The cells were counterstained with DAPI for Apoptosis rates were determined by Annexin V-/PI double staining 10 min

 C. Zhao, et al. Cancer Letters 481 (2020) 15–23 were calculated. for5min.Afterblockingwith5%bovineserumalbumin(BSA)/PBSfor 1 h, the cells were sequentially incubated with the primary antibodies 2.4. Annexin V/PI staining and Alexa Fluor-conjugated secondary (Abcam) at room temperature for 1 h. The cells were counterstained with DAPI for Apoptosis rates were determined by Annexin V-/PI double staining 10 min, and observed with a confocal laser microscope (Zeiss LSM510 as previously described[22]. Thesuitably treated cells wereharvested, Meta, Germany). washedwithabindingbuffer(Sigma-Aldrich),andthenincubatedwith 0.3μlAnnexinV-FITCandPIin100μlbindingbufferfor15mininthe 2.10. Xenograft model dark. The stained cells were observed under an inverted fluorescence microscope equipped with a digital camera (Axio Observer Z1, Zeiss, Nude BALB/c mice (male, 5-weeks old, weighing 18–22 g) were Germany). obtained from Guangdong Laboratory Animal Monitoring Institute (SCXK2008-2002), and housed under 12 h light/dark cycles with ad 2.5. Western blotting libitum access to food and water. All animal protocols were approved by the Institutional Animal Care and Use Committee of South China Western blotting was performed as previously described [23]. University of Technology. To establish the xenograft model, 6 Briefly, cells were lysed with RIPA buffer (Cell Signaling Technology), 1–10 × 10 HCT116 cells were subcutaneously inoculated into the andtheproteincontentinthelysates wasquantified.Equalamountsof flanks of nude mice, and tumor growth was measured every 3 days. protein per sample were separated by 6–15% SDS–PAGE and trans- After72hofinoculation, themicewereinjected intraperitoneally with ferred onto polyvinylidene difluoride (PVDF) membranes. After se- the vehicle (10% DMSO, 30% polyethylene glycol 400 and 60% 0.9% quential probing with the primary and horseradish peroxidase-con- NaCl), 5-Fu (30 mg/kg), prodigiosin (1 mg/kg), CQ (20 mg/kg), 5- jugated secondary antibodies, the positive bands were visualized using fu + CQ or prodigiosin + 5-Fu twice weekly for 21 days (6 doses in enhanced chemiluminescence detection reagents (Santa Cruz). total). At the end of the regimen, the mice were injected with 200 μl pentobarbital sodium (1%) and euthanized 30 min later. The tumors 2.6. Autophagicflux analysis were harvested, and their volumes were calculated as previously de- scribed [23]. SW480 cells were transfected with pMRX-IP-GFP-LC3B-RFP- LC3BΔG (Addgene plasmid # 84,572) using Lipofectamine 2000 re- 2.11. Immunohistochemistry agent according to the manufacturer\'s instructions. Forty-eight hours later, fresh medium containing 1 μg/ml puromycin was added and the The xenograft tumors werefixed in formalin, embedded in paraffin stabletransfectantswereselectedover7days.Thecellswereharvested and sectioned as previously described [24]. 

The sections were im- andresuspendedin50μlPBSbeforeacquiringontheFACSAccuriflow munostained using a MaxVision kit (Maixin Biol, Fuzhou, Fujian, cytometer (BD Biosciences) as per the manufacturer\'s instructions. The China) according to the manufacturer\'s instructions. The p###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-2.png####rimary anti- GFP/RFP mean fluorescence intensity (MFI) ratios of the treated sam- bodies were used as indicated along with 50 μl MaxVision TM reagent ples were normalized to that of the untreated (set at 100%) cck8 solubility. per slide. Color was developed using 0.05% diaminobenzidine and 0.03% H O in 50 mM Tris–HCl (pH 7.6), and the sections were 2 2 2.7. Real-time quantitative PCR counterstained with hematoxylin. Pre-immune rabbit serum was used as the negative control. 6 Total RNA was extracted from 5 × 10 cells using TRIzol reagent (Invitrogen) and 1 mg from each sample was reverse-transcribed using 2 Tigecycline.12. Statistical analyses aPrimeScriptRTreagentkit(TaKaRa).FiftynanogramstotalcDNAwas amplified using SYBR Premix Ex TaqII Kit (TaKaRa), and the relative The results of this study are presented as mean ± standard de- gene expression was analyzed by the comparative Ct method, with 18s viation of at least 3 independent experiments. Statistical analyses were ribosomal RNA as the endogenous control. The following primers were performedusingSPSSsoftware.

The Student 2-tailed t-testwasusedfor usedforRT-PCR:CTSBforward,5′-AACACGTCACCGGAGAGATGA- to compare groups. 3’;reverse,5′-CCCAGTCAGTGTTCCAGGAGTT-3′,CTSDforward,5′- CGA TGC CAA TCT CCC CGT AGTA-3’; reverse, 5′-CGA TGC CAA TCT 3. Results CCC CGT AGTA-3′, SQSTM1 forward, 5′-GAA CTC CAG TCC CTA CAG ATG CC-3’; reverse, 5′-CGG GAG ATG TGG GTA CAA GG-3′, 18s for- 3.1. Prodigiosin triggers autophagy in human CRC cells ward, 5′-AAA CGG CTA CCA CAT CCA AG-3’; reverse, 5′-CCT CCAATG GAT CCT CGT TA-3’. Theeffectofprodigiosinonautophagywasexaminedintermsofthe conversionofLC3B-ItoLC3B-II.AsshowninFig.1BandC,prodigiosin 2.8. RNA interference and transfection increased the amount of LC3B-II protein in both HCT116 and SW480cellsinadoseandtime-dependentmanner.Similarresultswere The siRNAs targeting BECN1, ATG5 and ATG7, and the scrambled observed with three more CRC cell lines and two gastric cancer cell controls were purchased from Shanghai GenePharma Co. Ltd. 

The CRC lines(Fig.1DandSupplementaryFig.1),indicatingthattheautophagy cells were transfected with 2 mg target or control siRNA using inducing effect of prodigiosin is not cell-type specific. Consistent with Lipofectamine 2000 reagent (Invitrogen) according to the manufac- the increase in LC3B-II levels, prodigiosin treatment also led to a dose turer\'s instructions. The cells were harvested 24 h after transfection for and time-dependent accumulation of EGFP-LC3 puncta in the the subsequent experiments. The siRNAs sequences are provided in SW480 cells (Fig. 1E and F). Table S1. 3.2. Prodigiosin inhibits the autophagicflux in CRC cells 2.9. Immunofluorescence The accumulation of LC3B-II is the result of increased autophago- Immunofluorescence was performed as previously reported [23]. some formation and poor autolysosomal maturation. SQSTM1 is an The suitably treated cells were washed with PBS, fixed with 4% par- autophagy receptor that sequesters ubiquitinated proteins into the au- aformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 tophagosomes by directly binding to LC3B AMG-176. Since it is degraded along 17