDS-PAGE and detected by western blot.

To examine the intracellular interaction between S1 and DNAJA3, HEK293T cells were transfected with the recombinant plasmids pCMV-S1-HA and pCMV-DNAJA3-Flag using Lipofectamine 2000 Reagents (Invitrogen, Carlsbad, CA, USA). The cells were lysed for Co-IP at 24 h post-transfection. The lysates were incubated with 2 mg of mouse anti-HA antibody, mouse anti-Flag antibody, or the same amount of mouse IgG as the control; rocked end-over-end overnight at 4 °C; and then mixed with Protein A+G agarose (Beyotime, Shanghai, China) and rocked for 6–8 h. The agarose beads were washed with phosphate buffered saline (PBS) and boiled in a 1 × SDS sample buffer. The supernatant was obtained and detected with Western blotting using rabbit anti-HA or Flag mAb.

Branched polyethylenimine (BPEI, MW = 1200, BPEI_1.2K) was purchased from Alfa Aesar (Shanghai, China). MAL–PEG–NH₂ (MW = 2000) was purchased from Yare Biotech Inc. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) and cisplatin (DDP) was purchased from Meilun Biotechnology Co. (Dalian, China). Dihydrothiophen-2(3H)-imine hydrochloride (2-IT) was purchased from Macklin (Shanghai, China). HA (MW = 50,000) was purchased from Sai Taisi Biotechnology Co. (Nanjing, China). iRGD peptides, siRNAs were purchased from Sangon Biotech Co. (Shanghai, China). The siRNAs were double-stranded and more stable in the external environment. Agarose and ethidium bromide was purchased from Beyotime (Shanghai, China).

In this study, oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and HPLC-purified. All synthesized oligonucleotides are listed in Table S1. Lambda exonuclease, T4 DNA ligase, exonuclease I, Phi29 DNA polymerase, HpaII restriction endonuclease, M.SssI were acquired from New England Biolabs (Beijing, China), and r Taq DNA Polymerase and T4 PNK were obtained from Takara Biotechnology (Dalian, China) Co., Ltd., and 5-Azacytidine (5-Aza) and 5-fluorouracil were purchased from Sigma–Aldrich. Agarose, 40% polyacrylamide (29:1), and nucleic acid dye Gel Red (10,000×) were purchased from Beyotime Biotechnology (Shanghai, China). SYBR^TM Gold was obtained from Life Technologies^TM (Eugene, OR, USA).

Soft agar dishes were precoated with 0.7% agarose (Beyotime) in RPMI-1640 medium. A549 and H1299 cells were plated at a density of 5000 cells/well in 0.35% agarose over the agar base and the medium was fleshed every 3 days with various concentrations of apatinib for 2 weeks, colonies with diameters > 50 μm were counted.