Chloroquine chq

The following stock solutions were used in the incubation experiments: manumycin A (MA; 10 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), MG132 (10 mM in DMSO; Selleck Chemicals, Houston, TX, USA), VER-155008 (VER; 20 mM in DMSO; Merck KGaA, Darmstadt, Germany), bortezomib (BTZ; 1.6 mM in DMSO; Selleckchem, Houston, TX, USA), bafilomycin A1 (BAF; 0.1 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), chloroquine (ChQ; 50 mM in DMSO; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Working solutions were freshly prepared from stock solutions prior to each experiment (in a culture medium or Optimem (Opti-MEM™ Reduced Serum Medium, GlutaMAX™ Supplement; Gibco, Thermo Fisher Scientific, Waltham, MA, USA)). Control cells were incubated with medium containing dimethyl sulfoxide (DMSO). All experiments were performed without the antibiotics addition.

HGPS and Control fibroblasts were treated with human ghrelin (1 nM; Bachem) every other day for up to 1 week, unless otherwise indicated. The lysosomal protein degradation inhibitor chloroquine (ChQ; 100 μM; Sigma‐Aldrich) was added to the cell culture medium 30 min prior to ghrelin treatment for 6 h.

Human CRC cell lines were purchased from the American Type Culture Collection (VA, USA). HCT-116 cells were cultured in DMEM (HyClone, Utah, USA) containing 10% fetal bovine serum (FBS; Gibco, NY, USA). SW480 cells were cultured in RPMI 1640 medium (HyClone, Utah, USA) containing 10% FBS. All cells were cultured in an incubator (Thermo, CA, USA) at a constant temperature of 37 °C and 5% CO₂. N-Acetylcysteine (NAC), Trolox, MLN4924, 5-Fu, and capecitabine were purchased from MedChemExpress LLC (Shanghai, China). MG132 was purchased from Santa Cruz Biotech (CA, USA). Chloroquine (CHQ) was purchased from Sigma (CA, USA). Rapamycin was purchased from Beyotime (Shanghai, China). Primary antibodies against SRSF3 and E-Cadherin were purchased from Abcam (Cambridge, UK). Primary antibodies against N-Cadherin, Caspase 3, DHCR24, CAT, Akt and phospho-Akt (Ser473) were purchased from Cell Signaling Technology (MA, USA). Primary antibodies against β-actin, mTOR, and phospho-mTOR (Ser2481) were purchased from Beyotime (Shanghai, China). Primary antibody against Vimentin was purchased from Proteintech (Wuhan, China). Primary antibody against Bcl-2 was purchased from Santa Cruz Biotech (CA, USA). Primary antibody against Flag was purchased from MBL Beijing Biotech (Beijing, China).