Fig 2: NDs API improves the ATO-based therapy in vivo. HepG2 tumor-bearing nude mice were administered intravenously (i.v.) with NS (200 μL), NDs (5 mg kg−1), ATO (0.75 mg kg−1), ATO (1.5 mg kg−1), NDs–0.75 mg kg−1 ATO mixture, or NDs–1.5 mg kg−1 ATO mixture daily for 5 consecutive days every week, with 13-week cycles. a Images of livers/tumors or excised tumors from treated tumor-bearing mice. b Weight ratio of tumors from treated mice (n = 10 for NS, 0.75 mg kg−1 ATO and 1.5 mg kg−1 ATO-treated groups, n = 8 for NDs–0.75 mg kg−1 ATO or NDs–1.5 mg kg−1 ATO-treated groups, n = 7 for NDs-treated group). **P < 0.01, ***P < 0.001 by t-test, significantly different from NS; #P < 0.05, ##P < 0.01 by t-test, significantly different from ATO. c Immunoblots of autophagy-related proteins LC3-II, p62 (left); semi-quantified analysis (n = 3) in tumor tissues from mice after treatment (right). GAPDH was used as the loading control. Normalized band densities were shown below each band. d Representative images of TUNEL stains and its quantification by Image J. Scale bar: 50 μm. ***P < 0.001 by t-test, significantly different from NS; ##P < 0.01, ###P < 0.001 by t-test, significantly different from ATO. e, f Survival curves (e, n = 10) and whole body weight curves (f) of nude mice after various treatments (n = 20). *P < 0.05; **P < 0.01, ***P < 0.001 by t-test or one-way analysis of variance (ANOVA) using SPSS, significantly different from NS; #P < 0.05, ##P < 0.01 by t-test or one-way analysis of variance (ANOVA) using SPSS, significantly different from ATO. g Immunostaining of LC3 and ATG13 in tumor tissues of NDs–1.5 mg kg−1 ATO-treated mice. Schematic showing (left) and imaging (right) indicate that LC3/Atg13 puncta are in the periphery and around the blood vessels inside the tumors. Scale bars: 20 μm. Error bars are s.d.

Fig 3: Effect of circ-PKD2 overexpression on the sensitivity of cisplatin.A–D Tumor volumes, weight, and tumor growth curves of subcutaneous implantation models of SCC-15 cells. E The expression of miR-646. F The expression of circ-PKD2. G qRT‐PCR results are showing the correlation between circ-PKD2 and miR-646. H IHC results show the expression of LC3B, P62, and ATG13 in the circ-PKD2 overexpressing and control groups. *p < 0.05, **p < 0.01.

Fig 4: Circ-PKD2 promotes Atg13-mediated autophagy by inhibiting miR-646 to increase the sensitivity of cisplatin.Schematic diagram depicting the proposed model in which circ-PKD2 regulates autophagy and cisplatin sensitivity.

Fig 5: ω-6 PUFAs activate autophagy and antioxidation via TOR-dependent and TOR-independent signaling pathways.The levels of total and phosphorylated raptor, TSC2, ULK1, ATG13, and TFEB and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with a TOR pathway inhibitor (500 nM RAPA) or activator (2 μM MHY1485) (n = 3). 17 gels were run and 17 blots were made. All of the blots were not stripped and re-probed. The blots of T-raptor, P-raptorSer792, T-TSC2, P-TSC2Ser1387, T-ULK1, P-ULK1Ser467, P-ULK1Ser555, P-ULK1Ser637, P-ULK1Ser757, T-ATG13, P-ATG13Ser355, T-TFEB, P-TFEBSer211, and T-Nrf2 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls (a, b). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with a TOR pathway inhibitor or activator. Scale bars, 50 μm. The same type of samples (LA group) was used in TOR pathway inhibitor (RAPA) group and autophagy inhibitor (3-MA) group (c, e). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with a TOR pathway inhibitor or activator (d, f). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a TOR pathway inhibitor or activator (n = 6) (g, h). The results are presented as the mean with SEM and were analyzed using Tukey’s test. Bars bearing the same letters are not significantly different among treatments (*P ≥ 0.05).